TW201114895A - Treatment using reprogrammed mature adult cells - Google Patents

Abstract

A method of treating various diseases, disorders, or conditions in patient using reprogrammed cells such as retrodifferentiated, transdifferentiated, or redifferentiated cells. The method comprises obtaining committed cells from the patient, retrodifferentiating the committed cells to obtain retrodifferentiated target cells, and administering the retrodifferentiated cells to the patient. In certain embodiments, the method comprises obtaining committed cells from the patient, transdifferentiating the committed cells to obtain transdifferentiated target cells, and administering the transdifferentiated target cells to the patient. The retrodifferentiated or transdifferentiated target cells repair or replenish tissue or cells in the patient.

Description

201114895 六、發明說明： 【發明所屬之技術領域】 在本文所引述之文件中引述或參照之所有文件與在本 文所述及之任何產品或倂入本文以供參考的任何文件中的 任何製造商指示、說明、產品規格及產品表一起藉此倂入 本文以供參考，並可用於本發明的實例中。 本發明關於一種利用已重新編程細胞，諸如已反分化 、已轉分化或已重新分化細胞治療病患中的各種疾病、病 症或症狀之方法。此方法包含從病患獲得定型細胞、反分 化定型細胞以獲得已反分化標的細胞及以已反分化細胞投 予病患。在某些具體例中，此方法包含從病患獲得定型細 胞，轉分化定型細胞以獲得已轉分化標的細胞及以已轉分 化標的細胞投予病患。已反分化或已轉分化標的細胞修復 或重新補充病患中的組織或細胞。 【先前技術】 幹細胞係以彼等經由有絲細胞***使彼等更新及分化 成多樣範圍的特別細胞類型之能力爲特徵。兩種廣泛類型 的哺乳類幹細胞爲從胚細胞之內細胞團分離之胚胎幹細胞 及在成人組織中發現的成人幹細胞。在發育胚胎中，幹細 胞可分化成所有的特別胚胎組織。在成年生物體中，幹細 胞及祖細胞重新補充特別細胞且維持再生器官（諸如血液 、皮膚或腸道組織）的正常轉變。 在發育胚胎中有大量的幹細胞，雖然幹細胞的量因發 -5- 201114895 育進展而降低。相較之下，成年生物體含有侷限於某些身 體部位的有限數量之幹細胞。 幹細胞的醫療應用具有變更多種疾病或病症之治療的 潛力。雖然一些成人幹細胞療法（諸如骨髓移植）已存在 ，但是醫學硏究人員利用幹細胞參與治療更廣泛的各種疾 病，尤其包括癌症、帕金森氏病、脊髓傷害、肌萎縮性脊 髓側索硬化症、多發性硬化症及肌肉損傷。此等療法可利 用幹細胞分化成治療疾病所必要之細胞類型的能力。 然而，有關於利用幹細胞成功治療此等病痛的不確定 性與可容易獲得幹細胞的議題。例如，造血幹細胞傳統係 從骨髓、生長因子流動的（mobilized)之末梢血液或臍 帶血（胎盤）分離萃取而來。造血幹細胞亦可從利用試管 內受精技術所獲得的胚胎萃取之胚胎幹細胞（ES)製備而 來。然而，從這些來源萃取是麻煩且有時危險的，並可受 到倫理議題的挑戰。可從這些來源獲得的幹細胞數量進一 步受到限制。而且，幹細胞可能經歷分化成治療病痛所必 要之細胞的困境。 【發明內容】 本發明關於已重新編程細胞修復病患中的組織或重新 補充病患中的組織或細胞的用途。例如，本發明關於從已 分化或定型細胞的反分化所獲得的已反分化細胞修復病患 中的組織或重新補充病患中的組織或細胞的用途。本發明 亦關於從已分化或定型細胞的轉分化所獲得的已轉分化細 -6 - 201114895 胞修復病患中的組織或重新補充病患中的組織或細胞的用 途。 此應用部分係基於從病患獲得的定型或體細胞可重新 編程以得到不同系譜之細胞及這些已重新編程細胞可投予 病患以修復或重新補充組織或細胞的本申請者之發現。重 新編程法的實例包括反分化法及轉分化法。 因此，申請者發現定型細胞可進行重新編程以得到不 同系譜之細胞。例如，定型細胞可進行反分化以得到已反 分化細胞，例如較低分化之細胞（諸如多能性幹細胞）， 且這些已反分化細胞可投予病患以修復或重新補充組織或 細胞。從病患獲得的定型細胞可進行轉分化以得到已轉分 化細胞，例如不同於定型細胞的系譜之細胞，且這些轉反 分化細胞可投予病患以修復或重新補充組織或細胞，作爲 另一實例。 本發明包含一種藉由已重新編程細胞投予病患而修復 或重新補充病患中的組織或細胞系譜之細胞的方法。本發 明特別包含一種修復或重新補充病患中的組織或細胞系譜 之細胞的方法，其包含（i)獲得定型細胞；（π)反分化 定型細胞以獲得已反分化標的細胞；及（in)以已反分化 標的細胞投予病患，其中已反分化標的細胞重新分化成細 胞系譜之細胞。這些已重新分化細胞可具有與定型細胞相 同的細胞系譜或不同的細胞系譜。 本發明亦包含一種修復或重新補充病患中的組織或細 胞系譜之細胞的方法，其包含（i)獲得定型細胞；（π) 201114895 轉分化定型細胞以獲得已轉分化標的細胞；及（no以已 轉分化標的細胞投予病患。 在一些具體例中，病患可遭受疾病、病症或症狀，其 包括（但不限於此）骨髓衰竭、血液學症狀、再生不全性 貧血、/3-地中海貧血、糖尿病、運動神經元疾病、帕金 森氏病、脊髓傷害、肌肉萎縮症、腎疾病、多發性硬化症 、充血性心臟衰竭、C型肝炎病毒、人類免疫缺乏病毒、 頭部創傷、脊髓傷害、肺疾病、抑鬱症、非阻塞性無精症 、男性更年期、更年期與***症、回春作用、硬皮病潰瘍 、牛皮癖、皺紋、肝硬化、自體免疫疾病、禿髮、色素沉 著性視網膜炎、角膜結晶失養症/失明、糖尿病及***症 。因此，在一些具體例中，已重新編程細胞可爲治療病患 中的再生不全性貧血、白血病、淋巴瘤或人類免疫缺乏病 毒之骨髓細胞。 在一些具體例中，已重新編程標的細胞，諸如已反分 化細胞、已轉分化標的細胞或已重新分化細胞可包括（但 不限於此）多能性幹細胞、多能性生殖細胞、造血幹細胞 、神經元幹細胞、上皮幹細胞、間葉幹細胞、內胚層與神 經外胚層幹細胞、生殖細胞、胚外細胞、胚胎幹細胞、腎 細胞、肺泡上皮細胞、內胚層細胞、神經元、外胚層細胞 、胰小島細胞、腺泡細胞、卵母細胞、***、造血細胞、 肝細胞、皮膚/角質化細胞、黑色素細胞、骨頭/骨細胞、 毛髮/真皮乳頭細胞、軟骨/軟骨細胞、脂細胞/脂肪細胞、 骨骼肌細胞、內皮細胞、心肌/心肌細胞及滋養層細胞。 -8- 201114895 在某些具體例中，定型細胞係從血液或相關組織（包 括骨髓）獲得。定型細胞可從全血獲得及/或可經由血漿 分離術獲得。血液可爲流動的（mobilized)或未流動的 (unmobilized )血液，。此等定型細胞包括（但不限於此 )T-細胞、B細胞、嗜酸性細胞、嗜鹼性細胞、嗜中性細 胞、巨核細胞、單細胞、紅血球、顆粒性細胞、肥胖細胞 、淋巴細胞、白血球、血小板及紅血細胞。或者，定型細 胞可從來自中樞神經系統或末梢神經系統之神經元組織、 肌肉組織或來自皮膚的表皮及/或真皮組織獲得。 在一些具體例中，定型細胞係從病患的血液或組織獲 得。在一些具體例中，定型細胞獲自的病患與被投予已重 新編程標的細胞，諸如已反分化標的細胞或已轉分化標的 細胞的病患爲相同的病患。 在一些具體例中，定型細胞係藉由定型細胞與劑接觸 而反分化。例如，定型細胞可以劑培育。在某些具體例中 ，劑接合介導在定型細胞表面上的抗原補獲、識別或表現 之受體。受體可爲第I型MHC抗原或第II型MHC抗原 。在一些具體例中，第I型抗原爲HLA-A受體、HLA-B 受體、HLA-C受體、HLA-E受體' HLA-F受體或HLA-G 受體及第II型抗原爲HLA-DM受體' HLA-DP受體、 HLA-DQ受體或HLA-DR受體。 在某些具體例中，劑可爲受體之抗體，諸如受體之單 株抗體。在一些具體例中，抗體爲單株抗體CR3/43及單 株抗體TAL 1B5。在另一具體例中，劑調節MHC基因表 201114895 現，諸如第1 +型MHC及/或第11 +型MHC表現。 在一些具體例中，已反分化細胞可在單獨的步驟中進 行重新分化。例如，已反分化細胞可藉由已反分化細胞與 生長因子接觸而重新分化，此生長因子包括（但不限於此 )鹼性纖維母細胞生長因子、表皮生長因子、顆粒性細胞 巨噬細胞群落刺激因子、幹細胞因子、介白素-1、-3、-6 及-7、鹼性纖維母細胞生長因子、表皮生長因子、顆粒性 細胞巨噬細胞群落刺激因子、顆粒性細胞群落刺激因子、 紅血球生成素、幹細胞因子及骨成形蛋白質。所得已重新 分化標的細胞接著可投予病患。 在本發明的一些具體例中，定型細胞可藉由將定型細 胞在特殊的培養條件下培養而轉分化。例如，定型細胞可 在與反分化劑結合的特殊類型之培養基中培養。這些培養 基的實例可包括杜爾貝克（Dulbecco’s)改良之伊格爾（ Eagle)培養基（DMEM)、伊思可夫（Iscove’s)改良之 杜爾貝克培養基（IMDM )等。組織培養基亦可包含分化 促進劑，諸如維生素及/或礦物質補充劑、氫皮質酮、地 塞松、沒-毓乙醇等。此外，額外的培養條件包括利用螯 合劑或抗生素在某些溫度或二氧化碳或氧濃度下培養及在 某些容器中培養。培養條件可決定所得已轉分化標的細胞 的類型。 本發明的一個觀點藉此爲一種獲得標的細胞的方法。 方法可包含獲得定型細胞及接著重新編程定型細胞。這些 方法如本申請案中所敘述。在一些具體例中，此方法可包 -10- 201114895 含反分化定型細胞。在其他的具體例中，此方法可包含轉 分化定型細胞。在又其他的具體例中，此方法可包含反分 化定型細胞及接著重新分化已反分化細胞。 本發明的另一觀點爲一或多種已重新編程標的細胞在 製備用於修復或重新補充病患中的組織或細胞系譜之細胞 ，或用於治療疾病或組織傷害之藥劑的用途。 本發明的又另一觀點爲一種治療在需要其之病患中的 疾病或組織傷害的方法。在某些具體例中，此方法包含獲 得定型細胞、重新編程定型細胞以獲得已重新編程標的細 胞及以已重新編程標的細胞投予病患。在一些具體例中， 標的細胞可經由反分化、轉分化及/或重新分化而重新編 程。在特殊的具體例中，標的細胞爲已反分化標的細胞、 已轉分化標的細胞及/或已重新分化標的細胞。獲得定型 細胞及重新編程定型細胞的方法如本申請案中所敘述。 在一些具體例中，已重新編程標的細胞，諸如已反分 化標的細胞、已轉分化標的細胞或已重新分化標的細胞可 經由注射、植入或輸注而投予。這些細胞可藉由非經腸、 肌肉內、靜脈內、皮下、眼內、口服、穿透皮膚注射或注 入脊髓液中而投予。在某些具體例中，已反分化標的細胞 或已轉分化標的細胞係在醫藥組成物中投予。醫藥組成物 可包含已反分化標的細胞或已轉分化標的細胞及至少一種 醫藥上可接受之賦形劑。 本發明的一個觀點爲用於投予已重新編程標的細胞， 諸如已反分化標的細胞或已轉分化標的細胞的醫藥組成物 -11 - 201114895 。醫藥組成物可包含一或多種類型的標的細 醫藥上可接受之載劑。醫藥組成物可隨意地 病患的佐劑及/或其他賦形劑。 本發明的另一觀點爲一種製備醫藥組成 法，其包含（i )獲得定型細胞，（i i )重新 以獲得已重新編程標的細胞，及（iii )隨意 編程標的細胞與一或多種醫藥賦形劑。在一 將已重新編程標的細胞與一或多種醫藥賦形 他的具體例中，已重新編程標的細胞未與一 形劑組合。獲得定型細胞及重新編程定型細 申請案中所敘述。 應注意在此揭示內容中及特別在申請I 段落中，術語，諸如"包含"（comprises comprising)及類似術語可具有歸諸於美國 :例如，彼等可意謂"包括〃 （includes including)及類似意義；且術語，諸如 '基 成〃 (consisting essentially of 及 consists )具有歸屬於美國專利法之意義，例如彼等 引述之要素：但排除出現於先前技藝中或影 本或新穎特徵之要素。 這些及其他具體例係由下列的詳細敘述 此明白且包含於其中。 詳細敘述 胞及至少一種 包括適合投予 物或藥劑之方 編程定型細胞 地組合已重新 些具體例中， 劑組合。在其 或多種醫藥賦 胞的方法如本 I利範圍及/或 、comprised、 專利法之意義 ，included 或 本上由…所組 essentially of 允許未明確地 響本發明的基 予以揭示或從 -12- 201114895 定義 如本文所使用之^定型細胞〃爲表現已分化特徵之細 胞。這些細胞常被認爲是成熟及特別細胞。實例包括白血 細胞、紅血細胞、上皮細胞、神經元及軟骨細胞。 如本文所使用之 '、非定型細胞〃爲不表現成熟的已分 化特徵之細胞。這些細胞常被認爲是未成熟及非特別細胞 。非定型細胞的實例爲幹細胞，其爲能夠自我更新（無限 制***）且分化（特別化）的未成熟細胞。 如本文所使用之％重新編程〃係指使第一細胞系譜之 定型細胞改變成不同的細胞類型之細胞的方法。此不同的 細胞類型可具有不同的細胞系譜。重新編程可經由諸如反 分化、轉分化或重新分化之方法發生。 如本文所使用之"已重新編程細胞"爲進行定型細胞 重新編程之細胞。已重新編程細胞可包括已反分化細胞、 已轉分化細胞及/或已重新分化細胞。 如本文所使用之"反分化〃爲使定型細胞，亦即成熟 的特別細胞回復成更原始的細胞階段之方法。"已反分化 細胞^爲從定型細胞的反分化所得之細胞。 如本文所使用之"轉分化〃爲使第一細胞系譜之定型 細胞改變成不同的細胞類型的另一細胞之方法。在一些具 體例中’轉分化可爲反分化與重新分化之組合。、已轉反 分化細胞〃爲從定型細胞的轉分化所得之細胞。例如，可 將定型細胞（諸如全血細胞）轉分化成神經元。 如本文所使用之w重新分化〃係指使非定型細胞或已 -13- 201114895 反分化細胞分化成更成熟的特別細胞之方法。、、已 化細胞〃係指從非定型細胞或已反分化細胞的重新 得之細胞。若已重新分化細胞係經由已反分化細胞 分化而獲得時，則已重新分化細胞可具有與進行反 定型細胞相同或不同的系譜。例如，可將定型細胞 白血細胞）反分化以形成已反分化細胞，諸如多能 胞’並接著可將已反分化細胞重新分化以形成淋巴 其具有與白血細胞（定型細胞）相同的系譜，或重 以形成神經元，其具有與白血細胞（定型細胞）不 譜。 如本文所使用之 ''標的細胞^爲已獲得供投予 修復或重新補充組織或細胞之細胞。例如，標的細 已重新編程標的細胞，諸如已反分化標的細胞或已 標的細胞，藉此將已反分化或已轉分化標的細胞投 定型細胞 如上所述，本發明的定型細胞爲表現已分化特 胞。定型細胞可包含任何關於抗原表現、補獲或識 份。例如，定型細胞可爲第1 +型MHC及/或第II， 細胞。 定型細胞亦可爲任何從未分化細胞衍生或可衍 胞。因此，在一個具體例中，定型細胞亦爲未分化 因此，以實例說明的定型細胞可爲淋巴幹細胞或骨 重新分 分化所 的重新 分化之 (諸如 性幹細 細胞， 新分化 同的系 病患以 胞可爲 轉分化 予病患 徵之細 別之組 y MHC 生之細 細胞。 髓幹細 -14- 201114895 胞，其相應於多能性幹細胞分化。 定型細胞可從生物材料衍生而來，諸如血液或相關組 織（包括骨髓或臍帶血）、從中樞神經系統或末梢神經系 統的神經元組織、肌肉組織或從皮膚的表皮及/或真皮（ 亦即例如經口刮削的方式）。生物材料可爲產後來源。 生物材料可利用適合於組織類型的本技藝中已知的方 法獲得。實例包括（但不限於此）切下、以針抽出、抹拭 及血漿分離術。 在特殊的具體例中，定型細胞係從全血或其處理產物 衍生而來，諸如血漿或膚色血球層，因爲其從受驗者移出 可以最低的醫療監護進行。血液樣品典型地以抗凝血劑（ 諸如肝素或檸檬酸鹽）處理。在生物樣品中的細胞可經處 理以富集某些細胞類型，移出某些細胞類型或從組織體解 離細胞。有用於純化且分離細胞的方法包括離心（諸如密 度梯度離心）、流動式細胞測量術及親和力層析術（諸如 以包含單株抗體的磁珠用於細胞表面標誌或篩選）（參閱 Vettese-Dadey, The Scientist 1 999, 13:21)。以實例說 明的Fi co 11-Hyp aque分離有用於移出紅血球及顆粒性細胞 以留下單核細胞，諸如淋巴細胞及單細胞。 可從血液衍生之定型細胞的實例包括（但不限於此） CFC-T 細胞、CFC-B 細胞、CFC-Eosin 細胞、CFC-Bas 細 胞、CFC-GM 細胞、CFC-Μ 細胞、CFC-MEG 細胞、BFC-E 細胞、CFC-E細胞、T細胞、B細胞、嗜酸性細胞、嗜鹼 性細胞、嗜中性細胞、單細胞、巨核細胞及紅血球。 -15- 201114895 以血液衍生之定型細胞可以彼之特殊抗原的表現鑑證201114895 VI. INSTRUCTIONS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION All documents cited or referenced in the documents cited herein, and any products described herein or incorporated herein by reference. The indications, descriptions, product specifications, and product tables are hereby incorporated by reference herein in its entirety in its entirety herein in its entirety in the extent of the disclosure. The present invention relates to a method of treating various diseases, diseases or symptoms in a patient using reprogrammed cells, such as cells that have been subjected to reverse differentiation, transdifferentiated or re-differentiated cells. The method comprises obtaining a committed cell from a patient, degrading the committed cell to obtain a cell having a dedifferentiated target, and administering the diseased cell to the patient. In some embodiments, the method comprises obtaining a committed cell from the patient, transdifferentiating the committed cell to obtain the transdifferentiated target cell, and administering the cell to the patient by the transdifferentiated target. Cells that have been dedifferentiated or have been transdifferentiated repair or replenish tissues or cells in the patient. [Prior Art] Stem cell lines are characterized by their ability to renew and differentiate into a wide range of specific cell types via mitotic cell division. Two broad types of mammalian stem cells are embryonic stem cells isolated from the inner cell mass of the blast cells and adult stem cells found in adult tissues. In developing embryos, stem cells can differentiate into all special embryonic tissues. In adult organisms, stem cells and progenitor cells replenish special cells and maintain normal transformation of regenerative organs such as blood, skin or intestinal tissue. There are a large number of stem cells in the developing embryos, although the amount of stem cells is reduced due to the progress of the development of -5-201114895. In contrast, adult organisms contain a limited number of stem cells that are confined to certain body parts. Medical applications of stem cells have the potential to alter the treatment of a variety of diseases or conditions. Although some adult stem cell therapies (such as bone marrow transplants) already exist, medical researchers use stem cells to participate in the treatment of a wider variety of diseases, including cancer, Parkinson's disease, spinal cord injury, amyotrophic lateral sclerosis, multiple Sclerosing and muscle damage. These therapies can exploit the ability of stem cells to differentiate into the cell types necessary to treat the disease. However, there are concerns about the uncertainty of using stem cells to successfully treat such pains and the availability of stem cells. For example, hematopoietic stem cell tradition is isolated and extracted from bone marrow, mobilized peripheral blood or cord blood (placenta). Hematopoietic stem cells can also be prepared from embryo-derived embryonic stem cells (ES) obtained by in vitro fertilization techniques. However, extracting from these sources is cumbersome and sometimes dangerous and can be challenged by ethical issues. The number of stem cells available from these sources is further limited. Moreover, stem cells may experience the dilemma of differentiating into cells necessary for the treatment of pain. SUMMARY OF THE INVENTION The present invention relates to the use of cells that have been reprogrammed to repair tissue in a patient or to replenish tissue or cells in a patient. For example, the present invention relates to the use of dedifferentiated cells obtained from the differentiation of differentiated or committed cells to repair tissue in a patient or to replenish tissue or cells in a patient. The present invention also relates to the use of tissues in a transdifferentiated -6 - 201114895 cell repaired patient obtained from transdifferentiation of differentiated or committed cells or replenishing tissues or cells in a patient. This application is based in part on the discovery that the patient obtained from the patient's stereotypes or somatic cells can be reprogrammed to obtain cells of different pedigrees and that these reprogrammed cells can be administered to the patient to repair or replenish the tissue or cells. Examples of reprogramming methods include inverse differentiation and transdifferentiation. Therefore, Applicants have discovered that committed cells can be reprogrammed to obtain cells of different pedigrees. For example, committed cells can be subjected to reverse differentiation to obtain dedifferentiated cells, such as less differentiated cells (such as pluripotent stem cells), and these dedifferentiated cells can be administered to a patient to repair or replenish tissue or cells. The committed cells obtained from the patient can be transdifferentiated to obtain transdifferentiated cells, such as cells different from the pedigree of the committed cells, and these transdifferentiated cells can be administered to the patient to repair or replenish the tissue or cells as another An example. The invention encompasses a method of repairing or replenishing cells of a tissue or cell lineage in a patient by reprogramming the cells into the patient. The invention particularly comprises a method of repairing or replenishing cells of a tissue or cell lineage in a patient comprising (i) obtaining a committed cell; (π) dedifferentiating the committed cell to obtain a cell having a dedifferentiated target; and (in) The patient is administered with the target cell which has been dedifferentiated, and the cell which has been dedifferentiated is re-differentiated into a cell of the cell lineage. These re-differentiated cells may have the same cell lineage or different cell lineage as the committed cells. The invention also encompasses a method of repairing or replenishing cells of a tissue or cell lineage in a patient comprising (i) obtaining a committed cell; (π) 201114895 transdifferentiating the committed cell to obtain a transdifferentiated target cell; and (no The patient is administered to the patient having the target of transdifferentiation. In some embodiments, the patient may suffer from a disease, disorder or symptom including, but not limited to, bone marrow failure, hematological symptoms, aplastic anemia, Thalassemia, diabetes, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney disease, multiple sclerosis, congestive heart failure, hepatitis C virus, human immunodeficiency virus, head trauma, spinal cord Injury, lung disease, depression, non-obstructive azoospermia, male menopause, menopause and infertility, rejuvenation, scleroderma ulcer, psoriasis, wrinkles, cirrhosis, autoimmune disease, alopecia, hyperpigmentation Retinitis, corneal dystrophies/blindness, diabetes, and infertility. Therefore, in some specific cases, reprogrammed cells can be treated Bone marrow cells of patients with aplastic anemia, leukemia, lymphoma or human immunodeficiency virus. In some specific cases, the target cells have been reprogrammed, such as dedifferentiated cells, transdifferentiated cells, or re-differentiated cells. These may include, but are not limited to, pluripotent stem cells, pluripotent germ cells, hematopoietic stem cells, neuronal stem cells, epithelial stem cells, mesenchymal stem cells, endoderm and neuroectodermal stem cells, germ cells, extraembryonic cells, embryonic stem cells , kidney cells, alveolar epithelial cells, endoderm cells, neurons, ectoderm cells, pancreatic islet cells, acinar cells, oocytes, sperm, hematopoietic cells, hepatocytes, skin/keratinocytes, melanocytes, bones/ Bone cells, hair/dermal papilla cells, cartilage/chondrocytes, lipocytes/fatty cells, skeletal muscle cells, endothelial cells, myocardial/cardiomyocytes, and trophoblast cells. -8- 201114895 In some specific examples, committed cell lines Obtained from blood or related tissues (including bone marrow). Stem cells can be obtained from whole blood and/or Obtained via plasmapheresis. The blood may be mobilized or unmobilized blood. Such committed cells include, but are not limited to, T-cells, B cells, eosinophils, basophils , neutrophils, megakaryocytes, single cells, red blood cells, granular cells, obese cells, lymphocytes, white blood cells, platelets, and red blood cells. Alternatively, the committed cells can be derived from neuronal tissues or muscles from the central nervous system or the peripheral nervous system. Tissue or epidermal and/or dermal tissue from the skin is obtained. In some embodiments, the committed cell line is obtained from the blood or tissue of the patient. In some embodiments, the patient from which the committed cell was obtained has been reintroduced The target cells, such as cells that have been dedifferentiated or those that have been transdifferentiated, are the same patient. In some embodiments, the committed cell line is dedifferentiated by contact of the committed cells with the agent. For example, committed cells can be incubated. In some embodiments, the agent conjugates a receptor that mediates antigen recruitment, recognition or expression on the surface of the committed cell. The receptor may be a type I MHC antigen or a type II MHC antigen. In some embodiments, the type I antigen is an HLA-A receptor, an HLA-B receptor, an HLA-C receptor, an HLA-E receptor 'HLA-F receptor or an HLA-G receptor, and a type II The antigen is the HLA-DM receptor 'HLA-DP receptor, HLA-DQ receptor or HLA-DR receptor. In some embodiments, the agent can be an antibody to the receptor, such as a monoclonal antibody to the recipient. In some embodiments, the antibody is monoclonal antibody CR3/43 and monoclonal antibody TAL 1B5. In another embodiment, the agent modulates the MHC gene table 201114895, such as MHC class 1 + and / or MHC class 11 +. In some embodiments, the dedifferentiated cells can be re-differentiated in a separate step. For example, cells that have been dedifferentiated can be re-differentiated by contact of the dedifferentiated cells with growth factors including, but not limited to, basic fibroblast growth factor, epidermal growth factor, and granular macrophage colony. Stimulating factors, stem cell factors, interleukin-1, -3, -6 and -7, basic fibroblast growth factor, epidermal growth factor, granulocyte macrophage community stimulating factor, granulocyte community stimulating factor, Erythropoietin, stem cell factor and bone forming protein. The resulting re-differentiated target cells can then be administered to the patient. In some embodiments of the invention, the committed cells can be transdifferentiated by culturing the committed cells under specific culture conditions. For example, committed cells can be cultured in a special type of medium that binds to a retrodifferentiator. Examples of such a medium may include Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Durbeck medium (IMDM), and the like. The tissue culture medium may also contain a differentiation promoting agent such as a vitamin and/or mineral supplement, hydrocorticosterone, dexamethasone, sputum-free ethanol, and the like. In addition, additional culture conditions include culturing with certain chelating agents or antibiotics at certain temperatures or carbon dioxide or oxygen concentrations and culturing in certain containers. The culture conditions determine the type of cells that have been transdifferentiated. One aspect of the invention is thereby a method of obtaining target cells. The method can comprise obtaining a committed cell and then reprogramming the committed cell. These methods are as described in this application. In some embodiments, the method may comprise a retroprecipitation-type cell comprising -10-201114895. In other embodiments, the method can comprise transdifferentiating committed cells. In still other embodiments, the method can comprise re-differentiating the committed cells and then re-differentiating the dedifferentiated cells. Another aspect of the invention is the use of one or more reprogrammed cells for the preparation of a cell for repairing or replenishing a tissue or cell lineage in a patient, or for treating a disease or tissue injury. Yet another aspect of the invention is a method of treating a disease or tissue injury in a patient in need thereof. In some embodiments, the method comprises obtaining a committed cell, reprogramming the committed cell to obtain a reprogrammed target cell, and administering the cell to the patient with the reprogrammed target. In some embodiments, the subject cells can be reprogrammed via reverse differentiation, transdifferentiation, and/or re-differentiation. In a specific embodiment, the target cells are cells that have been dedifferentiated, cells that have been transdifferentiated, and/or cells that have been re-differentiated. Methods for obtaining committed cells and reprogramming committed cells are as described in this application. In some embodiments, the target cells that have been reprogrammed, such as cells that have been re-segmented, cells that have been transdifferentiated, or cells that have been re-differentiated, can be administered via injection, implantation, or infusion. These cells can be administered by parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, intradermal injection or injection into the spinal fluid. In some embodiments, the dedifferentiated target cell or the transdifferentiated target cell line is administered in a pharmaceutical composition. The pharmaceutical composition may comprise cells that have been dedifferentiated or cells that have been transdifferentiated and at least one pharmaceutically acceptable excipient. One aspect of the present invention is a pharmaceutical composition for administering a cell that has been reprogrammed, such as a cell that has been de-differentiated or a cell that has been transdifferentiated -11 - 201114895. The pharmaceutical composition may comprise one or more types of standard pharmaceutically acceptable carriers. The pharmaceutical composition is optionally admixed with the patient's adjuvant and/or other excipients. Another aspect of the invention is a method of preparing a pharmaceutical composition comprising (i) obtaining a committed cell, (ii) re-acquiring a reprogrammed target cell, and (iii) randomly programming the target cell with one or more pharmaceutical excipients . In a specific example of reprogramming a labeled cell with one or more drugs, the reprogrammed target cells are not combined with a sizing agent. Obtain the shaped cells and reprogram the styling as described in the application. It should be noted that in this disclosure and particularly in the application paragraph I, terms such as "comprises comprising" and similar terms may have the meaning attributed to the United States: for example, they may mean "including 〃 (includes And the like, and terms such as 'consisting essentially of and consists' have the meaning attributed to the US patent law, such as the elements cited therein: but exclude elements that appear in prior art or in the form of a novel or novel feature. . These and other specific examples are apparent from and are included in the following detailed description. DETAILED DESCRIPTION The combination of cells and at least one of which is suitable for administration of the agent or agent. The combination of programmed cells has been re-established in some specific examples. The method of constituting a cell or a plurality of medicinal cells, such as the scope of the present invention and/or the meaning of the patent law, included or otherwise, is essential to allow the disclosure of the present invention to be unrecognized or from -12. - 201114895 Defining a cell type as used herein is a cell that exhibits differentiated characteristics. These cells are often considered to be mature and specific cells. Examples include white blood cells, red blood cells, epithelial cells, neurons, and chondrocytes. As used herein, a non-shaped cell is a cell that does not exhibit mature, differentiated characteristics. These cells are often considered to be immature and non-specific cells. An example of a non-shaped cell is a stem cell which is an immature cell capable of self-renewal (infinite division) and differentiation (specialization). % reprogramming 〃 as used herein refers to a method of altering a committed cell of a first cell pedigree to a cell of a different cell type. This different cell type can have a different cell lineage. Reprogramming can occur via methods such as reverse differentiation, transdifferentiation, or re-differentiation. As used herein, "reprogrammed cells" cells for reprogramming committed cells. Cells that have been reprogrammed can include dedifferentiated cells, transdifferentiated cells, and/or re-differentiated cells. As used herein, "anti-differentiation" is a method of restoring committed cells, i.e., mature, specialized cells, to a more primitive cellular stage. " already differentiated cells ^ are cells derived from the anti-differentiation of committed cells. "Transdifferentiation" as used herein is a method of altering a committed cell of a first cell lineage into another cell of a different cell type. In some specific cases, 'transdifferentiation can be a combination of dedifferentiation and re-differentiation. The transdifferentiated cells are cells obtained from transdifferentiation of committed cells. For example, committed cells (such as whole blood cells) can be transdifferentiated into neurons. As used herein, w re-differentiation refers to a method of differentiating non-shaped cells or cells that have been differentiated into more mature specific cells. , Cellular sputum refers to a cell that has been regenerated from a non-shaped cell or a dedifferentiated cell. If the re-differentiated cell line is obtained by differentiation of the dedifferentiated cells, the re-differentiated cells may have the same or different pedigree as the transfected cells. For example, the committed cell white blood cells can be dedifferentiated to form a dedifferentiated cell, such as a pluripotent cell' and then the dedifferentiated cells can be re-differentiated to form lymph, which has the same pedigree as white blood cells (type cells), or Heavy to form neurons, which have no spectrum with white blood cells (type cells). The ''target cell' as used herein is a cell that has been obtained for administration to repair or replenish tissue or cells. For example, the target fine has reprogrammed the target cells, such as cells that have been dedifferentiated or labeled cells, thereby degrading or transdifferentiating the target cell-derived cells as described above, and the committed cells of the present invention are characterized by differentiation. Cell. The committed cells can contain any indication, recruitment or recognition of the antigen. For example, the committed cells can be Type 1 + MHC and/or II, cells. The committed cells can also be derived from any undifferentiated cells or can be derivatized. Therefore, in one specific example, the committed cells are also undifferentiated. Therefore, the exemplified cells can be re-differentiated by lymphocytic cells or bone re-differentiation (such as sexual stem cells, newly differentiated patients). The cells can be transdifferentiated into a fine group of y MHC cells. The medullary stem-14- 201114895 cells, which correspond to pluripotent stem cell differentiation. The committed cells can be derived from biological materials, such as Blood or related tissue (including bone marrow or cord blood), neuronal tissue from the central nervous system or peripheral nervous system, muscle tissue or from the epidermis and/or dermis of the skin (ie, for example, by oral scraping). The biomaterial can be Postpartum Sources Biomaterials can be obtained using methods known in the art that are suitable for tissue types. Examples include, but are not limited to, excision, needle extraction, wipes, and plasmapheresis. In particular embodiments, A committed cell line derived from whole blood or a processed product thereof, such as a plasma or skin color layer, because it can be removed from the subject with the lowest Monitoring is performed. Blood samples are typically treated with an anticoagulant such as heparin or citrate. Cells in a biological sample can be processed to enrich certain cell types, remove certain cell types or dissociate cells from tissue. Methods for purifying and isolating cells include centrifugation (such as density gradient centrifugation), flow cytometry, and affinity chromatography (such as magnetic beads containing monoclonal antibodies for cell surface markers or screening) (see Vettese- Dadey, The Scientist 1 999, 13:21). The Fi Co 11-Hyp aque separation illustrated by the examples is used to remove red blood cells and granular cells to leave monocytes, such as lymphocytes and single cells. Examples of committed cells include, but are not limited to, CFC-T cells, CFC-B cells, CFC-Eosin cells, CFC-Bas cells, CFC-GM cells, CFC-Μ cells, CFC-MEG cells, BFC-E cells. , CFC-E cells, T cells, B cells, eosinophils, basophils, neutrophils, single cells, megakaryocytes, and red blood cells. -15- 201114895 Blood-derived committed cells To show a particular antigen it's assurance

。例如，B 細胞爲 CD19+、CD21+、CD22 +及 DR +細胞。T 細胞爲CD2+、CD3+及CD4 +或CD8 +細胞。未成熟的淋巴 細胞爲CD4 +及CD8 +細胞。活化T細胞爲DR +細胞。自然 殺手細胞（NK)爲CD56 +及CD16 +細胞。T淋巴細胞爲 CD7 +細胞。白血球爲CD45 +細胞。顆粒性細胞爲CD13 +及 CD33 +細胞。單細胞巨噬細胞爲CD14 +及DR +細胞。 在某些具體例中，定型細胞可爲B淋巴細胞（活化或 非活化）、T淋巴細胞（活化或非活化）、來自巨噬細胞 單細胞系譜之細胞、能夠表現第I型或第Π型抗原之有 核細胞、可經誘發以表現第I型或第Π型抗原之細胞或 除核細胞（亦即不含有核之細胞，諸如紅血細胞）。 在可替換之具體例中，定型細胞可選自包含大顆粒淋 巴細胞、無抗原標記之淋巴細胞及自然殺手細胞之細胞群 中任一者，每個細胞表現CD56及/或CD16細胞表面受體 〇 因爲定型細胞爲基本的初代培養物，所以可能必要以 適合的營養素補給細胞群體以維持生存力。適合的培養條 件爲熟習本技藝者已知。但是細胞群體的處理較佳地儘可 能在從病患移出生物樣品之後立即開始，典型地在1 2小 時之後，較佳地在2至4小時之類。細胞生存力可利用已 知的技術查驗，諸如錐藍質排除或碘化丙錠。 已反分化細胞 -16- 201114895 反分化爲重新編程法的類型，藉此使細胞結構及功能 漸進性改變以引起較不特別細胞。反分化可自然發生，其 中細胞可於活體內進行反應於組織損傷的有限之逆分化。 或者，反分化可利用美國申請序號08/594,164，現爲美國 專利第6,090,625號、美國申請序號09/742,520，現爲美 國專利第7，1 12,440號、美國申請序號1 0/140,978，現爲 美國專利第7,220,412號、美國申請序號1 0/150,789，現 爲美國專利第7,4 1 0,773號及美國申請序號09/853,1 88中 所述之方法誘發，將全部倂入本文以供參考。 本發明的已反分化細胞可包括（但不限於此）多能性 幹細胞、淋巴幹細胞、骨髓幹細胞、神經元幹細胞、骨骼 肌衛星細胞、上皮幹細胞、內胚層幹細胞、間葉幹細胞及 胚胎幹細胞。 在特殊的具體例中，定型細胞係從血液衍生且反分化 以形成造血細胞系譜的已分化細胞。這些已分化細胞的實 例包括（但不限於此）多能性幹細胞、淋巴幹細胞及骨髓 幹細胞。 定型細胞可藉由細胞與可操作接合細胞的劑（agent )接觸而反分化。接著培育細胞以允許那些已以劑操作接 合之細胞經由反分化法進展且最終未經分化。 接觸步驟可包含劑與表面抗原接合在定型表面上。劑 可與定型細胞以直接接合或間接接合起作用。直接接合的 實例係在定型細胞具有至少一個細胞表面受體在其細胞表 面上時，諸如具有同源區域之鏈（常被發現具有相同 -17- 201114895 或類似序列之區域），諸如那些可在B細胞上發現的區域 ，且其中劑直接接合細胞表面受體。另一實例係在定型細 胞具有細胞表面受體在其細胞表面上時，諸如具有同源區 域之鏈，諸如那些可在T細胞上發現的區域，且其中 劑直接接合細胞表面受體。 間接接合的實例係在定型細胞具有至少兩個細胞表面 受體在其細胞表面上且劑與受體之一的接合影響接著誘發 定型細胞反分化的其他受體時。 用於定型細胞反分化之劑可爲化學化合物或組成物。 例如，劑可使細胞表面受體能夠接合在定型細胞表面上。 在某些具體例中，劑可操作接合在定型細胞表面上存在的 受體，該受體可以定型細胞表現，諸如能夠以定型細胞表 現的受體。 例如，劑可包括（但不限於此）環腺苷單磷酸鹽（ CAMP ) 、CD4分子、CD8分子、一部分或全部的T-細胞 受體、配體（固定或游離）、肽、T-細胞受體（TCR)、 抗體、交叉反應抗體、單株抗體或多株抗體中任一或多者 。亦可利用生長因子，諸如造血生長因子，例如紅血球生 成素及顆粒性細胞-單細胞群落刺激因子（GM-CSF)。 若劑爲抗體、交叉反應抗體、單株抗體或多株抗體， 則劑可爲下列任一或多者之抗體、交叉反應抗體、單株抗 體或多株抗體中任一或多者··第Π型MHC抗原之々-鏈 、MHC HLA-DR抗原之/3-鏈、第I型或第II型MHC抗 原之α-鏈、HLA-DR抗原之α-鏈、第II型MHC抗原或 -18- 201114895 第I型MHC抗原之α-與yS-鏈。抗體的實例爲 由DaKo供應）。 術語"抗體〃可包括各種斷片（不論是否由 解切割或重組體技術所衍生）及保留結合活性之 諸如Fab、F ( ab’）2及scFv抗體，與其模擬物 質體。亦包括基因工程變種作爲抗體，其中一些 列已藉由例如置換胺基酸殘基而修改以增強結合 抗體已以不同的物種到達希望根據本發明的方法 胞的生物體，以降低免疫副作用的可能性（此實 人化"小鼠單株抗體）。 用於實現定型細胞轉化成已反分化細胞之劑 以定型細胞的細胞外起作用。例如，定型細胞可 可操作接合之受體及劑可操作接合受體。 例如，受體可爲細胞表面受體。細胞表面受 實例包括（但不限於此）第I型及第II型MHC 體可包含α ·組份及/或Θ -組份，作爲第I型5 MHC受體的例子。 受體可包含具有同源區域之/3 -鏈，例如至 域的HLA-DR之冷-鏈。 或者或另外，受體可包含具有同源區域之| 如至少同源區域的HLA-DR之α-鏈。受體可爲 相容性複合物（MHC )之第I型或第II型抗原 具體例中，細胞表面受體可包括（但不限於此： 受體、DM受體、DP受體、DQ受體、HLA-A受 CR3/43 ( 蛋白質分 衍生物， 或生物同 胺基酸序 ，或其中 治療其細 例爲"擬 較佳地可 包含以劑 體的特殊 受體。受 .第 Π型 少问源區 2 _鏈，例 主要組織 。在某些 )HLA-DR 體、HLA- -19- 201114895 B受體、HLA-C受體、HLA-E受體、HLA-F受體或HLA- G受體。在一些具體例中，細胞表面受體可爲η LA-DR受 體。 劑可爲受體之抗體，諸如受體之單株抗體。 劑的實例可爲一種調節Μ H C基因表現之劑，諸如第 1 +型MHC及/或第11 +型MHC表現。 在某些具體例中，劑可與生物反應改良劑結合使用。 生物反應改良劑的實例包括（但不限於此）烷基化劑、免 疫調節劑、生長因子、細胞介素、細胞表面受體、激素、 核酸、核苷酸序列、抗原或肽。例如，烷基化劑可爲或可 包含環磷酸醯胺。 其他的生物反應改良劑可包括能夠向上調節第I型及 /或第II型MHC抗原表現之化合物，在一些具體例中， 其可允許劑與MHC受體結合以更有效運作。 當可使任何細胞類型表現第I型及/或第II型MHC抗 原時，此可提供一種用於反分化各種廣泛的細胞類型之方 法，不論此等細胞類型是否構成表現第I型及/或第II型 MHC抗原。 定型細胞通常以劑培育至少2小時，典型地介於2與 24小時之間，較佳地介於2與1 2小時之間。培育典型地 在從約室溫或例如約22t到至多約37°C，包括33°C下執 行。反分化程序的進展可藉由移出一小份樣品及利用顯微 鏡及/或流動式細胞測量術檢查細胞而定期查驗。或者， 裝置可包含用於線上監控反分化程序進展的追蹤設備。 -20- 201114895 除了利用反分化劑以外，定型細胞可在自體血漿或血 清中或在胎兒血清或馬血清中培養。定型細胞可隨意地以 抗凝血劑、螯合劑或抗生素培養。培育細胞的溫度範圍可 延伸至18-40°C且亦可包括4-10%之C02及/或10-35%之 〇2。此外，培育可發生在經塗佈或未經塗佈之血液袋、組 織培養袋或塑膠容器中。 某些類型的已反分化細胞可藉由利用特殊的培養條件 反分化而獲得。例如，定型細胞可藉由在與反分化劑結合 之杜爾貝克改良之伊格爾培養基（DMEM )、非必需胺基 酸（NEAA) 、L-麩醯胺酸（L-glu)及/3 -毓乙醇（2冷ME )中培養定型細胞而反分化成多能性細胞。定型細胞亦可 最初暴露於螯合劑。 定型細胞可利用與反分化劑結合之DMEM (低葡萄糖 )及 L-glu或 DMEM (低葡萄糖）、L-glu、2沒ME及 NEAA培養以獲得間葉細胞，作爲另一實例。再者，亦可 將抗生素健大黴素（gentamycin)用於細胞培養中。 已轉分化細胞 已轉分化細胞係藉由與反分化劑結合之組織培養基培 養定型細胞而獲得。定型細胞由此進行轉分化，其中定型 細胞轉變成另一細胞類型的細胞；在一些具體例中，定型 細胞轉變成不同系譜之細胞。 經由轉分化所獲得的標的細胞類型取決於培養條件。 這些條件係根據組織培養基類型、各種分化促進劑的存在 -21 - 201114895 /不存在、不同的血清存在/不存在、培育溫度、氧或二氧 化碳的存在/不存在及用於培育的箱子或容器類型而變更 〇 用於轉分化的組織培養基的實例包括（但不限於此） 伊思可夫改良之杜爾貝克培養基（IMDM )、杜爾貝克改 良之伊格爾培養基（DMEM )、伊格爾最低必需（eme ) 培養基、α -最低必需培養基（α - Μ Ε Μ )、洛斯維（ Roswell )公園紀念所（RPMI ;發展出培養基的場所） 1 640、Ham-F-12、E199、MCDB、Leibovitz L-1 5 及威廉 斯（Williams )培養基E或任何商業上調配之組織培養基 〇 分化促進劑包括抗凝血劑、螯合劑及抗生素。此等劑 的實例可爲下列之一或多者：維生素、礦物質或其衍生物 ’諸如維生素A (視黃醇）、維生素B3、維生素C (抗壞 血酸鹽）、抗壞血酸2-磷酸鹽、維生素D2、維生素D3、 維生素K、視黃酸、菸鹼醯胺、鋅或鋅化合物及鈣或鈣化 合物：天然或合成激素，諸如氫皮質酮及地塞松；胺基酸 或其衍生物，諸如L-麩醯胺酸（L-glu)、乙二醇四乙酸 (EGTA)、脯胺酸及非必需胺基酸（NEAA):化合物或 其衍生物，諸如毓基十六烷醇、二丁基環腺苷單磷酸 鹽（db-cAMP)、單硫代甘油（MTG)、腐肉鹼、二甲亞 楓（DMSO )、次黃嘌呤、腺嘌呤、弗斯可林（forskolin )、西洛他醯胺（cilostamide)及3 -異丁基-1-甲基黃嘿 呤；核苷及其類似物，諸如5-氮雜胞嘧啶核苷；酸或其 -22- 201114895 鹽類，諸如抗壞血酸、丙酮酸鹽、黑海綿酸（okadic acid )、亞麻油酸、乙二胺四乙酸（EDTA )、抗凝血檸檬酸 鹽葡萄糖式 A(ACDA)、二鈉EDTA、丁酸鈉及甘油磷 酸鹽：抗生素或藥物，諸如G418、健大黴素、本西法林 (Pentoxifylline ) ( 1- ( 5-酮己基）-3,7-二甲基黃嘿玲） 及美洒辛（indomethacin);及蛋白質，諸如組織血纖維 蛋白溶酶原活化劑（TPA)。 這些分化促進劑可被用於獲得特殊類型的標的細胞。 例如，維生素B3可被用於得到腺泡細胞，諸如胰小島細 胞；氫皮質酮或地塞松可被用於得到間葉來源或上皮來源 之細胞（例如，腎上皮細胞、皮膚及相關結構，諸如真皮 乳頭細胞）：及Θ -巯基十六烷醇可被用於得到外胚層細 胞，諸如神經元細胞，包括CNS之附層細胞。 培養基可含有自體血榮、血小板、血清（諸如胎兒血 液取樣）或哺乳動物來源之血清（諸如馬血清）。此外， 轉化法可發生在血液袋、支架、組織培養袋或塑膠組織培 養容器內部。組織培養容器可爲附著或未附著之組織培養 谷器’可以諸如明膠、膠原、基底膜基質（matrigel)或 細胞外基質之劑塗佈或未經塗佈，以促進附著或漂浮，其 取決於所需之組織類型或欲製備之特別細胞而定。 額外的培養條件包括溫度，其可介於約1 〇與約6〇t 之間或介於約1 8與約4(TC之間；二氧化碳（C02 )濃度 ’其可介於約〇與約20%或約4與約10%之間：及氧（ 〇2)濃度’其可介於約0與約50%或約1〇與約35%之間 -23- 201114895 用於獲得標的細胞且與反分化劑結合使用之方法的實 例討論於表1中。 表 1 與反分化劑結合以獲得各種類型的標的細胞之方法 標的細胞類型 與反分化劑結合使用之培養條件 額外的培養條件 多能性幹細胞或多 能性祖細胞之異質 群體 • DMEM、NEAA、L-glu、2 /3 ME •定型細胞暴露於螯合 劑或抗凝血劑之前 •抽出反分化劑，僅以 培養基稀釋祖細胞 間葉幹細胞 •DMEM (低葡萄糖）、L-glu; 或 .DMEM (低葡萄糖）、L-glu、2 /3 ME ' NEAA •健大黴素 •多能性幹細胞 •卵母細胞 •*** •用於卵母細胞之RPMI 1640、隨 意具有 NEAA、L-glu、2/3ME :或 •用於卵母細胞之ΕΜΕ培養基、 視黃醇、L-glu、丙酮酸鈉、乳 酸鈉、NEAA ;或 •用於***之DMEM、Hams F12 、維生素C、維生素E、視黃酸 、視黃醇、丙酮酸鹽、隨意具有 本西法林；或 •用於上列多能性幹細胞且具有列 於下一欄用於***或卵母細胞之 額外培養條件的培養條件 GAIC •用於***之培育可在 約30-32°C下及用於 卵母細胞之培育可在 約 38-39°C下 •細胞可在反分化及重 新分化前暴露於螯合 劑，諸如EDTA及 EGT •用於***，可包括加 入黑海綿酸、DMSO 及鋅或鋅化合物； Gamete 100可被用作 爲替代的基本培養基 •用於卵母細胞，可包 括加入dp-cAMP、二 鈉EDTA、弗斯可林 、西洛他醯胺及次黃 嘿哈 •用於卵母細胞，培養 基199可被用作爲替 代的基本培養基 腎細胞 • DMEM、Hams F12及氫皮質酮 ，隨意具有維生素K;或 •用於上歹0多能性幹細胞之培養條 件，具有氫皮質酮； -24- 201114895 •肺泡上皮細胞 •內胚層細胞 • DMEM、NEAA、L-glu、隨意 具有2ΘΜΕ、菸鹼醯胺；或 •用於上列多能性幹細胞之培養條 件，具有菸鹼醯胺、隨意具有地 塞松、視黃酸、db-cAMP ;或 • MDM、L-glu、抗壞血酸、 MTG •具有抗生素G418及 以基底膜基質塗佈之 培養容器 •神經元細胞 •外胚層細胞 •具有 Hams F12 之 DMEM、 NEAA、2；SME、隨意具有腐肉 鹼、視黃酸、L-glu、氫皮質酮 、抗壞血酸鹽；或 •用於上列多能性幹細胞之培養條 件，隨意具有腐肉鹼、視黃酸、 氫皮質酮、抗壞血酸鹽 胰小島細胞 腺泡細胞 .DMEM、Hams Π2、維生素 B3 :或 •具有維生素B3之RPMI 1640 ; •用於上列多能性幹細胞之培養條 件，具有維生素B3 (菸鹼醯胺 )、隨意具有地塞松 造血細胞 • IMDM、隨意具有氫皮質酮；或 • IMDM、L麩醯胺酸及MTG ; •用於上列多能性幹細胞之培養條 件，具有取代2沒ME之MTG、 隨意具有維生素 •培育可在33°C下 •培育可在室溫下以擴 大在培養基中的巨核 細胞 •已分化細胞可在轉化 前暴露於螯合劑，以 擴大在培養基中的紅 血球祖細胞 • RPMI1640可被用作 富集淋巴祖細胞之基 本培養基 •丁酸鈉及/或5·氮雜 胞嘧啶核苷可加到培 養基中，以促進原始 紅血球分化 肝的肝細胞 • DMEM或IMDM或α -最低必需 培養基、L-glu、隨意具有^塞 松、L抗壞血酸-2-磷酸鹽及菸鹼 醯胺；或 •威廉斯培養基E、丙酮酸鈉、地 塞松；或 .用於上列多能性幹細胞之培養條 件，具有地塞松 -25- 201114895 皮膚/角質化細胞 • HamsF12、DMEM (3 : 1 之培 養基）、氫皮質酮、L-glu、隨 意具有腺嘌Π令；或 •具有 L-glu 之 E199 或 DMEM、 隨意具有氫皮質酮及腺嘌呤；或 •用於上列多能性幹細胞之培養條 件，具有氫皮質酮、隨意具有鈣 或鈣化合物或抗壞血酸鹽 •可具有健大黴素作爲 抗生素 •可在36.4t下培育 黑色素細胞 • MCDB 及 Leibovitz L-15、TPA 、隨意具有3-異丁基-1-甲基黃 嚷哈；或 •培養基199及氣皮質酮 骨細胞/骨頭 • DMEM、/3-甘油磷酸鹽、地塞 松、抗壞血酸鹽及L-glu，隨意 具有維生素D3 ;或 •用於上列多能性幹細胞之培養條 件，具有甘油磷酸鹽、地塞松及 抗壞血酸鹽，隨意具有維生素 d3 真皮乳頭細胞/毛髮 •威廉斯培養基E、L-glu、氫皮 質酮及/或維生素D2、腺嘌呤及 亞麻油酸；或 • DMEM、作爲基本培養基的 HamFl2，L-glu、氫皮質酮及/ 或維生素D2、腺嘌呤及亞麻油 酸；或 •用於上列多能性幹細胞之培養條 件，具有氫皮質酮、維生素d2 、腺嘌呤及亞麻油酸 軟骨細胞/軟骨 • DMEM、丙酮酸鹽、抗壞血酸 鹽2-磷酸鹽、地塞松及脯胺酸 :或 •用於上列多能性幹細胞之培養條 件，具有抗壞血酸鹽2-磷酸鹽 、地塞松及脯胺酸 脂肪細胞/脂細胞 • DMEM、地塞松及美洒辛；或 •地塞松及美洒辛 -26- 201114895 骨骼肌細胞 • DMEM、低葡萄糖、隨意具有 氫皮質酮、地塞松、L-麩胺醯胺 及丙酮酸鈉；或 •作爲基本培養基之DMEM及 Ham F12 或 ΠΟ，或 DMEM 及 培養基199 ;或 • €於上列多能性幹細胞之培養條 件’具有葡萄糖、健大黴素及低 血清 •可具有健大黴素作爲 抗生素 •可具有低血清濃度 •可以具有明膠之培養 容器 •可在上升至39°C之溫 度下 •可包括加入5-氮雜胞 嘧啶核苷 血液容器（內皮細 胞） • DMEM、NEAA及2ySME ;或 • IMDM及地塞松 心肌/心肌細胞 • 4 : 1之DMEM及M199培養基 或DMEM (低葡萄糖）、L-glu 、NEAA ;或具有抗壞血酸之 DMEM (低葡萄糖）及/或 DMSO ;或 •用於上列多能性幹細胞之培養條 件，具有抗重力培養環境或在振 動環境中 •以明膠塗佈之培養容 器用於完全分化及去 血漿之自體血清或血 小板 •完全分化可於蓋玻片 載片上觀察到 滋養層細胞 •以減去反分化劑之相同培養基連 續稀釋之DMEM、L-glu、 2/3ME、NEAA ;或 • RPMI 1640、2ySME、丙酮酸鈉 及L-麩胺醯胺 •可被用於生產分化細 胞成爲中胚層細胞及 外胚層細胞（包括生 殖幹細胞及祖細胞） 必要之自體生長因子 及激素 以對應之培養基接著稀釋抽出反分化劑之培養條件用 於表1中每一細胞類型所述之培養條件特別造成更增強之 反分化。 在培養期間，隨意含有各種分化促進劑的培養基可藉 由加入更多培養基稀釋，但是不以反分化劑。不受理論限 制’稀釋似乎增加分化，因爲細胞變得較不濃稠及增生刺 激因子較不濃縮。因此，加入培養基可進一步增強轉分化 且影響在細胞系譜內獲得什麼樣類型的細胞。例如，若標 的細胞爲神經元，則加入培養基可造成朝向更成熟的神經 •27- 201114895 元發育移動而不是神經元祖細胞（二者具有相同的系譜） 。藉由逐漸降低血清濃度而達成的培養基連續稀釋僅骨骼 肌祖細胞分化，作爲前進分化的另一實例。 已重新分化細胞 已重新分化細胞可被用於獲得標的細胞，其係藉由將 已反分化細胞定型或重新分化成標的細胞類型。此可藉由 已反分化細胞與生長因子接觸而執行。例如，視黃酸已被 用於分化幹細胞成爲神經元細胞。接著藉由與骨髓基質細 胞株及IL7共同培養的甲基纖維素已被用於分化幹細胞成 爲淋巴細胞先驅（NisiUni等人之Int Immuno 1 994，6: 909-916) 。Le Page (New Scientist Dec. 16，2000)指導 幹細胞可分化成肺上皮細胞。Bischoff ( Dev Biol 1 986, 115: 129-39)指導如何分化肌衛星細胞成爲成熟的肌纖 維。神經元先驅細胞可以鹼性纖維母細胞生長因子及表皮 生長因子擴張（Nakafuku 與 Nakamura，J. Neurosci Res 1995，41 : 153-168)。造血幹細胞可利用許多生長因子， 包括GM-CSF、紅血球生成素、幹細胞因子及介白素（11-1、IL-3、IL-6)擴張，參閱 Metcalf( Nature 1989，339: 27_30 )以檢視這些各種因子。. For example, B cells are CD19+, CD21+, CD22+, and DR+ cells. T cells are CD2+, CD3+ and CD4+ or CD8+ cells. Immature lymphocytes are CD4 + and CD8 + cells. Activated T cells are DR+ cells. Natural killer cells (NK) are CD56+ and CD16+ cells. T lymphocytes are CD7 + cells. White blood cells are CD45+ cells. Granular cells are CD13 + and CD33 + cells. Single cell macrophages are CD14+ and DR+ cells. In some embodiments, the committed cells can be B lymphocytes (activated or non-activated), T lymphocytes (activated or non-activated), cells from the macrophage single cell lineage, capable of expressing type I or type III A nucleated cell of an antigen, a cell which can be induced to express a type I or a scorpion-type antigen, or a nucleated cell (i.e., a cell which does not contain a nucleus, such as red blood cells). In an alternative embodiment, the committed cells can be selected from any of the group consisting of large particle lymphocytes, antigen-free lymphocytes, and natural killer cells, each cell exhibiting a CD56 and/or CD16 cell surface receptor. Because the committed cells are basic primary cultures, it may be necessary to replenish the cell population with suitable nutrients to maintain viability. Suitable culture conditions are known to those skilled in the art. However, the treatment of the cell population is preferably started as soon as possible after removal of the biological sample from the patient, typically after 12 hours, preferably 2 to 4 hours. Cell viability can be assayed using known techniques, such as cone blue exclusion or propidium iodide. Dedifferentiated cells -16- 201114895 Dedifferentiation is a type of reprogramming whereby tissue structure and function are progressively altered to cause less specific cells. Dedifferentiation can occur naturally, in which cells can react in vivo to a limited inverse differentiation of tissue damage. Alternatively, the anti-differentiation can be utilized in U.S. Patent Application Serial No. 08/594,164, issued to U.S. Patent No. 6,090,625, U.S. Serial No. 09/742,520, U.S. Patent No. 7,1,12,440, U.S. Serial No. 10/140,978, U.S. Patent No. 7,220, 412, U.S. Patent Application Serial No. No. No. No. No. No. No. No. No. No. No. No. No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No No. The dedifferentiated cells of the present invention may include, but are not limited to, pluripotent stem cells, lymphocyte stem cells, bone marrow stem cells, neuronal stem cells, skeletal muscle satellite cells, epithelial stem cells, endoderm stem cells, mesenchymal stem cells, and embryonic stem cells. In a particular embodiment, the committed cell line is derived from blood and is inversely differentiated to form differentiated cells of the hematopoietic cell lineage. Examples of such differentiated cells include, but are not limited to, pluripotent stem cells, lymphocyte stem cells, and bone marrow stem cells. The committed cells can be counter-differentiated by contact of the cells with an agent that is operable to engage the cells. The cells are then incubated to allow those cells that have been manipulated by the agent to progress via the reverse differentiation process and ultimately to be undifferentiated. The contacting step can comprise attaching the agent to the surface antigen on the styling surface. The agent can act in direct or indirect engagement with the committed cells. An example of direct ligation is when a committed cell has at least one cell surface receptor on its cell surface, such as a strand having a homologous region (often found to have the same region of -17-201114895 or a similar sequence), such as those The region found on B cells, and where the agent directly binds to cell surface receptors. Another example is when a committed cell has a cell surface receptor on its cell surface, such as a strand having a homologous region, such as those found on T cells, and wherein the agent directly binds to a cell surface receptor. An example of indirect ligation is when the committed cells have at least two cell surface receptors on their cell surface and the binding of the agent to one of the receptors affects other receptors that subsequently induce dedifferentiation of the committed cells. The agent for dedifferentiating the committed cells may be a chemical compound or a composition. For example, the agent enables cell surface receptors to bind to the surface of the committed cells. In certain embodiments, the agent is operably linked to a receptor present on the surface of the committed cell, which receptor can be characterized by a committed cell, such as a receptor capable of expressing it as a committed cell. For example, the agent may include, but is not limited to, a cyclic adenosine monophosphate (CAMP), a CD4 molecule, a CD8 molecule, a part or all of a T-cell receptor, a ligand (fixed or free), a peptide, a T-cell Any one or more of a receptor (TCR), an antibody, a cross-reactive antibody, a monoclonal antibody, or a polyclonal antibody. Growth factors such as hematopoietic growth factors such as erythropoietin and granulocyte-single cell community stimulating factor (GM-CSF) can also be utilized. If the agent is an antibody, a cross-reactive antibody, a monoclonal antibody, or a plurality of antibodies, the agent may be any one or more of any one or more of the following antibodies, cross-reactive antibodies, monoclonal antibodies, or polyclonal antibodies. 々-chain of MHC-type antigen, α-chain of MHC HLA-DR antigen, α-chain of MHC antigen of type I or type II, α-chain of HLA-DR antigen, MHC antigen of type II or 18- 201114895 α- and yS-chain of MHC class I antigen. An example of an antibody is supplied by DaKo). The term "antibody" can include various fragments (whether derived from decleavage or recombinant techniques) and retaining binding activities such as Fab, F(ab')2 and scFv antibodies, and mimicking their plastids. Genetically engineered variants are also included as antibodies, some of which have been modified by, for example, replacement of amino acid residues to enhance the potential for binding of antibodies to different organisms to the organisms of the desired method according to the invention, to reduce the potential for immune side effects. Sex (this is a humanized " mouse monoclonal antibody). An agent for effecting the transformation of committed cells into dedifferentiated cells functions as an extracellular cell of a committed cell. For example, a constituting cell can be operably linked to a receptor and the agent is operably linked to the receptor. For example, the receptor can be a cell surface receptor. Examples of cell surface receptors include, but are not limited to, Type I and Type II MHC bodies may comprise an alpha component and/or a quinone component as an example of a Type I 5 MHC receptor. The receptor may comprise a cold-chain of a HLA-DR having a homologous region, such as a domain of HLA-DR. Alternatively or additionally, the receptor may comprise an alpha-chain of HLA-DR having a homologous region, such as at least a homologous region. The receptor may be a type I or type II antigen of a compatible complex (MHC). In particular, cell surface receptors may include, but are not limited to, receptors, DM receptors, DP receptors, DQ receptors. Body, HLA-A is regulated by CR3/43 (protein derivative, or bio-amino acid sequence, or a fine example of which is treated as "specially intended to contain a specific receptor for the agent. Type of source region 2 _ chain, the main organization. In some) HLA-DR, HLA- -19- 201114895 B receptor, HLA-C receptor, HLA-E receptor, HLA-F receptor or HLA-G receptor. In some embodiments, the cell surface receptor may be a η LA-DR receptor. The agent may be an antibody to the receptor, such as a monoclonal antibody of the receptor. An example of the agent may be a regulatory Μ HC Agents for gene expression, such as MHC class 1 + and / or MHC class 11 +. In some embodiments, agents can be used in combination with bioreactor modifiers. Examples of bioreactor modifiers include (but are not limited to) An alkylating agent, immunomodulator, growth factor, interleukin, cell surface receptor, hormone, nucleic acid, nucleotide sequence, antigen or peptide. The alkylating agent may be or may comprise cyclic guanidinium phosphate. Other biological response modifiers may include compounds that are capable of upregulating the expression of MHC class I and/or type II antigens, and in some embodiments, may allow The agent binds to the MHC receptor for more efficient operation. When any cell type can be expressed as a type I and/or type II MHC antigen, this provides a means for counter-differentiating a wide variety of cell types, regardless of such Whether the cell type constitutes a MHC class I and/or type II. The committed cells are typically incubated with the agent for at least 2 hours, typically between 2 and 24 hours, preferably between 2 and 12 hours. Incubation is typically performed from about room temperature or, for example, about 22 t up to about 37 ° C, including 33 ° C. The progress of the reverse differentiation procedure can be achieved by removing a small sample and using a microscope and/or flow cytometry. The cells are examined periodically and periodically. Alternatively, the device may contain tracking devices for online monitoring of the progress of the anti-differentiation program. -20- 201114895 In addition to the use of the anti-differentiation agent, the committed cells may be in autologous plasma or serum or in fetal blood. Cultured in serum or horse serum. The committed cells can be arbitrarily cultured with anticoagulants, chelating agents or antibiotics. The temperature range of the cells can be extended to 18-40 ° C and can also include 4-10% of CO 2 and / or Between 10 and 35% 〇 2. In addition, incubation can occur in coated or uncoated blood bags, tissue culture bags or plastic containers. Certain types of dedifferentiated cells can be obtained by using special culture conditions. Obtained by dedifferentiation. For example, the committed cells can be obtained by using Dürbeck modified Eagle's medium (DMEM), non-essential amino acid (NEAA), L-glutamic acid (L-) in combination with the anti-differentiation agent. The gliotype cells were cultured in glu) and /3 - hydrazine ethanol (2 cold ME) and dedifferentiated into pluripotent cells. The committed cells can also be initially exposed to a chelating agent. The committed cells can be cultured using DMEM (low glucose) and L-glu or DMEM (low glucose), L-glu, 2 no ME, and NEAA in combination with a differentiation promoter to obtain mesenchymal cells, as another example. Furthermore, the antibiotic gentamycin can also be used in cell culture. Transdifferentiated cells Transdifferentiated cell lines are obtained by culturing committed cells in tissue culture medium in combination with a retrodifferentiator. The committed cells are thus transdifferentiated, wherein the committed cells are transformed into cells of another cell type; in some embodiments, the committed cells are transformed into cells of different pedigrees. The type of target cell obtained via transdifferentiation depends on the culture conditions. These conditions are based on the type of tissue culture medium, the presence of various differentiation enhancers - 21, 201114895 / absence, different serum presence/absence, incubation temperature, presence/absence of oxygen or carbon dioxide, and the type of box or container used for cultivation. Examples of tissue culture media used for transdifferentiation include, but are not limited to, ISK-modified Durbuker's medium (IMDM), Durbeck's modified Eagle's medium (DMEM), and Eagle's lowest Essential (eme) medium, α-minimum essential medium (α - Μ Ε Μ ), Roswell Park Memorial (RPMI; site where the medium was developed) 1 640, Ham-F-12, E199, MCDB, Leibovitz L-1 5 and Williams Medium E or any commercially formulated tissue culture medium differentiation enhancer includes anticoagulants, chelating agents, and antibiotics. Examples of such agents may be one or more of the following: vitamins, minerals or derivatives thereof such as vitamin A (retinol), vitamin B3, vitamin C (ascorbate), ascorbic acid 2-phosphate, vitamin D2 , vitamin D3, vitamin K, retinoic acid, nicotinamide, zinc or zinc compounds and calcium or calcium compounds: natural or synthetic hormones such as hydrocorticosterone and dexamethasone; amino acids or derivatives thereof, such as L - glutamic acid (L-glu), ethylene glycol tetraacetic acid (EGTA), proline and non-essential amino acids (NEAA): compounds or derivatives thereof, such as mercapto cetyl alcohol, dibutyl Cyclic adenosine monophosphate (db-cAMP), monothioglycerol (MTG), carnitine, dimethyl sulfoxide (DMSO), hypoxanthine, adenine, forskolin, cilostazol An amide (cilostamide) and 3-isobutyl-1-methylxanthine; a nucleoside and an analogue thereof, such as a 5-azacytidine nucleoside; an acid or a -22- 201114895 salt thereof, such as ascorbic acid, acetone Acid salt, okadic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), anticoagulant citric acid Glucose A (ACDA), disodium EDTA, sodium butyrate and glycerol phosphate: antibiotics or drugs such as G418, gentamicin, pentoxifylline (1-(5-ketohexyl)-3,7 - dimethylxanthine) and indomethacin; and proteins such as tissue plasminogen activator (TPA). These differentiation enhancers can be used to obtain a particular type of target cell. For example, vitamin B3 can be used to obtain acinar cells, such as pancreatic islet cells; hydrocorticosterone or dexamethasone can be used to obtain mesenchymal or epithelial-derived cells (eg, renal epithelial cells, skin, and related structures, For example, dermal papilla cells: and Θ-mercaptohexadecanol can be used to obtain ectodermal cells, such as neuronal cells, including adherent cells of the CNS. The medium may contain autologous blood, platelets, serum (such as fetal blood samples) or serum from mammalian sources (such as horse serum). In addition, the transformation process can occur inside a blood bag, stent, tissue culture bag or plastic tissue culture container. The tissue culture vessel may be coated or uncoated with an agent such as gelatin, collagen, matrigel or extracellular matrix for adhering or unattached tissue culture to promote attachment or floatation, depending on The type of tissue required or the particular cell to be prepared. Additional culture conditions include temperature, which may be between about 1 Torr and about 6 〇t or between about 18 and about 4 (TC; carbon dioxide (C02) concentration, which may be between about 〇 and about 20 % or between about 4 and about 10%: and oxygen (〇2) concentration 'which may be between about 0 and about 50% or between about 1 and about 35% -23- 201114895 for obtaining target cells and Examples of methods in which the anti-differentiator is used in combination are discussed in Table 1. Table 1 Methods for Combining with Differentiation Agents to Obtain Various Types of Target Cells Standard Cell Types Combined with Anti-Differentiation Agents Culture Conditions Additional Culture Conditions Pluriability Heterogeneous population of stem cells or pluripotent progenitors • DMEM, NEAA, L-glu, 2 / 3 ME • Prior to exposure to chelating agents or anticoagulants • Extraction of anti-differentiators, dilution of progenitor mesenchymes only in medium Stem cells • DMEM (low glucose), L-glu; or .DMEM (low glucose), L-glu, 2 / 3 ME ' NEAA • Jiandamycin • Pluripotent stem cells • Oocytes • Sperm • For eggs RPMI 1640 of parental cells, optionally with NEAA, L-glu, 2/3ME: or • for oocytes Sputum medium, retinol, L-glu, sodium pyruvate, sodium lactate, NEAA; or • DMEM for sperm, Hams F12, vitamin C, vitamin E, retinoic acid, retinol, pyruvate, optionally Benximalin; or • Culture conditions for the above listed pluripotent stem cells with additional culture conditions listed in the next column for sperm or oocytes • For sperm culture at about 30-32 ° C Lower and for oocyte culture can be at about 38-39 ° C • Cells can be exposed to chelating agents such as EDTA and EGT before dedifferentiation and re-differentiation • For sperm, can include the addition of black sponge acid, DMSO And zinc or zinc compounds; Gamete 100 can be used as an alternative basic medium • for oocytes, including dp-cAMP, disodium EDTA, forskolin, cilostaamine and hypoxanthine • For oocytes, medium 199 can be used as an alternative to the basic medium kidney cells • DMEM, Hams F12 and hydrocorticosterone, optionally with vitamin K; or • culture conditions for upper 歹 0 pluripotent stem cells, with hydrogen Corticosterone; -24- 201114895 • Alveolar epithelial cells • Endoderm cells • DMEM, NEAA, L-glu, random 2 ΘΜΕ, nicotinic amide; or • Culture conditions for the above listed pluripotent stem cells, with nicotine amide, freely Cesson, retinoic acid, db-cAMP; or • MDM, L-glu, ascorbic acid, MTG • Culture medium with antibiotic G418 and basement membrane matrix • Neuronal cells • Ectodermal cells • DMEM with Hams F12 , NEAA, 2; SME, optionally with carnitine, retinoic acid, L-glu, hydrocorticosterone, ascorbate; or • for the culture conditions of the above listed pluripotent stem cells, optionally with carnitine, retinoic acid, Hydrocorticosterone, ascorbate pancreatic islet cell acinar cells. DMEM, Hams Π 2, vitamin B3: or • RPMI 1640 with vitamin B3; • for the culture conditions of the above listed pluripotent stem cells, with vitamin B3 (nicotine 醯Amine), ad libitar with hematopoietic cells • IMDM, optionally with hydrocorticosterone; or • IMDM, L-glutamic acid and MTG; • for the culture conditions of the above pluripotent stem cells, with Generation 2 without MT MTG, random vitamins • Incubation at 33 ° C • Incubation at room temperature to expand megakaryocytes in the culture medium • Differentiated cells can be exposed to chelating agents prior to transformation to expand in the medium Red blood cell progenitor cells • RPMI1640 can be used as a basic medium for enrichment of lymphoid progenitor cells • Sodium butyrate and/or 5-aza-cytosine can be added to the culture medium to promote hepatocytes differentiated from the original red blood cells. DMEM or IMDM or α-minimum essential medium, L-glu, arbitrarily with sesamone, L ascorbate-2-phosphate and nicotinamide; or • Williams medium E, sodium pyruvate, dexamethasone; For the culture conditions of the above pluripotent stem cells, with dexamethasone-25- 201114895 skin / keratinocytes • HamsF12, DMEM (3: 1 medium), hydrocorticosterone, L-glu, random adenine Or; E199 or DMEM with L-glu, optionally with hydrocorticosterone and adenine; or • Culture conditions for the above listed pluripotent stem cells, with hydrocorticosterone, random calcium or calcium compounds or resistance to Acid salt • can have gentamicin as an antibiotic • Can grow melanocytes at 36.4t • MCDB and Leibovitz L-15, TPA, optionally with 3-isobutyl-1-methylxanthine; or • Culture medium 199 And corticosterone osteocytes/bone • DMEM, /3-glycerophosphate, dexamethasone, ascorbate and L-glu, optionally with vitamin D3; or • for the culture conditions of the above pluripotent stem cells, with glycerol Phosphate, dexamethasone and ascorbate, optionally with vitamin d3 dermal papilla cells/hairs • Williams medium E, L-glu, hydrocorticosterone and/or vitamin D2, adenine and linoleic acid; or • DMEM, as Basic medium of HamFl2, L-glu, hydrocorticosterone and / or vitamin D2, adenine and linoleic acid; or • for the culture conditions of the above listed pluripotent stem cells, with hydrocorticosterone, vitamin d2, adenine and Linoleic acid chondrocytes/cartilage • DMEM, pyruvate, ascorbate 2-phosphate, dexamethasone and proline: or • for the culture conditions of the above pluripotent stem cells, with ascorbate 2- Acid, dexamethasone and proline adipocytes/lipid cells • DMEM, dexamethasone and mersin; or • dexamethasone and mersin-26- 201114895 skeletal muscle cells • DMEM, low glucose, random Hydrocorticosterone, dexamethasone, L-glutamine and sodium pyruvate; or • DMEM and Ham F12 or sputum as the basic medium, or DMEM and medium 199; or • Culture of the pluripotent stem cells listed above Condition 'with glucose, gentamicin and low serum• can have gentamicin as an antibiotic • can have a low serum concentration • can have a gelatin culture container • can be raised to a temperature of 39 ° C • can include 5 - aza-cytosine nucleoside blood container (endothelial cells) • DMEM, NEAA and 2ySME; or • IMDM and dexamethasone myocardium/cardiomyocytes • 4:1 DMEM and M199 medium or DMEM (low glucose), L-glu , NEAA; or DMEM with ascorbic acid (low glucose) and / or DMSO; or • for the culture conditions of the above listed pluripotent stem cells, with anti-gravity culture environment or in a vibrating environment • culture with gelatin coating Autologous serum or platelets for complete differentiation and plasma removal • Complete differentiation can be observed on coverslip slides in trophoblast cells • DMEM, L-glu, 2/3ME serially diluted in the same medium minus dedifferentiators , NEAA; or • RPMI 1640, 2ySME, sodium pyruvate and L-glutamine; can be used to produce differentiated cells into mesoderm cells and ectoderm cells (including germline and progenitor cells) essential autogrowth factors And the culture conditions in which the hormone is extracted with the corresponding medium and then diluted to extract the anti-differentiation agent for the culture conditions described in each cell type in Table 1 particularly cause a more enhanced reverse differentiation. During the culture, the medium optionally containing various differentiation promoters can be diluted by adding more medium, but not by a differentiation inhibitor. Without being bound by theory, dilution appears to increase differentiation as cells become less dense and proliferative stimulating factors are less concentrated. Therefore, the addition of the medium further enhances the transdifferentiation and affects what type of cells are obtained within the cell lineage. For example, if the target cells are neurons, the addition of culture medium can cause developmental movement toward more mature nerves rather than neuronal progenitors (both have the same pedigree). The medium achieved by gradually decreasing the serum concentration serially dilutes only the skeletal muscle progenitor cells to differentiate as another example of advancing differentiation. Re-differentiated cells Re-differentiated cells can be used to obtain target cells by typing or re-differentiating the dedifferentiated cells into the target cell type. This can be performed by contacting the dedifferentiated cells with growth factors. For example, retinoic acid has been used to differentiate stem cells into neuronal cells. Methylcellulose co-cultured with bone marrow stromal cells and IL7 has been used to differentiate stem cells into lymphocyte precursors (Nisi Uni et al., Int Immuno 1 994, 6: 909-916). Le Page (New Scientist Dec. 16, 2000) directs stem cells to differentiate into lung epithelial cells. Bischoff (Dev Biol 1 986, 115: 129-39) directs how to differentiate muscle satellite cells into mature muscle fibers. Neuronal precursor cells can be expanded with basic fibroblast growth factor and epidermal growth factor (Nakafuku and Nakamura, J. Neurosci Res 1995, 41: 153-168). Hematopoietic stem cells can be expanded using a number of growth factors, including GM-CSF, erythropoietin, stem cell factor, and interleukin (11-1, IL-3, IL-6), see Metcalf ( Nature 1989, 339: 27_30) for review. These various factors.

Potocnik 等人（EMBO J 1 994，1 3: 5274-83 )甚至證 明利用低氧（5% )條件分化幹細胞成爲造血細胞。 已重新分化細胞可具有與衍生出已反分化細胞之定型 細胞相同的系譜。或者，已重新分化細胞可具有與衍生出 -28- 201114895 已反分化細胞之定型細胞不同的系譜。例如，B淋巴細胞 可反分化成CD34 + CD38-HLA-DR·幹細胞、此幹細胞接著 可循著B細胞系譜（相同的系譜）或淋巴系譜（不同的系 譜）重新分化或定型。 標的細胞 本發明的標的細胞爲已重新編程細胞，其可藉由如上 述之反分化、轉分化或重新分化而獲得。根據本發明，標 的細胞可包括（但不限於此）多能性幹細胞、淋巴幹細胞 、脊髓幹細胞、神經元幹細胞、骨骼肌衛星細胞、上皮幹 細胞、內胚層與神經外胚層幹細胞、生殖細胞、胚外與胚 胎幹細胞、間葉幹細胞、腎細胞、肺泡上皮細胞、內胚層 細胞、神經元、外胚層細胞、胰小島細胞、腺泡細胞、卵 母細胞、***、造血細胞、肝細胞、皮膚/角質化細胞、 黑色素細胞、骨頭/骨細胞、毛髮/真皮乳頭細胞、軟骨/軟 骨細胞、脂細胞/脂肪細胞、骨骼肌細胞、內皮細胞、心 肌/心肌細胞及滋養層細胞。 如上所述，定型細胞及/或已反分化細胞係在特殊的 條件下培養以誘發反分化及/或轉分化及/或重新分化及獲 得標的細胞。培養定型細胞及/或已反分化細胞期間不以 特別的時間長度控制，但反而由測定已產出之標的細胞控 制。 已反分化、已轉分化或已重新分化細胞的產出及數量 變化的測定可藉由監控向下調節系譜相關標誌或轉錄因子 -29 * 201114895 表現之定型細胞的相對數量變化及/或具有標的細胞之細 胞表面標誌特徵之細胞的相對數量變化來執行。或者或另 外，可監控具有定型細胞而非標的細胞典型的細胞表面標 誌之細胞降低的數量。例如’標的細胞可爲胚胎幹細胞’ 其以許多階段特異性標誌特徵化，諸如POU5F1 ( OCT-4 )、TERT、KLF4、UTF1、SΟX2、Nanog 或階段特異性胚 胎標誌3與4(SSEA-3與SSEA-4)、高分子量醣蛋白 TRA-1-60與TRA-1-81及驗性磷酸酶（Andrews等人之 Hybridoma 1 984，3 : 3 4 7-3 6 1 ； Kannagi 等人之 EMBO J 1 983，2 : 2355-236 1 ； Fox 等人之 Dev Biol 1 984，103 : 263 -266 ； Ozawa 等人之 Cell Differ 1 985, 1 6: 1 69- 1 73 ) 。彼等亦不表現SSEA-1，其之存在爲分化指標。已知其 他類型的幹細胞之其他標誌，諸如神經上皮幹細胞之 Nestein (J Neurosci 985，5: 3310)。間葉幹細胞對例 如 SH2、SH3、CD29、CD44、CD71、CD90、CD 1 06 ' CD120a 及 CD124 爲陽性，而對 CD34、CD45 及 CD14 爲 陰性。多能性幹細胞爲CDS^DITTdr細胞（其他有用的 標誌爲CD38·及CD36+)。淋巴幹細胞爲DR+、CD34 +及 TdT +細胞（亦爲CD38+)。骨髓幹細胞爲CD34+、DR+、 CD13+、CD33+、CD7 +及 TdT +細胞。 標的細胞額外的細胞標誌可經由微陣列分析發現。此 分析可包含從已反分化及/或已轉分化及/或已重新分化之 標的細胞分離RNA、以染料標記分離之RNA及使分離之 RNA與微陣列雜交。微陣列可包含代表全基因體的基因 -30- 201114895 或寡核苷酸，或可包含引導至特殊的器官系統、組織系統 、疾病、病理學等之基因或寡核苷酸。細胞標誌可藉由其 使基因/寡核苷酸展現高訊號強度而予以鑑證且由此在標 的細胞中向上調節或向下調節。接著可應用此訊息以測定 由微陣列分析鑑證存在之標誌或甚至標誌群或圖案爲基準 之標的細胞。 標的細胞的確認亦可利用許多試管內檢定法執行，諸 如CFC檢定法（亦參閱實例）。非常原始的造血幹細胞 常利用長期培養引發細胞（LTC-IC )檢定法測量（Eaves 等人之 J Tiss Cult Meth 1991，1 3 : 55-62 ) » LTC-IC 持續 造血5至12週。 用於其他細胞類型（諸如中樞神經系統、胰、肝、腎 、皮膚等之細胞）的細胞培養可持續，直到標的細胞出現 以免疫組織化學、流動式細胞測量術 '微陣列或反轉錄聚 合酶鏈反應（RT-PCR )之特徵化爲止，其爲本技藝中已 知的技術。此亦可包括功能檢定法，例如植入免疫缺乏宿 主或矯正或改善如本文所觀察之下列臨床症狀。 標的細胞可以顯示在標的細胞中獲取之新系譜特異性 轉錄因子、蛋白質及訊號之微陣列或RT-PCR鑑證。例如 ’轉化成外胚層系譜之標的細胞的已反分化幹細胞可表現 諸如 Nestin、Criptol、isll、LHX1 及 / 或 EN1 之基因，若 進一步分化成神經元，則表現神經絲（NF )。另一方面 ’轉化成內胚層系譜之標的細胞的細胞可表現諸如sox7 、soxl 7、Nodal、PDX1 及/或 F0XA2 之基因；但是進一 -31 - 201114895 步朝向胰藏的胰小島細胞分化之標的細胞可表現諸如胰島 素（INS)及神經顆粒素（neurog ) 3 ( NGN3 )之基因。 朝向所欲標的細胞轉化可特別伴隨向下調節與進行轉化之 原起始群體有關聯的成熟轉錄因子。 另外，產出之標的細胞的測定可藉由識別標的細胞的 特殊結構及/或型態特徵，例如細胞形狀、尺寸等而被發 現。這些特徵爲本發明的標的細胞技藝中已知。 一旦所欲細胞類型的相對數量增加到適合的濃度時， 其可爲例如低至〇. 1 %或高至5 %，則可以許多方式利用 所得變更之細胞群體。至於所形成之標的細胞（例如，多 能性幹細胞）的數量，重要的是鑑定幹細胞的增生能力。 雖然在一些環境下所形成之幹細胞或其他已反分化細胞數 量看似少量，但是硏究顯示僅50多能性造血幹細胞可在 小鼠施予者中重組成整個造血系統。因此，治療效用不需 要形成大量細胞。 定型細胞轉化成已反分化、已轉分化或已重新分化標 的細胞亦可藉由與醫藥載劑或稀釋劑混合的劑投予病患而 於活體內進行。然而，在許多例子中，較佳的是反分化、 轉分化或重新分化係在試管內/活體外執行。 在試管內獲得的經治療之細胞群體接著可以最少的處 理予以利用。例如，彼等可與醫藥上可接受之載劑或稀釋 劑簡單地混合且投予需要幹細胞之病患。 然而，可能希望富集已反分化、已轉分化或已重新分 化標的細胞的細胞群體或純化來自細胞群體的細胞。此可 -32- 201114895 利用許多方法方便地執行（參閱 Vattese-Dadey，The Scientist 1 999, 1 3 )。例如，細胞可利用層析術及/或流動 式細胞測量術以細胞表面標誌爲基準純化。但是既不常需 要亦不希望大規模純化來自細胞群體的已反分化、已轉分 化或已重新分化標的細胞，因爲在群體中存在的其他細胞 (例如，基質細胞）可維持幹細胞生存力及功能。 流動式細胞測量術爲一種使混合之群體內的細胞特徵 化與挑選細胞之完整建立的可信賴且有效力的技術。因此 ，純化或分離設備可包含流動式細胞測量儀。流動式細胞 測量術係以液體懸浮液中的粒子之物理特徵爲基準操作， 在以光束審訊時可以區別粒子。此等粒子當然可爲細胞。 物理特徵包括細胞尺寸及結構或以結合螢光分子之單株抗 體束縛之細胞表面標誌，其成爲近年來非常流行的特徵。Potocnik et al. (EMBO J 1 994, 1 3: 5274-83) even demonstrated the differentiation of stem cells into hematopoietic cells using hypoxic (5%) conditions. The re-differentiated cells may have the same pedigree as the committed cells from which the dedifferentiated cells have been derived. Alternatively, the re-differentiated cells may have a different pedigree from the committed cells from which the dedifferentiated cells of -28-201114895 have been derived. For example, B lymphocytes can be dedifferentiated into CD34 + CD38-HLA-DR· stem cells, which can then be re-differentiated or stereotyped following the B cell lineage (same pedigree) or lymphoid profile (different pedigree). Target Cells The subject cells of the present invention are reprogrammed cells which can be obtained by reverse differentiation, transdifferentiation or re-differentiation as described above. According to the present invention, the target cells may include, but are not limited to, pluripotent stem cells, lymphoid stem cells, spinal cord stem cells, neuronal stem cells, skeletal muscle satellite cells, epithelial stem cells, endoderm and neuroectodermal stem cells, germ cells, extraembryonic With embryonic stem cells, mesenchymal stem cells, kidney cells, alveolar epithelial cells, endoderm cells, neurons, ectodermal cells, pancreatic islet cells, acinar cells, oocytes, sperm, hematopoietic cells, hepatocytes, skin/keratinization Cells, melanocytes, bone/osteocytes, hair/dermal papilla cells, cartilage/chondrocytes, lipocytes/fatty cells, skeletal muscle cells, endothelial cells, myocardial/cardiomyocytes, and trophoblast cells. As described above, committed cells and/or dedifferentiated cell lines are cultured under specific conditions to induce retrodifferentiation and/or transdifferentiation and/or re-differentiation and to obtain target cells. The culture of committed cells and/or dedifferentiated cells is not controlled for a particular length of time, but instead is controlled by assaying the cells that have been produced. Changes in the output and quantity of cells that have been de-differentiated, transdifferentiated, or re-differentiated cells can be monitored by monitoring down-regulated pedigree-associated markers or transcription factors -29 * 201114895 The relative amount of cells of the cell surface marker characteristic of the cell is performed to perform. Alternatively or additionally, the amount of cell reduction with cell surface markers typical of committed cells, but not the target cells, can be monitored. For example, 'the target cell can be an embryonic stem cell' which is characterized by a number of phase-specific markers, such as POU5F1 ( OCT-4 ), TERT, KLF4, UTF1, SΟX2, Nanog or phase-specific embryo markers 3 and 4 (SSEA-3 and SSEA-4), high molecular weight glycoprotein TRA-1-60 and TRA-1-81 and phosphatase (Andrews et al. Hybridoma 1 984, 3: 3 4 7-3 6 1 ; Kannagi et al. EMBO J 1 983, 2: 2355-236 1 ; Fox et al. Dev Biol 1 984, 103: 263-266; Ozawa et al., Cell Differ 1 985, 1 6: 1 69- 1 73 ). They also do not express SSEA-1, and their existence is an indicator of differentiation. Other markers of other types of stem cells are known, such as Nestein of neuroepithelial stem cells (J Neurosci 985, 5: 3310). Mesenchymal stem cells are positive for SH2, SH3, CD29, CD44, CD71, CD90, CD 1 06 'CD120a and CD124, and negative for CD34, CD45 and CD14. The pluripotent stem cells are CDS^DITTdr cells (other useful markers are CD38· and CD36+). Lymphocytes are DR+, CD34+ and TdT+ cells (also CD38+). Bone marrow stem cells are CD34+, DR+, CD13+, CD33+, CD7+ and TdT+ cells. Additional cell markers for the target cells can be found via microarray analysis. This assay may comprise isolating RNA from cells that have been dedifferentiated and/or transdifferentiated and/or re-differentiated, isolating the RNA isolated by dye labeling, and hybridizing the isolated RNA to the microarray. The microarray may comprise a gene -30-201114895 or an oligonucleotide representing a whole genome, or may comprise a gene or oligonucleotide that directs to a particular organ system, tissue system, disease, pathology, and the like. The cell marker can be verified by which the gene/oligonucleotide exhibits a high signal intensity and thereby up-regulated or down-regulated in the target cell. This message can then be applied to determine cells identified by microarray analysis for the presence of a marker or even a marker population or pattern. Confirmation of target cells can also be performed using a number of in-vitro assays, such as the CFC assay (see also examples). Very primitive hematopoietic stem cells are often measured using the Long Term Culture Initiating Cell (LTC-IC) assay (Eaves et al. J Tiss Cult Meth 1991, 13: 55-62) » LTC-IC continues to hematopoietic for 5 to 12 weeks. Cell culture for other cell types (such as cells of the central nervous system, pancreas, liver, kidney, skin, etc.) can be sustained until the target cells appear to be immunohistochemical, flow cytometry' microarray or reverse transcriptase Characterization of the chain reaction (RT-PCR), which is a technique known in the art. This may also include functional assays, such as implanting an immunodeficient host or correcting or ameliorating the following clinical symptoms as observed herein. The target cells can display a new pedigree-specific transcription factor, a microarray of proteins and signals, or RT-PCR forensics obtained in the target cells. For example, the dedifferentiated stem cells transformed into the cells of the ectodermal pedigree may express genes such as Nestin, Criptol, isll, LHX1 and/or EN1, and if further differentiated into neurons, the neurofilaments (NF) are expressed. On the other hand, cells that are transformed into the cells of the endoderm pedigree may express genes such as sox7, soxl 7, Nodal, PDX1, and/or F0XA2; however, the target cells that differentiate toward the pancreatic islet cells of the pancreas in the -31 - 201114895 step Genes such as insulin (INS) and neurotrophin 3 (NGN3) can be expressed. Transformation to the desired cell can be particularly accompanied by down-regulation of the mature transcription factor associated with the original starting population for transformation. In addition, the determination of the target cells of the production can be found by identifying the particular structure and/or type characteristics of the target cells, such as cell shape, size, and the like. These features are known in the subject art of the invention. Once the relative amount of cell type desired is increased to a suitable concentration, which can be, for example, as low as 0.1% or as high as 5%, the resulting altered cell population can be utilized in a number of ways. As for the number of cells (e.g., pluripotent stem cells) formed, it is important to identify the proliferative capacity of stem cells. Although the number of stem cells or other dedifferentiated cells formed in some environments appears to be small, studies have shown that only 50 pluripotent hematopoietic stem cells can reconstitute the entire hematopoietic system in mouse donors. Therefore, the therapeutic effect does not require the formation of a large number of cells. The transformation of committed cells into cells which have been subjected to reverse differentiation, transdifferentiation or re-differentiation may also be carried out in vivo by administration to a patient by mixing with a pharmaceutical carrier or diluent. However, in many instances, it is preferred that the anti-differentiation, transdifferentiation or re-differentiation is performed in vitro/in vitro. The treated cell population obtained in the test tube can then be utilized with minimal treatment. For example, they can be simply mixed with a pharmaceutically acceptable carrier or diluent and administered to a patient in need of stem cells. However, it may be desirable to enrich a population of cells that have been dedifferentiated, have been transdifferentiated or have been reclassified, or purified cells from a population of cells. This can be easily implemented in many ways - 32- 201114895 (see Vattese-Dadey, The Scientist 1 999, 1 3 ). For example, cells can be purified by chromatography and/or flow cytometry based on cell surface markers. However, it is neither necessary nor desirable to large-scale purification of cells that have been dedifferentiated, transdifferentiated or re-differentiated from a cell population, as other cells present in the population (eg, stromal cells) can maintain stem cell viability and function. . Flow cytometry is a reliable and effective technique for characterizing cells within a mixed population and for the integrity of selected cells. Thus, the purification or separation device can comprise a flow cytometer. Flow cytometry operates on the basis of the physical characteristics of the particles in the liquid suspension, and the particles can be distinguished when the beam is interrogated. These particles can of course be cells. Physical characteristics include cell size and structure or cell surface markers bound by a single antibody that binds to fluorescent molecules, which has become a very popular feature in recent years.

Kreisseg 等人（J Hematother 1994，3: 263-89)陳述 "多參數流動式細胞測量術成爲測定造血幹細胞及祖細胞 之選擇工具，因爲抗-CD34單株抗體的可利用率"。 Kreisseg進一步敘述以流動式細胞測量術定量且特徵化 CD34表現細胞的槪括技術。Korbling等人（Bone Marrow Transplant 1 994，1 3: 649-54 )進一步指導以免疫吸附及 接著以HLA-DR表現爲基準之流動式細胞測量術純化 CD34 +細胞。如上述所討論，CD34 +爲與幹細胞/祖細胞有 關的有用標誌。流動式細胞測量術亦可用於挑選以其他的 物理特徵爲基準之幹細胞。例如’ visser等人（Blood Cells 1 9 8 0, 6 : 3 9 1 -407 )指導幹細胞可以彼之尺寸及結構 -33- 201114895 化程度爲基準分離。Gr〇gan等人（Blood Ce丨】s 1980，6: 625-44 )亦指導"可從簡單的造血組織挑選具有高且可證 實之純度的可存活幹細胞"。 不但以存在的細胞表面標誌或其他的物理性質爲基準 選擇細胞（正向選擇）’而且細胞群體可利用負向準則富 集純化。例如，具有系譜特異性標誌之細胞（諸如CD4 ' C D 8、C D 4 2及C D 3 )可可藉由流動式細胞測量術或親和 力層析術從細胞群體移出。 非常有用的純化細胞技術包含利用與磁珠連結之抗體 或其他親和配體。將磁珠以細胞群體培育且捕獲具有與親 合配體結合之細胞表面標誌（諸如CD34 )的細胞。將含 有細胞的樣品試管放入磁樣品濃縮器中，磁珠在此被吸引 至試管內部。在一或多次清洗階段之後，有興趣的細胞已 部分或實質上完全從其他的細胞純化。當使用以棄置液相 代替清洗以磁珠束縛之細胞的負向選擇格式時，則保存液 相及接著有效地從細胞群體移出以磁珠束縛之細胞。 這些以親和配體爲基準之純化方法可與適合其之標誌 已經特徵化或可特徵化的任何細胞類型使用。Urbankova 等人（J Chromatogr B Biomed Appl 1 996，687 : 449-52 ) 指導以重力場流體分離法從小鼠骨髓懸浮液製備造血幹細 胞之微製備作用。Urbankova等人進一步建議使小鼠骨髓 的幹細胞特徵化的方法，因爲這些細胞比骨髓中的其他細 胞更大，而因此有可能使這些細胞從混合物分離。因此， 除了細胞表面標誌以外的物理參數可用幹細胞純化/富集 -34- 201114895 包含由本發明的方法所產生之已重新編程標的細胞（ 諸如已反分化、已轉分化或已重新分化標的細胞）的細胞 群體及/或已純化之已重新編程標的細胞（諸如已反分化 、已轉分化或已重新分化標的細胞）可利用已知的技術維 持於試管內。典型地使用以哺乳動物血清（諸如FBS )及 隨意以自體血漿補充之最低生長培養基，諸如Hanks、 RPMI 1 640、杜爾貝克最低必需培養基（DMEM )或伊思 可夫改良之杜爾貝克培養基，以提供適合於細胞的生長環 境。幹細胞可在餵養層（諸如基質細胞層）上培養（參閱 Deryugina 等人之 Crit Rev Immunology 1 993，13 : 115- 1 50 )。咸信基質細胞分泌維持祖細胞於未分化狀態之因 子。用於幹細胞之長期培養系統由Dexter等人（J Cell Physiol 1 9 7 7, 9 1 : 3 3 5 )及 Dexter 等人（Acta Haematol 1979，62: 299)所敘述。 例如，Lebkowski 等人(Transplantation 1992，53: 1011-9)指導人類CD34 +造血細胞可利用使用共價固定於 聚苯乙烯表面上之單株抗體爲基準之技術純化且以此方法 純化之CD34 +細胞可維持大於85%之生存力。Lebkowski 等人（J Hematother 1 993，2 : 3 3 9-42 )亦指導如何分離及 培養人類 CD34+細胞。亦參閱 Haylock 等人（ Immunomethods 1 9 9 4, 5: 2 1 7-25 )以檢視各種方法。 可將包含幹細胞之細胞群體及包含幹細胞之純化製品 冷凍/冷凍保存供未來使用。適合於冷凍細胞及接著使彼 -35- 201114895 等復活之技術本爲本技藝中已知。 在一個觀點中，反分化、轉分化或重新分化發生於來 自或於膚色血球層樣品中的細胞。術語"膚色血球層〃意 謂在未凝結血液離心或允許靜置時在紅細胞與血漿之間形 成的白色細胞層。 治療方法 本發明的已重新編程標的細胞（諸如已反分化標的細 胞、已轉分化標的細胞及已重新編程標的細胞）可與各種 組份組合以產生本發明的組成物。組成物可與一或多種醫 藥上可接受之載劑或稀釋劑組合以產生醫藥組成物（其可 供人類或動物使用）。適合的載劑及稀釋劑包括（但不限 於此）等滲壓食鹽水溶液，例如磷酸鹽緩衝之食鹽水。本 發明的組成物可以直接注射投予。組成物可經調配用於非 經腸、肌肉內、靜脈內、皮下、眼內、口服、穿透皮膚投 予或注入脊髓液中。 包含標的細胞的組成物可藉由注射或植入而輸入。細 胞可於懸浮液中輸送或嵌進支撐基質中，諸如天然及/或 合成的生物可降解基質。天然基質包括（但不限於此）膠 原基質。合成的生物可降解基質包括（但不限於此）聚酐 及聚乳酸。這些基質可提供活體內脆裂細胞支撐。 組成物亦可包含本發明的已反分化或已轉分化或已重 新分化標的細胞及至少一種醫藥上可接受之賦形劑、載劑 或媒劑。 36- 201114895 輸送亦可爲經控制之輸送，亦即經可從數分鐘至數小 時或數天之期間輸送。輸送可爲全身性（例如，藉由靜脈 內注射）或引導至特殊的關注位置。細胞可利用微脂體轉 移而引入活體內。 標的細胞可以每公斤從lxl 05至lxl 07個細胞之劑量 投予。例如，可以14x1 06個CD3 4 +細胞投予70公斤病患 供組織重組。劑量可爲本申請案中所列之標的細胞的任何 組合。 本發明的方法可用於治療各種疾病、症狀或病症。此 等症狀包括（但不限於此）骨髓衰竭、血液學症狀、再生 不全性貧血、/S -地中海貧血、糖尿病、運動神經元疾病 、帕金森氏病、脊髓傷害、肌肉萎縮症、腎疾病、肝疾病 、多發性硬化症、充血性心臟衰竭、C型肝炎病毒、人類 免疫缺乏病毒、頭部創傷、肺疾病、抑鬱症、非阻塞性無 精症、男性更年期、更年期與***症、回春作用、硬皮病 潰瘍、牛皮癬、皺紋、肝硬化、自體免疫疾病、禿髮、色 素沉著性視網膜炎、角膜結晶失養症/失明或與組織退化 有關聯的任何病症。 再生不全性貧血爲罕見但是致命的骨髓病症，以全血 球減少症及細胞過少性骨髓爲表徵（Young等人之Blood 2006，108: 2509-2519)。病症可由表現Thl細胞激素（ 尤其以造血幹細胞室爲標的之r-干擾素）之活化I型細 胞毒性T細胞的免疫介導性病理生理學所引起，導致骨髓 衰竭及於是造血官能不足（Bacigalupo 等人之 -37- 201114895Kreisseg et al. (J Hematother 1994, 3: 263-89) stated that "multiparameter flow cytometry is the tool of choice for the determination of hematopoietic stem and progenitor cells because of the availability of anti-CD34 monoclonal antibodies". Kreisseg further describes a technique for quantifying and characterizing CD34 expressing cells by flow cytometry. Korbling et al. (Bone Marrow Transplant 1 994, 1 3: 649-54) further directed the purification of CD34+ cells by immunosorbent and subsequent flow cytometry based on HLA-DR expression. As discussed above, CD34+ is a useful marker associated with stem/progenitor cells. Flow cytometry can also be used to select stem cells based on other physical characteristics. For example, 'visser et al. (Blood Cells 1 9 8 0, 6: 3 9 1 - 407) direct stem cells to be separated based on their size and structure -33- 201114895. Gr〇gan et al. (Blood Ce丨) s 1980, 6: 625-44) also teaches "survivable stem cells with high and verifiable purity can be selected from simple hematopoietic tissue". Not only cells (positive selection) are selected based on the presence of cell surface markers or other physical properties, and the cell population can be enriched and purified using negative criteria. For example, cells with pedigree-specific markers (such as CD4 'C D 8, C D 4 2 and C D 3 ) can be removed from the cell population by flow cytometry or affinity chromatography. A very useful purification cell technique involves the use of antibodies or other affinity ligands linked to magnetic beads. The magnetic beads are grown in a population of cells and capture cells having a cell surface marker (such as CD34) that binds to the affinity ligand. The sample tube containing the cells is placed in a magnetic sample concentrator where the magnetic beads are attracted to the inside of the test tube. After one or more wash stages, the cells of interest have been partially or substantially completely purified from other cells. When a negative selection format is used in which the liquid phase is used instead of washing the cells bound by the magnetic beads, the liquid phase is preserved and then the cells bound by the magnetic beads are effectively removed from the cell population. These purification methods based on affinity ligands can be used with any cell type that is suitable for characterization or characterization. Urbankova et al. (J Chromatogr B Biomed Appl 996, 687: 449-52) directed the preparation of hematopoietic stem cells from a mouse bone marrow suspension by gravity field fluid separation. Urbankova et al. further suggest a method of characterizing stem cells of mouse bone marrow because these cells are larger than other cells in the bone marrow, and thus it is possible to separate these cells from the mixture. Thus, physical parameters other than cell surface markers can be purified/enriched with stem cells -34-201114895 Contains reprogrammed target cells (such as cells that have been dedifferentiated, transdifferentiated or re-differentiated) produced by the methods of the invention The cell population and/or purified reprogrammed target cells (such as cells that have been dedifferentiated, transdifferentiated or re-differentiated) can be maintained in vitro using known techniques. Minimal growth media, such as Hanks, RPMI 1 640, Durbek minimal essential medium (DMEM), or Isker's modified Durbeck medium, are typically used in mammalian serum (such as FBS) and optionally in autologous plasma. To provide a growth environment suitable for cells. Stem cells can be cultured on a feeding layer, such as a stromal cell layer (see Deryugina et al., Crit Rev Immunology 1 993, 13: 115-150). The stromal cells secreted to maintain the progenitor cells in an undifferentiated state. Long-term culture systems for stem cells are described by Dexter et al. (J Cell Physiol 197 7 , 9 1 : 3 3 5 ) and Dexter et al. (Acta Haematol 1979, 62: 299). For example, Lebkowski et al. (Transplantation 1992, 53: 1011-9) direct human CD34+ hematopoietic cells to be purified by this technique using a monoclonal antibody covalently immobilized on the surface of polystyrene. Cells can maintain greater than 85% viability. Lebkowski et al. (J Hematother 1 993, 2: 3 3 9-42) also teach how to isolate and culture human CD34+ cells. See also Haylock et al. (Immunomethods 1 9 9 4, 5: 2 1 7-25) for a variety of methods. Cell populations comprising stem cells and purified preparations comprising stem cells can be frozen/frozen for future use. Techniques suitable for freezing cells and subsequent resurrection of He-35-201114895 are known in the art. In one aspect, reverse differentiation, transdifferentiation, or re-differentiation occurs in cells from a sample of the skin layer of the skin color. The term "skin layer is a layer of white cells formed between red blood cells and plasma when the uncondensed blood is centrifuged or allowed to stand. Methods of Treatment The reprogrammed target cells of the invention, such as cells that have been de-differentiated, cells that have been transdifferentiated, and cells that have been reprogrammed, can be combined with the various components to produce a composition of the invention. The composition can be combined with one or more pharmaceutically acceptable carriers or diluents to produce a pharmaceutical composition (which can be used by humans or animals). Suitable carriers and diluents include, but are not limited to, isotonic saline solutions such as phosphate buffered saline. The composition of the present invention can be administered by direct injection. The composition can be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular, oral, penetrating skin administration or injection into the spinal fluid. The composition comprising the target cells can be input by injection or implantation. The cells can be delivered in a suspension or embedded in a support matrix, such as a natural and/or synthetic biodegradable matrix. Natural matrices include, but are not limited to, collagen bases. Synthetic biodegradable matrices include, but are not limited to, polyanhydrides and polylactic acids. These matrices provide fragile cell support in vivo. The composition may also comprise cells of the present invention that have been subjected to reverse differentiation or transdifferentiation or re-differentiation and at least one pharmaceutically acceptable excipient, carrier or vehicle. 36- 201114895 Conveying can also be controlled delivery, that is, it can be delivered from minutes to hours or days. Delivery can be systemic (e. g., by intravenous injection) or directed to a particular location of interest. Cells can be introduced into the living body by transfer of the liposome. The target cells can be administered at a dose of from 1 x 105 to 1 x 107 cells per kilogram. For example, a 70 kg patient can be administered 14 x 106 CD3 4 + cells for tissue reorganization. Dosages can be any combination of the subject cells listed in this application. The methods of the invention can be used to treat a variety of diseases, conditions or conditions. These symptoms include, but are not limited to, bone marrow failure, hematological symptoms, aplastic anemia, /S-thalassemia, diabetes, motor neuron disease, Parkinson's disease, spinal cord injury, muscular dystrophy, kidney disease, Liver disease, multiple sclerosis, congestive heart failure, hepatitis C virus, human immunodeficiency virus, head trauma, lung disease, depression, non-obstructive azoospermia, male menopause, menopause and infertility, rejuvenation , scleroderma ulcer, psoriasis, wrinkles, cirrhosis, autoimmune disease, alopecia, pigmented retinitis, corneal dystrophy/blindness, or any condition associated with tissue degeneration. Incomplete aplastic anemia is a rare but fatal bone marrow disorder characterized by hypopocytopenia and hypoxic bone marrow (Young et al., Blood 2006, 108: 2509-2519). The condition can be caused by the immune-mediated pathophysiology of activated type I cytotoxic T cells expressing Th1 cytokines, especially r-interferons marked by the hematopoietic stem cell compartment, leading to bone marrow failure and thus insufficient hematopoietic function (Bacigalupo et al.人之-37- 201114895

Hematology 2007，23-28)。雖然大部分的再生不全性貧 血病患可以從HLA-吻合之兄弟姊妹所獲得的幹細胞移植 而治療（Locasciulli 等人之 Haematologica. 2007; 92: 1 1 -1 8 )，但是以此進路延伸至較年長的病患或那些沒有 家族施予者的病患仍有大的挑戰。儘管在HLA-吻合之異 體幹細胞移植後尙可行的存活率，但是由於用於避免移植 物對抗宿主疾病（GVDH )之免疫抑制體系而使程序帶有 潛在的風險。例如，具有或不具有抗胸腺細胞球蛋白（ ATG )之高劑量環磷醯胺導致延長的免疫抑制期，使病患 易有機會感染。其他的潛在風險爲移植失敗，其可在幹細 胞移植後接著發生數週或數月（Gottdiener等人之Arch Intern Med 1981，1 4 1 : 758-763 ； Sanders 等人之 Semin Hematol 1991，28 : 244-249 )。而且，移植失敗的風險隨 著幹細胞移植前所接受之輸血次數而增加。 地中海貧血係以減低組成血紅素的血球蛋白鏈之一的 合成速度爲表徵之正染色體隱性遺傳性血液病。因此，常 由於調節基因的突變而使正常的血球蛋白生產不足，其引 起異常的血紅素分子形成，造成貧血。不同類型的地中海 貧血包括α地中海貧血、Θ地中海貧血及6地中海貧血， 其分別影響α血球蛋白、Θ血球蛋白及<5血球蛋白的產生 。治療包括長期輸血、鐵螯合劑、脾切除術及異體造血移 植。然而，由於缺乏HLA吻合之骨髓施予者而使長期輸 血不適用於大部分的病患，而異體造血細胞移植與許多可 能的倂發症有關聯，諸如感染及移植物對抗宿主疾病。 • 38 - 201114895 糖尿病爲引起異常高的血糖濃度（高血糖症）之症候 群。糖尿病係指由於體內的胰島素分泌或胰島素作用有缺 陷而導致高的血液葡萄糖濃度的疾病類別。糖尿病典型地 被分成兩種類型：以減少胰島素生產爲表徵之第I型糖尿 病或以對抗胰島素效應爲表徵之第II型糖尿素。兩種類 型皆導致高血糖症，其主要引起通常與糖尿病有關聯的徵 候，例如尿量太多、造成補償口渴且流體攝取增加、視覺 模糊 '不明原因的體重下降、嗜睡及能量代謝變化。糖尿 病被認爲是不會痊癒的慢性疾病。治療選擇侷限於胰島素 注射、運動、適當的膳食或對患有第2型糖尿病之病患的 一些藥劑，例如那些促進胰臟的胰島素分泌、降低由肝臟 所產生的葡萄糖、增加細胞對胰島素的敏感度等之藥劑。 運動神經元疾病係指影響運動神經元的神經病症類別 。此等疾病包括肌萎縮性脊髓側索硬化症（ALS )、原發 性脊髓側索硬化症（PLS )及漸進性肌肉萎縮症（PMA) 。ALS係以上與下運動神經元二者退化爲表徵，其停止訊 息至肌肉及引起其變弱且最終萎縮。PLS爲只影響上運動 神經元的罕見運動神經元疾病，其造成平衡困難、腿部虛 弱與僵直、痙攣及脾臟問題。PM Α爲只影響下運動元神 經的ALS次型，其可造成肌肉萎縮、束化及虛弱。沒有 任何已知的運動神經元疾病痊癒療法。咸信減輕對運動神 經元損傷的利魯唑（riluzole)已被證實爲用於ALC之藥 劑，雖然其緩慢降低ALS的進展而不是改善其影響。PLS 的治療僅著重於徵狀，諸如可減輕痙攣的貝可芬（ -39- 201114895 baclofen)及可降低痛性痙攣的奎寧（quinine)。 帕金森氏病（PD)係以喪失黑質紋狀體路徑爲表徵 之神經退化性病症，從黑質內的多巴胺能神經元退化所引 起。PD的原因未知，但是與多巴胺能（酪胺酸羥化酶（ TH )陽性）中腦神經元的漸進性死亡有關聯，誘發運動 障礙。於是PD係以肌肉僵硬、震顫、動作遲緩及可能的 運動不能症爲特徵。因此，目前沒有任何滿意的帕金森氏 病痊癒療法或預防或治療帕金森氏病或其徵候的治療。與 疾病有關聯的運動障礙之對症療法包含口服投予二羥苯基 丙胺酸（L-DOPA )，其可導致實質改善的運動功能，但 是其效果在多巴胺能神經元進展退化時減低。替代策略包 括神經元移植，其係以從植入紋狀體中的細胞供給之多巴 胺可取代喪失的黑質紋狀體的槪念爲基準，及基因療法， 可藉由引入負責L-D0PA之酵素用於代替在受影響之紋狀 體中的多巴胺，或多巴胺合成法，諸如藉由引入可避免 TH-陽性神經元死亡或刺激在損傷之黑質紋狀體系統中的 再生及功能復原之潛在神經保護分子。 脊髓傷害係以脊髓及特別爲神經纖維損傷爲特徵，引 起傷害位置以下的部分或全部的肌肉或神經障礙。此等損 傷可經由骨折、脫位、壓碎或壓縮脊椎骨中之一或多個的 脊椎創傷’或經由關節炎、癌症、發炎或椎間盤退化所造 成之非創傷性傷害而發生。雖然在脊髓傷害後的治療可包 含藥劑’諸如甲基去氫皮質醇，其爲減輕神經細胞的損傷 及降低傷害區域發炎的皮質類固醇，或控制疼痛與肌肉痙 -40- 201114895 攣之藥劑，以及固定脊椎或以手術移除突出的椎間盤或任 何可能正在損傷脊椎之物體，但是沒有任何已知逆轉脊髓 損傷的方法。 肌肉萎縮症（MD )係指使骨骼肌變弱的遺傳性肌肉 疾病組別。MD可以漸進性肌肉虛弱、肌肉蛋白質缺陷、 肌肉細胞凋亡及組織萎縮爲特徵。有超過100種展現MD 特徵之疾病，雖然有9種疾病被歸類成MD，特別爲裘馨 型（Duchenne)、貝克型（Becker)、肢帶型、先天性、 顏面肩胛肱骨型、肌強直型、眼咽型、末梢型症及艾梅氏 (Emery-Dr ei fuss )型。沒有任何已知的 MD痊癒療法， 亦沒有任何特殊的治療。物理療法可維持肌肉張力及手術 可用於改善生活品質。徵候（諸如肌僵直）可進一步以藥 劑治療，但是沒有任何長期治療。 腎疾病係指損傷腎且降低其作用之能力的症狀，該作 用包括從血液移出廢棄物和過多的水、調節電解質、血壓 、酸鹼平衡及再吸收葡萄糖與胺基酸。腎疾病的兩種主要 原因爲糖尿病及高血壓，雖然其他的原因包括腎小球性腎 炎' 狼瘡及腎畸形與阻塞。沒有任何腎疾病的痊癒療法， 由此使醫療專注於減慢疾病的進展及治療疾病的原因，諸 如經由控制血糖與高血壓及監控膳食；治療疾病倂發症， 例如藉由著重於體液滯留、貧血、骨疾病：及代替喪失的 腎功能，諸如經由透析或移値。 多發性硬化症爲自體免疫症狀，其中免疫系統侵襲中 樞神經系統，導致脫髓鞘作用。MS影響腦及脊髓中的神 -41 - 201114895 經細胞彼此相通的能力，因爲身體本身的免疫系統 損傷圍繞神經元軸突的髓鞘脂。當喪失髓鞘脂時， 不再可能有效地傳導訊號。此可導致各種經常進展 及認知失能的神經徵候。沒有任何M S的痊癒療法 嘗試在侵襲後（突發或惡化的MS徵候）恢復功能 新的侵襲及避免失能。例如，以皮質類固醇治療可 止侵襲，同時在侵襲初期以干擾素治療已顯示出降 MS發展的機會。 人類免疫缺乏病毒（HI V )爲可導致後天免疫 (AIDS )的慢病毒屬，其中免疫系統開始衰竭的 HIV主要感染在人類免疫系統中的活力細菌，諸如 細胞、巨噬細胞及樹狀細胞。HIV感染係由直接病 感染細胞、增加感染細胞凋亡率或由識別感染細胞 細胞毒素淋巴細胞殺死感染的CD4+ T細胞而導 CD4+ T細胞濃度。目前沒有任何HIV或AIDS疫 癒療法。HIV感染的治療係由高活性抗反轉錄病毒 HAART所組成。目前的HAART選擇爲至少三種有 種類型的抗反轉錄病毒劑之藥物所組成的組合（或 酒〃）。這些類別典型地爲兩種核苷類似的反轉錄 劑（NARTI或NRTI)加上蛋白酶抑制劑或非核苷 酶抑制劑（NNRTI )。 充血性心臟衰竭係指其中心臟不可以泵送足夠 至身體其他器官的症狀。此症狀可從冠狀動脈疾病 肌梗塞所引起在心臟上的瘢痕組織、高血壓、心臟 侵襲及 則軸突 成身體 ;治療 、避免 幫助終 低臨床 缺乏症 症狀。 輔助T 毒殺死 的CD8 致低的 苗或座 療法或 至少兩 ，雞尾 酶抑制 反轉錄 的血液 、由心 瓣膜疾 -42 - 201114895 病、心臟缺陷及心臟瓣膜感染所造成。治療計劃典型地由 休息、適當的膳食、改善的每日活動及藥物（諸如血管緊 縮素轉化酵素（ACE )抑制劑、石阻斷劑、洋地黃、利尿 劑、血管舒張劑）所組成。然而，治療計劃不逆轉心臟的 損傷或症狀。 C型肝炎爲C型肝炎病毒所引起之肝臟中的感染疾病 。C型肝炎可進展成瘢痕（纖維變性）及增劇之瘢痕（肝 硬化）。肝硬化可導致肝衰竭及其他的倂發症，諸如肝癌 。目前的治療包括使用聚乙烯二醇化（pegylated)干擾素 α與抗病毒藥物利巴韋林（ribavirin )之組合。成功率可 取決於病毒基因型而在5 0-80%之間變動。 頭部創傷係指可能或可能不造成腦部傷害的頭部傷害 。頭部創傷的常見原因包括交通意外、居家與職場意外、 跌倒及襲擊。各種類型的問題可從頭部創傷引起，包括頭 骨骨折、頭皮撕裂傷、硬腦膜下血腫（在硬膜下出血）、 硬膜上血腫（在硬膜下與頭骨之間出血）、腦部挫傷（腦 部瘀傷）、腦震盪（由於創傷而暫時性功能喪失）、昏迷 或甚至死亡。頭部創傷的治療隨傷害類型而變動。若腦部 損傷，沒有任何快速的設備固定腦部及損傷常因可利用的 治療設備而爲不可逆。 肺疾病爲呼吸系統疾病的廣義術語，其包括肺、胸腔 、支氣管、氣管、上呼吸道及用於呼吸的神經與肌肉。肺 疾病的實例包括阻塞性肺部疾病（其中支氣管變窄）、侷 限性或纖維化肺疾病（其中肺喪失順應性且造成不完全的 -43- 201114895 肺擴張及增加肺硬度）、呼吸道感染（可由常見的感冒或 肺炎引起）、呼吸腫瘤（諸如那些由癌症所造成的腫瘤） 、胸腔疾病及肺部血管疾病（其影響肺部循環）。肺疾病 治療係根據疾病類型而變動，但是可包括藥劑（諸如皮質 類固醇與抗生素）、氧、呼吸器通氣、放射療法及手術。 抑鬱症爲精神性病症，其係以伴隨低自我形象及對一 般歡樂活動喪失興趣或樂趣的低情緒爲特徵。抑鬱症在生 物學上係由變更腦部多重部位的活動而隨著發生，包括縫 核，其爲羥色胺來源的上腦幹中的小核群；視叉上核，其 控制生物節律，諸如睡眠和醒來週期；下視丘-腦垂腺-腎 上腺軸，其爲身體對各種應激物的反應期間活動的結構鏈 :中腦腹側被蓋區，其被認爲負責腦的 > 報償〃迴路；阿 肯伯氏核（nucleus accumbens )，其被吾等視爲在報償、 笑、樂趣、成癮及害怕中扮演一角色；及前扣帶皮質區， 其因負面經驗而活動。抑鬱症的治療包括增加在腦中的細 胞外羥色胺量的抗抑鬱藥、運動及心理治療。然而，這些 治療的效力仍舊被懷疑。 非阻塞性無精症爲雄性的醫學症狀，由於具有***生 成作用的問題而在其***中不具任何可測量的***濃度。 此常由賀爾蒙不平衡所引起，並可使用修復不平衡之藥劑 治療。 男性更年期爲中年男性所經歷之似更年期症狀，其包 含減少激素睪固酮及去氫表雄固酮的產生。治療包括激素 替代療法及運動。 •44- 201114895 硬皮病爲影響結締組織的慢性自體免疫疾病。皮膚硬 化爲疾病最常見的表現，雖然其可影響全身的結締組織。 沒有任何已知的硬皮病直接痊癒療法。 牛皮癬爲造成在皮膚上出現鱗狀紅斑的慢性自體免疫 疾病。牛皮癬的原因與過度生長的皮膚細胞有關連。一種 假設暗示其與遷移至真皮且觸動誘發皮膚細胞快速產生的 細胞激素釋放之T-細胞有關連。牛皮癖的治療包括靶定 T-細胞的藥物。 色素沉著性視網膜炎爲漸進性視網膜失養症類型，其 中光受體或視網膜色素上皮細胞異常且導致視覺喪失。用 於治療色素沉著性視網膜炎的醫療法有限。 本文所述之症狀可以特殊類型的標的細胞或該等類型 之組合治療。在較佳的具體例中，本文所述之症狀可藉由 輸注表2中所槪述之細胞類型來治療。 -45- 201114895 表2 利用已重新編程，亦即已反分化或已轉分化或已重新分化標的細胞的各種症狀之治 療體系 症狀 治療細胞類型 (個別類型或其組合） 再生不全性貧血 造血細胞 冷-地中海貧血 造血細胞 糖尿病 間葉幹細胞、多能性幹細胞及/或胰小島細胞 運動神經元疾病 多能性幹細胞、肺泡上皮細胞、外胚層細胞及/ 或神經元 帕金森氏病 多能性幹細胞及/或神經元 脊髓傷害 多能性幹細胞及/或神經元 肌肉萎縮症 多能性幹細胞、間葉幹細胞及/或骨骼肌細胞 腎疾病 腎細胞、間葉幹細胞及/或多能性幹細胞 多發性硬化症 多能性幹細胞、間葉幹細胞及/或神經元 充血性心臟衰竭 心肌細胞、間葉幹細胞、多能性幹細胞及/或內 皮細胞 C型肝炎病毒 造血細胞、多能性幹細胞'間葉幹細胞及/或肝 的肝細胞 人類免疫缺乏病毒 造血細胞 頭部創傷 多能性幹細胞及/或神經元 肺疾病 多能性幹細胞、間葉幹細胞、肺泡上皮細胞及/ 或內皮細胞 抑鬱症 多能性幹細胞及/或神經元 非阻塞性無精症或男性更年期 多能性幹細胞、冬能件牛殖細胞及/或*** 更年期與***症 多能性幹細胞'卵母細胞、多能件牛殖細胞、 回春作用 多能性生殖細胞、多能性幹細胞、角質化細胞 、真皮乳頭細胞、神經元、***、卵母細胞、 軟骨細胞、心肌細胞、骨骼肌細胞、神經元、 內皮細胞及/或黑色素細胞 硬皮病潰瘍 多能性幹細胞、內皮細胞、角質化細胞及/或間 葉幹細胞 牛皮癖 間葉幹細胞及/或多能件幹細胞 皺紋 _閛葉幹細胞、角質化細胞及/或多能性幹細胞 肝硬化 肝的肝細胞'間葉幹細胞、內皮細胞、多能性 幹細胞 自體免疫疾病 間葉幹細胞及/或冬能性幹細胞 禿髮 真皮乳頭細胞及/或n色||細|^ 色素沉著性視網膜炎或角膜結 晶失養症/失明 神經兀及/或多能性幹細胞 -46- 201114895 病患可經由以實例方式的下列步驟治療如上述之症狀 1)將Fistula套管***病患手臂中； 2 )利用自動化系統，諸如COB E® Spectra裝置（ Gambro PCT )經由血漿分離術採收白血細胞； 3) 從病患的白血細胞生產自體已反分化幹細胞； 4) 將自體已反分化幹細胞清洗及接著經靜脈內輸注 至病患中； 5) 監控病患的進展，包括採取血液試驗及評鑑傷害 區域。 本發明現將以實例方式的下列非限制性實例進一步敘 述’其進一步例證本發明，而不意圖亦不應被解釋成限制 本發明的範圍。 【實施方式】 實例1 材料及方法 此臨床硏究評鑑在暴露於造血誘發培養條件後輸注個 齊1量之自體已重新標程細胞至4位患有再生不全性貧血 之病患中3小時的安全性。 此臨床硏究係由King Edward Memorial (KEM)醫 %之丨侖理委員會許可且與免疫血液學硏究所（IIH)共同 € ί乍執行。病患必需符合表3中槪述之準則。結果招收四 β胃胃Μ重貧血（3位男性）及先天再生不全性貧血（1 -47- 201114895 位女性）的病患進行硏究。選擇這4位病患且由IIH/KEM 人員監控。病患的臨床及治療史記述於表4中，同時將彼 之CD 3 4 +細胞輸注劑量顯示於表5中》 表3 納入準則 每一準則 皆必要 1-絕對嗜中性細胞計數<0.5x109/L 2-血小板計數<20x109/L 3-校正後網狀紅血球之貧血< 1 % 準則中 僅一者爲必要 4-骨髓細胞性<25% 5-骨髓細胞性<50%，具有少於30%之造血細胞 6-在診斷前的3個月內評估之受驗者 7-受驗者先前未接受免疫抑制療法 將病患輸予2單位以輻射照射之紅血細胞濃厚液及4 單位血小板以維持彼之血紅素濃度大於8公克/公合及血 小板計數大於50,000。利用Cobe Spectra血駿分離機及 白血細胞分離套組（二者皆來自 Gambro BCT)處理2-3 次病患總血量而使病患經血漿分離。血漿分離術包含以用 於靜脈通路之單管腔導管的頸及肘前靜脈導管***術。 細胞分裝係在收集1 50-200毫升膚色血球層後經無菌 收集供CD34分析。隨後使膚色血球層在造血誘發培養條 件下接受重新編程。簡言之，重新編程程序包含在3 0毫 升伊思可夫改良之培養基中稀釋的1 000微克純化之 CR3/43 (尤其以 DakoCytomation 爲 TriStem Corp.所製備 )經無菌加入白血細胞袋中。接著將細胞袋在維持在3 7 。(：及5 % C02下的無菌組織培養培育器中培育3小時"在 -48 - 201114895 完成重新編程法後，分析轉化之細胞的CD 3 4 +細胞含量。 隨後將細胞利用Cobe細胞處理器2991以食鹽水溶液清洗 兩次。一經攪拌且再懸浮於食鹽水溶液時’細胞懸浮液係 利用輸注設備在重力下經由頸靜脈輸注於病患中。在輸注 自體已重新編程細胞前及後連續監控病患的活力徵象’包 括CBC計數。 表4 再生不全性貧血病患直到自體人類已重新編程幹細胞（HRSC)輸注之臨床及治療史 病患 直到HRSC輸注時 之臨床史__ 直到RSC輸注時之治療 病患A : 25 歲男性嚴重 的再生不全 性貧血 •在2002年被診斷爲SAA •呈現虛弱且呼吸困難的徵候 •時常發作直腸與牙床出血及嘔吐 •每月分別接受4及2單元之血液及血 小板 •出現患黃疸嚴重的眼睛充血 •接受8個月的同化類 固醇（T^bMeiwbol) 試驗*沒有任何顯著 的改善 病患B : 26 歲女性先天 再生不全性 貧血 •在2004年1月被診斷爲先天再生不 全性貧血 •呈現6個月期間的經血排出量過多的 徵候 .病患僅在RSC輸注前分別接受4及6 單元之紅血細胞濃厚液及血小板 •從輸注RSC前3個月 起接受淨血劑，沒有 任何反應 病患C : 19 歲男性非常 嚴重的再生 不全性貧血 •在2003年被診斷爲非常嚴重的再生 不全性貧血 •呈現貧血、虛弱且費力時呼吸困難 •由於嚴重的嗜中性細胞減少症而持續 10-15天伴有寒顫之發燒 •多次發作感染、嘔吐及紫斑 •臀膿瘍 •由於腦部出血而昏厥兩次 •每兩週分別接受5及2單元之血液及 血小板 •接受環孢靈（ cyclosporine)療法 6 個月 ，在2003年3月結束，沒 有任何效果 病患D : 35 歲男性非常 嚴重的再生 不全性貧血 •貧血、虛弱、呼吸困難 •由於嚴重嗜中性細胞減少症而伴有寒 顫之發燒且多次發作感染及出血 •在3年前被診斷出 .每月分別接受4及2單元之血液及血 小板 •接受環孢靈及抗結核 病療法。6個月沒有免 疫抑制反應 -49- 201114895 所有的臨床監控皆由ΠΗ/KEM人員進行。在輸注前 或後的輸血需求係由接受任何單元之血液樣品後所獲得的 輸血記錄決定。輸血單元僅在獲得病患及作爲見證的直接 親屬同意之後利用。在病患輸血前，所有的血液樣品皆在 Tata記念醫院以輻射照射。病患在輸注已重新編程細胞前 及後亦被詢問關於彼之福祉的問題。所有的病患皆攜帶彼 之症狀及所有實驗室與臨床追蹤的影本或正本記錄。這些 病患的監控期原先設定爲2年，但後來經延長。病患在輸 注後第1個月內留在無菌的正壓室內。 表5 病患、輸注日期、重量與身高、收集之單核細胞及輸妇 Ε之CD34+細胞 病患ID及輸注日期 重量 身局 收集之 單核細胞 接受之CD34+/公斤 病患A 7/6/2004 48 152 lxlO9 11.7χ106 病患B 7/14/2004 38 153 3.6x109 20χ106 病患C 7/27/2004 52 166 3·9χ109 25x106 病患D 8/17/2004 52.5 160 4·3χ109 23χ106 將1百萬細胞根據製造商指示以下列的單株抗體盤（ 全部皆來自DakoCytomation)染色： 第1盤係由同型負對照組IgGl-FITC、IgGl-PE-Cy5 及IgGl-RPE共軛物所組成 第2盤係由抗人類CD45-FITC及CD34-RPE-Cy5所 -50- 201114895 組成 第3盤係由抗人類CD38-FITC及CD34-RPE-Cy5所 組成 第4盤係由CD61-FITC及CD34-RPE-Cy5所組成 第5盤係由CD33/13RPE及CD7-FITC所組成 第6盤係由CD45及血型糖蛋白（Glycophorin) -A-RPE所組成 第7盤係由抗人類CD3-FITC及CD19-RPE所組成 細胞分析係利用BD細胞Quest軟體以FACSCalibur 系統（BD bioscience)執行。 關於單株檢定法，將輸注已重新編程細胞前及後之病 患的骨髓單核細胞（MNC)接種在根據製造商指示（Stem Cell Technologies )以重組生長因子補充之 meth〇Cult GFH4434中。利用相位差轉換顯微鏡評鑑及以時間評分成 爲造血細胞群落的分化作用。 在程序前及後連續監控病患CBC、肝酵素及血紅素變 異。在出院後，病患CBC、肝酵素、血紅素變異及末梢血 液染色體核型鑑定與G-染色體條紋帶係以再確認爲目的 而由獨立的實驗室監控。這些試驗常在輸注自體已重新編 程細胞後執行。 分析在輸注自體已重新編程細胞前及後的末梢血液樣 品。在初次自體已重新編程幹細胞療法後第一年以6個月 間隔及2年後以每年爲基準重複此試驗。另外，分析在輸 注前的已重新編程細胞以硏究細胞的穩定性，此分析亦在 -51 - 201114895 3小時轉化步驟與已轉化細胞最長1個月的長期培養建立 後執行。染色體核型鑑定與G-染色體條紋帶係由第三個 獨立實驗室監控。 骨髓抹片及環鑿區係在輸注自體已重新編程細胞前及 後執行》此試驗係在自體已重新編程細胞後1 4-20天及隨 後以每年爲基準執行。 所有的抹片及環鑿區皆在輸注已重新編程幹細胞前及 後利用掛在攝錄像機的顯微鏡掃描。 結果 所有的病患皆耐受於血漿分離術及個別已重新編程幹 細胞輸注程序，沒有任何不良事件。病患A及病患D係 在個別輸注已重新編程造血幹細胞（RHSC )後獨立輸血 (參閱表6及7)。血小板、嗜中性細胞及紅血細胞植入 期接著分別發生在輸注病患A及病患D後3及6天。胎 兒血紅素切換註明在病患A及病患D中（參閱表4及5 ) ，但未註明在病患B及病患C中（沒有數據顯示）。在 輸注前，肝酵素在病患A及病患D中上升，儘管對HCV 爲陰性（如以ELISA所測量）。肝酵素在輸注RHSC後 正常化且在輸注RHSC後4年達到正常濃度。病患B及C 分別在輸注後2年及6個月死亡。胎兒Hb切換註明在病 患〇〇 1及病患〇〇4中（參閱表6及7 )。這兩位病患在個 別輸注自體HRSC後展現長期植入期（engraftment)。 -52- 201114895 表6 在輸注自體HRSC前及後嚴重的再生不全性貧血之病患A的血液常規檢査、血紅素 變異及肝酵素 血液試驗 Nadir 輸注前 07/19/ 2004 輸注後 05/26/ 2005 輸注後 03/07/ 2006 輸注後 01/05/ 2008 輸注後 WBC[103///L] 1.3 3.4 3.1 3.7 5.4 HB[g/dLl 2.6 7.1 9 12 14 RBC[106//zL] 0.9 2 2.3 2.9 3.86 RETIC[%] 1 4 1.7 MCVrfLl 101.5 101.5 121.74 100 101.4 MCHiPsl 33.6 35.5 39.13 30.8 36.3 MCHC[g/dLl 33.8 35 32.14 30.8 35.8 血小板 [103//zL] 18 26 37 58 189 嗜中性細胞 [103/^L] 0.32 1.000 1.054 1.184 1.994 ESO[103//zL] 0.62 0.74 0.65 BASO [103///L] 0 0 0.054 LYMPH [103///L] 1.500 1.922 2.220 2.214 MONO [103///L] 0.500 0.062 0.222 0.324 HBA[g/dLl 5.1 6.7 9.2 12 HBA2[g/dll 0.13 0.14 0.24 0.39 HBF[g/dll ND 1.2 2.4 2.6 1.82 SGOT[U/Ll 150 127 74 32 30 SGPT[U/L1 145 181 49 37 44 WBC=白血細胞；HB=血紅素；RBC=紅血細胞；RETIC=網狀紅血球；MCV=平均血 球容積；MCH=平均血球紅血素；MCHC=平均血球紅血素濃度；ESO=嗜酸性細胞； BASO=嗜鹼性細胞；LYMPH=淋巴細胞；MONO=單細胞；HBA=血紅素A ; HBA2= 血紅素A2 ; HBF=胎兒血紅素；SGOT=血清麩胺酸草乙酸轉胺酶；SGPT=血清麩胺 酸丙酮酸轉胺酶 -53- 201114895 表7 在輸注自體HRSC前及後嚴重的再生不全性貧血之病患D的血液常規檢査、血 _ 變異及肝酵素 — '、 血液試驗 Nadir 輸注前 09/28/ 2004 輸注後 05/30/ 2005 輸注後 03/07/ 2006 輸注後 01/05/ 2008 輸注後 WBC[103//zL] 1.7 3 2.2 2.7 4 HB 3 11.1 7.7 11.5 13 RBC 3.6 2.4 3.2 3.45 RETIC[%] 2.4 3.4 1.7 MCV 90.3 108.33 113 110.3 MCH 30.9 32 35.9 37.8 MCHC 34.2 29.62 31.9 34.2 血小板 [103///L] 5 30 20 25 68 嗜中性細胞 [103/^L] 0.1 0.72 0.50 1.0 1.24 ESO[103//iL] 0.90 0.22 0.54 0.64 BASO [103//zL] 0 0 0 0 LYMPH [l03/βL] 2.01 1.67 1.59 1.88 MONO [103//zL] 0.18 0.44 0.54 0.12 HBA 10.6 7.13 10.33 12.1 HBA2 0.33 0.19 0.28 0.36 HBF 0.11 0.2 0.38 0.9 0.42 SGOT 160 46 46 69 34 SGPT 150 27 32 60 46 WBC=白血細胞；HB=血紅素；RBC=紅血細胞：RETIC=網狀紅血球；MCV=平均血 球容積；MCH=平均血球紅血素；MCHC=平均血球紅血素濃度；ESO=嗜酸性細胞； BASO嗜鹼性細胞；LYMPH:淋巴細胞；MONO單細胞；HBA:血紅素A ; HBA2: 血紅素A2 : HBF=胎兒血紅素；SGOT=血清麩胺酸草乙酸轉胺酶；SGPT=血清麩胺 酸丙酮酸轉胺酶 -54- 201114895 在誘發造血重新編程前及後3小時的血漿分離之 細胞的流動式細胞測量術顯示於圖1中。在造血重新 後所產生之C D 3 4陽性細胞數量陳列於表4中。在造 新編程前及後的血漿分離之單核細胞的代表性流動式 測量術顯示顯著增加表現及不表現CD45、CD38及 的CD34陽性細胞數量（圖1 )。在輸注時，CD34細 環末梢血液3-6天且隨後在可持續的濃度下分化成骨 胞，如顯著增加表現具有及不具有CD7之CD33 & 1 細胞所描述，具有高的前及側散射（參閱圖2)。在 的病患中觀察到此重新編程圖案。 簡直沒有任何群落從輸注接種於甲基纖維素細胞 物後的自體HRSC前之病患骨髓抽出物形成（參閱圖 。僅在遭受先天再生不全性貧血之病患B中的克隆檢 顯示出壓抑的造血作用。然而，在輸注後14天至20 病患獲得的所有骨髓抽出物以溫和上升的爆發群落属 紅血球（BFU-紅血球）數量造成多種造血群落至正 圍。在輸注自體HRSC後14-20天的骨髓抹片及環鑿 圖3 )顯示在各種分化階段中以所有病患中的成熟及 熟巨核細胞顯著增加與基準線相比的骨髓細胞數量。 種分化階段中的紅血球增生亦註明於所有的樣品中。 在所有的病患中輸注自體HRSC前及後（在病患 病患D中經長達4年以上）所獲得的末梢血液或骨 品中沒有任何染色體核型與G-染色體條紋帶圖案的 (圖 4 )。 單核 編程 血重 細胞 CD7 胞循 髓細 3之 所有 培養 3 ) 定法 天從 ί位-常範 區（ 未成 在各 Α及 髓樣 變化 -55- 201114895 在自體HRSC輸注於再生不全性貧血病患後，原始（ CD38陰性）及定型（CD38陽性）CD34細胞在重新編程 爲骨髓細胞前循環於末梢循環3天（圖2) »在輸注於病 換A、病患B及病患D後接著發生3天骨髓植入期。另 一方面’在以流動式細胞測量術分析時，病患003接著在 第20天發生骨髓植入期。在未使用任何預調理體系的個 別輸注HRSC導致4位患有再生不全性貧血之病患中之2 位有長植入期。病患A及B顯示出長植入期而在輸注 HRSC後沒有任何輸血。在此病患中的嗜中性細胞、紅血 細胞及血小板的植入期伴隨切換或增加的Hb F血紅素濃 度（表6及7 )。此未註明於其他2位死亡的病患中。在 這兩位病患中的Hb F切換確認輸注之HRSC趨向幼期Hb 基因型的重新編程能力，由此使植入期及重組如以臍帶血 幹細胞移植物所觀察（Elhasid等人之Leukemia 2000，14 :93 1 -934 ; Locatelli 等人之 Bone Marrow Transplant 1996， 18: 1095-101) 〇 保存染色體數量及條紋帶的長植入値明確地反映在造 血條件中輸注HRSC的安全性，其中克隆演變（clonal evolution)不是傳統療法的罕見事件。 重要的是自體HRSC能夠在沒有使用任何免疫抑制體 系之嚴重的再生不全性貧血病患子集中有長植入期及存活 率，恰如那些以同基因幹細胞所見。 總結，在輸注後1 4天，已輸注病患的骨髓分析顯示 出隨著脂細胞及基質細胞（彼等爲嚴重的再生不全性貧血 -56- 201114895 骨髓的優勢佔有者）下降而增加骨髓細胞性及在各種分化 階段的骨髓細胞、紅血球與巨核細胞系譜。隨著對應增加 之血紅素濃度與網狀紅血球濃度而顯著增加在骨髓中的紅 血細胞團。亦穩定增加胎兒血紅素，其爲改善鐮狀細胞貧 血及/3地中海貧血的重要組份。此外，顯著改善紅血細胞 指數’如以紅血細胞尺寸、血紅素含量及濃度所測定。已 重新編程細胞在輸注後展現正常的染色體核型及基因穩定 性。最後’在3位以上遭受再生不全性貧血之病患中觀察 到植入期及長期重建群體。 實例2 材料及方法 自體已重新編程造血細胞（標的細胞）係在患有沒地 中海貧血的2 1位病患中測試。1 9位病患有重度/3 -地中 海貧血及2位病患有中度地中海貧血。中度地中海貧血病 患中之一是地中海貧血/Hb E變異（常見源自於東方及印 度之病患中）及另一位患有地中海貧血/鐮狀細胞貧血。 病患係藉由處理2 - 3次病患總血量而血漿分離。自體 已重新編程細胞係經由重新編程白血細胞直到獲得標的細 胞而產生，如以彼等如上述之區別特徵所指明。病患係經 由靜脈內輸注於頸靜脈中或手臂或大腿的靜脈中而投予自 體已重新編程細胞。 結果 -57- 201114895 當與基準線相比時，在已重新編程細胞輸注於患有β 地中海貧血之病患後未觀察到任何毒性或相反的副作用’ 如以活力徵象監控、心臟超音波影像、骨密度、肝與腎酵 素（包括染色體核型及G-染色體條紋帶）所測量。當與 基準線相比時，在輸注已重新編程細胞後現近9個月的重 度/3地中海貧血之病患中有令人滿意且顯著平均減少（5 〇 %)的輸血需求。在輸注已重新編程細胞後現近9個月的 兩位中度/3地中海貧血（一位是地中海貧血/ΗΒ Ε及另一 位是地中海貧血/鐮狀細胞貧血）的地中海貧血病患不依 賴輸血。 當與基準線相比時，在輸注已重新編程細胞後的平均 重量及身高明顯較大，且脾及/或肝腫大的地中海貧血病 患的器官尺寸正常化。當與基準線相比時，在輸注已重新 編程細胞後患重度及中度地中海貧血之病患中的絕對平均 胎兒血紅素濃度顯著增加（圖5 )。當與基準線相比時， 由改善紅血細胞尺寸、血紅素含量（圖6 )及濃度（圖7 )所反映之平均紅血細胞指數亦顯著改善。 最後，在輸注已重新編程細胞後顯著降低地中海貧血 病患的平均血清鐵蛋白（用於鐵質超載之生物標誌）（圖 8)。鐵質超載爲患有地中海貧血、鐮狀細胞貧血及任何 以輸血性鐵質超載誘發病症之病患死亡率及發病率的主要 原因。 實例3 -58- 201114895 材料及方法 兩位患有糖尿病之病患係藉由處理2-3次彼 而血漿分離。自體已重新編程間葉幹細胞、多能 及胰小島細胞（標的細胞）係經由重新編程已血 白血細胞直到發育出標的細胞而獲得，如以彼等 區別特徵所指明。病患係經由靜脈內輸注於頸靜 或大腿的靜脈中而投予自體已重新編程細胞。 結果 在輸注自體已重新編程細胞後，由病患合成tt 胰島素濃度，如以禁食及90分鐘之進食刺激之c 量。此正常的c-肽濃度在輸注自體已重新編程細肪 至多3個月（圖9)。另外，代表血糖控制的Hb 度係在輸注自體已重新編程細胞後正常化（圖1< 如，在輸注前具有大於10%之Hb A1C的病患在接 新編程細胞後現具有5.8%之Hb A1C。此外，當與 相比時，這些病患的血液葡萄糖濃度在輸注後似ΐ 常濃度。另外，經註明在輸注已重新編程細胞後方 由糖尿病病患的胰島素攝取/注射。 實例4 材料及方法 四位患有肌萎縮性脊髓側索硬化症（ALS ) 受自體已重新編程細胞。此疾病之診斷不包含特 總血量 幹細胞 分離之 上述之 或手臂 丨正常的 -肽所測 ί後維持 A1C濃 ))。例 丨受已重 〖基準線 =達到正 :大減少 病患接 性生物 -59- 201114895 標誌。此疾病係藉由其他類似病症的臨床排除來診斷。 病患係藉由處理2-3次彼之總血量而血漿分離。自體 已重新編程多能性幹細胞、肺泡上皮細胞及神經元（標的 細胞）係經由重新編程已血漿分離之白血細胞直到發育出 標的細胞而獲得，如以彼等如上述之區別特徵所指明。病 患係經由靜脈內輸注於頸靜脈或手臂或大腿的靜脈中而投 予自體已重新編程細胞》 結果 在遭受ALS且以自體已重新編程細胞治療之病患中 ，在肺功能試驗（PFT )中有顯著的改善。在此肺功能試 驗中的障礙爲導致早期死亡率的原因之一。大部分的病患 經歷較不僵直的肢體及頸部，且記述一些病患的脾功能改 善。其他的病患顯示出彼之行走能力與抬頭功能的改善。 實例5 材料及方法 四位患有帕金森氏病之病患係藉由處理2-3次彼之總 血量而血漿分離。自體已重新編程多能性幹細胞及神經元 (標的細胞）係經由重新編程已血漿分離之白血細胞直到 發育出標的細胞而獲得，如以彼等如上述之區別特徵所指 明。病患係經由靜脈內輸注於頸靜脈或手臂或大腿的靜脈 中而投予自體已重新編程細胞。 -60- 201114895 結果 其中疾病的發抖效應明顯的病患經歷顯著減少的搖動 及用於控制其病症所攝取之慣例藥劑。第1位病患每天服 用4粒息寧（Sinemet)(多巴胺調節劑），但在輸注後4 個月一天僅服用1粒。 實例6 材料及方法 兩位患有脊髓傷害之病患係藉由處理2-3次彼之總血 量而血漿分離。自體已重新編程多能性幹細胞及神經元（ 標的細胞）係經由重新編程已血漿分離之白血細胞直到發 育出標的細胞而獲得，如以彼等如上述之區別特徵所指明 。病患係經由靜脈內輸注於頸靜脈或手臂或大腿的靜脈中 而投予自體已重新編程細胞。 結果 一位病患失去追縱。另一病患爲具有C5-C6脊髓傷害 的四肢麻痺患者。此病患不能坐起來或在床上旋轉其軀幹 。在治療後，其能夠坐直非常長的時間且能夠在床上轉動 其身體。其亦能夠在靠著牆壁支撐其身體後單獨站立。其 進一步能夠扭動其腳趾且記述其膀胱的敏感度。 在輸注已重新編程細胞前及後的M RI分析顯示在輸 注已重新編程細胞後略微縮減損害規模。其開始主動進行 物理療法且通常感覺比以前更好。 -61 - 201114895 實例7 材料及方法 兩位患有肌肉萎縮症（M D )之病患係以自體已重新 編程多能性幹細胞、間葉幹細胞及骨骼肌細胞（標的細胞 )治療，該等細胞係經由重新編程已血漿分離之白血細胞 直到發育出標的細胞而獲得，如以彼等如上述之區別特徵 所指明。第1位病患係遭受肢帶型MD，其係指其中受到 最嚴重影響的肌肉通常是那些臀部及肩部的MD類別，而 第2位病患遭受奈蒙林型（nemelin) MD。 病患係藉由處理2-3次彼之總血量而血漿分離。自體 已重新編程細胞係經由根據本發明的重新編程而產生。病 患係經由靜脈內輸注於頸靜脈或手臂或大腿的靜脈中而投 予自體已重新編程細胞。 結果 與MD有關聯的肌肉萎縮症可藉由監控肌肉酵素肌胺 酸磷酸轉化酶（CPK )濃度而測量。此酵素係反應已重新 編程細胞的輸注而降低（圖11)。乳酸鹽脫氫酶（在組 織破壞期間上升的酵素）亦降低（圖11)。病患亦經歷 降低的肝酵素丙胺酸轉胺酶（ALT)及天冬胺酸轉胺酶（ AST )，二者皆與肝細胞及骨骼肌發炎及傷害有關聯。 而且，當與基準線相比時，病患顯示出改善的病患發 病率，如以輸注已重新編程細胞前及後的病患cam記錄及 -62- 201114895 在輸注已重新編程細胞前及後的肺功 實例8 材料及方法 患有腎疾病之病患係以自體已重 、間葉幹細胞及腎細胞（標的細胞） 由重新編程已血漿分離之白血細胞直 獲得，如以彼等如上述之區別特徵所 理2-3次彼之總血量而血漿分離。自 經由根據本發明的重新編程而產生。 注於頸靜脈或手臂或大腿的靜脈中而 細胞。 結果 接受自體已重新編程細胞輸注之 誌濃度的改善，其爲建康者的腎功能 、血清肌酸酐及BUN或尿素。例如 在以自體已重新編程細胞治療後24 功能（參閱表8 )。病患顯示出增加 氧的紅血細胞中的蛋白分子）及似, IGF-1)(―種生長因子）濃度。病 素（一種有機化合物）、肌酸酐（其 酸鹽之分解產物）、尿酸（其爲尿中 及磷（其爲骨頭中發現的礦物質）； 試驗中所測定。 新編程多能性幹細胞 治療，該等細胞係經 到發育出標的細胞而 指明。病患係藉由處 體已重新編程細胞係 病患係經由靜脈內輸 投予自體已重新編程 病患經歷各種流體標 之指標，諸如排尿量 1 7 5歲男性糖尿病患 個月顯示出改善的腎 的血紅素（其爲攜帶 夷島素生長因子-1 ( i、亦顯示出降低的尿 爲肌肉中的肌胺酸磷 分泌的有機化合物） 大量的這些標誌爲差 -63- 201114895 的腎功能指標。病患進一步顯示出降低的醣化血紅素（ HbAlC )，其爲血紅素形式且常被用作爲遭受糖尿病之人 類的血漿葡萄糖濃度的指標。 同樣地，5 1歲男性糖尿病患在治療後1 2個月展現出 類似的改善（參閱表8)。此病患顯示出增加的血紅素濃 度及降低的肌酸酐、HbAlC及血尿素氮（BUN)(其爲血 液中以尿素形式的氮量之測量）濃度。 表8 在兩位糖尿病男性病患中的腎功能標誌 75 1 糖g 歲男性 瞒患 51歲男性 糖尿病患 基準線 治療後 24個月 基準線 治療後 12個月 血紅素[g/dl] 8 12.9 12 14.5 尿素[g/dl] 190 38 n/a n/a 肌酸酉干[mg/dl] 12 1.9 15 4.5 尿酸[mg/dl] 8.5 4 n/a n/a 磷[mg/dl] 6.1 3.8 n/a n/a HbAlC[%] 14.4 6.1 12 6 IGF-1「_11 45 112 n/a n/a BUNfmg/dll n/a n/a 79 19Hematology 2007, 23-28). Although most patients with aplastic anemia can be treated with stem cell transplants obtained from HLA-matched brothers and sisters (Locasciulli et al., Haematologica. 2007; 92: 1 1 -1 8 ), this approach extends to Older patients or those who do not have family-based donors still have major challenges. Despite the viable viability after HLA-matched allogeneic stem cell transplantation, the procedure carries potential risks due to the immunosuppressive system used to avoid graft versus host disease (GVDH). For example, high doses of cyclophosphamide with or without antithymocyte globulin (ATG) result in prolonged periods of immunosuppression, making patients susceptible to infection. Other potential risks are graft failure, which can occur weeks or months after stem cell transplantation (Gottdiener et al. Arch Intern Med 1981, 141: 758-763; Sanders et al. Semin Hematol 1991, 28: 244) -249). Moreover, the risk of graft failure increases with the number of blood transfusions received prior to stem cell transplantation. Thalassemia is a positive recessive hereditary blood disease characterized by a reduced rate of synthesis of one of the hemoglobin chains that make up heme. Therefore, normal blood globulin production is often insufficient due to mutation of the regulatory gene, which causes abnormal hemoglobin molecules to form, resulting in anemia. Different types of Mediterranean anemia include alpha thalassemia, thalassemia, and thalassemia, which affect alpha blood globulin and sputum globulin, respectively. <5 production of blood globulin. Treatment includes long-term blood transfusion, iron chelator, splenectomy, and allogeneic hematopoiesis. However, long-term transfusions are not suitable for most patients due to the lack of HLA-matched bone marrow donors, and allogeneic hematopoietic cell transplantation is associated with many possible complications, such as infections and grafts against host disease. • 38 - 201114895 Diabetes is a syndrome that causes abnormally high blood glucose levels (hyperglycemia). Diabetes refers to a class of diseases in which high blood glucose concentrations are caused by defects in insulin secretion or insulin action in the body. Diabetes is typically divided into two types: Type I diabetes, characterized by reduced insulin production, or Type II saccharide urea, which is characterized by an anti-insulin effect. Both types cause hyperglycemia, which primarily causes signs that are often associated with diabetes, such as too much urine, distracting thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. Diabetes is considered a chronic disease that does not heal. Treatment options are limited to insulin injections, exercise, appropriate diets, or some agents for patients with type 2 diabetes, such as those that promote pancreatic insulin secretion, reduce glucose produced by the liver, and increase cellular sensitivity to insulin. A drug such as a degree. Motor neuron disease refers to the category of neurological disorders that affect motor neurons. These diseases include amyotrophic lateral sclerosis (ALS), primary lateral sclerosis (PLS), and progressive muscular atrophy (PMA). Both the ALS system and the lower motor neurons degenerate into a characterization that stops the message to the muscle and causes it to weaken and eventually shrink. PLS is a rare motor neuron disease that affects only upper motor neurons, which causes difficulties in balance, weakness and stiffness in the legs, spasms, and spleen problems. PM Α is an ALS subtype that only affects the lower motor neuron, which can cause muscle atrophy, beaming and weakness. There are no known healing treatments for motor neuron diseases. The riluzole, which relieves the damage to the sacred neurons, has been shown to be a drug for ALC, although it slowly slows the progression of ALS rather than improving its effects. The treatment of PLS focuses only on symptoms such as bevacant (-39-201114895 baclofen) which reduces sputum and quinine which reduces painful sputum. Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of the nigrostriatal pathway, caused by degeneration of dopaminergic neurons in the substantia nigra. The cause of PD is unknown, but it is associated with progressive death of brain neurons in dopaminergic (tyrosine hydroxylase (TH)-positive), which induces dyskinesia. The PD is then characterized by muscle stiffness, tremors, bradykinesia, and possible motor insufficiency. Therefore, there is currently no satisfactory treatment for Parkinson's disease healing or prevention or treatment of Parkinson's disease or its signs. Symptomatic therapies associated with disease-related dyskinesia include oral administration of dihydroxyphenylalanine (L-DOPA), which results in substantially improved motor function, but whose effect is reduced when dopaminergic neurons progress. Alternative strategies include neuronal transplantation, which is based on the dopamine supply from the cells implanted in the striatum to replace the loss of the nigrostriatal striatum, and gene therapy, which can be introduced by L-D0PA. Enzymes are used to replace dopamine in the affected striatum, or dopamine synthesis, such as by introducing a regenerative and functional recovery that prevents TH-positive neuron death or stimulation in the damaged nigrostriatal system. Potential neuroprotective molecule. Spinal cord injury is characterized by spinal cord and, in particular, nerve fiber damage, causing some or all of the muscle or neurological disorders below the injury site. Such injuries can occur through fractures, dislocations, crushing or compressing one or more of the spinal cords of the vertebrae' or non-traumatic injuries caused by arthritis, cancer, inflammation or disc degeneration. Although treatment after spinal cord injury may include agents such as methyl dehydrocortisol, which are used to reduce nerve cell damage and reduce inflammation in the injured area, or to control pain and muscle spasm - 201114895 The spine is fixed or surgically removed from the protruding disc or any object that may be damaging the spine, but there is no known method of reversing spinal cord injury. Muscular atrophy (MD) refers to a group of hereditary muscle diseases that weaken skeletal muscle. MD is characterized by progressive muscle weakness, muscle protein defects, muscle cell apoptosis, and tissue atrophy. There are more than 100 diseases that exhibit MD characteristics, although 9 diseases are classified as MD, especially Duchenne, Becker, limb band, congenital, facial scapula, muscle rigidity Type, eye-pharyngeal, peripheral type and Emery-Dr ei fuss type. There are no known MD healing therapies and there are no special treatments. Physical therapy maintains muscle tone and surgery can be used to improve quality of life. Signs (such as muscle stiffness) can be further treated with a drug, but without any long-term treatment. Kidney disease refers to the symptoms of the ability to damage the kidney and reduce its effects, including removing waste and excess water from the blood, regulating electrolytes, blood pressure, acid-base balance, and reabsorption of glucose and amino acids. The two main causes of kidney disease are diabetes and high blood pressure, although other causes include glomerulonephritis, lupus and kidney malformations and obstruction. There is no cure for kidney disease, thereby allowing the medical focus to slow the progression of the disease and the causes of the disease, such as by controlling blood sugar and high blood pressure and monitoring the diet; treating disease complications, for example by focusing on fluid retention, Anemia, bone disease: and replaces lost kidney function, such as via dialysis or migration. Multiple sclerosis is an autoimmune condition in which the immune system invades the central nervous system, leading to demyelination. MS affects the brain and the gods in the spinal cord -41 - 201114895 The ability of cells to communicate with each other because the body's own immune system damages myelin lipids surrounding neuronal axons. When the myelin is lost, it is no longer possible to transmit the signal effectively. This can lead to a variety of neurological signs of frequent progression and cognitive disability. Healing therapies without any M S Try to restore function after the invasion (sudden or worsening MS signs) new invasiveness and avoid disability. For example, treatment with corticosteroids can prevent invasion, while treatment with interferon in the early stages of invasion has shown an opportunity to reduce MS development. Human immunodeficiency virus (HI V ) is a lentivirus genus that causes acquired immunity (AIDS), in which HIV, which begins to fail in the immune system, primarily infects viable bacteria in the human immune system, such as cells, macrophages, and dendritic cells. HIV infection leads to CD4+ T cell concentration by directly infecting cells, increasing the rate of apoptotic cells, or killing infected CD4+ T cells by recognizing infected cell cytotoxic lymphocytes. There are currently no HIV or AIDS episodes. The treatment of HIV infection consists of the highly active antiretroviral HAART. The current HAART selection is a combination of at least three types of antiretroviral agents (or wine cellars). These classes are typically two nucleoside-like reverse transcripts (NARTI or NRTI) plus a protease inhibitor or a non-nucleosidase inhibitor (NNRTI). Congestive heart failure is a condition in which the heart cannot pump enough to other organs of the body. This symptom can be caused by coronary artery disease, myocardial infarction, scar tissue on the heart, high blood pressure, heart attack and axon formation; treatment, avoiding the symptoms of low clinical deficiency. Attenuated T-poisoned CD8-induced low seedling or seat therapy or at least two, chicken tail enzymes inhibit reverse transcription of blood, caused by heart valve disease, heart defects, and heart valve infections. Treatment plans typically consist of rest, proper diet, improved daily activities, and medications such as angiotensin-converting enzyme (ACE) inhibitors, stone blockers, digitalis, diuretics, and vasodilators. However, the treatment plan does not reverse the damage or symptoms of the heart. Hepatitis C is an infectious disease in the liver caused by hepatitis C virus. Hepatitis C can progress to scars (fibrosis) and increase scars (cirrhosis). Cirrhosis can lead to liver failure and other complications such as liver cancer. Current treatments include the use of a combination of pegylated interferon alpha and the antiviral drug ribavirin. The success rate can vary between 50 and 80% depending on the viral genotype. Head trauma refers to head injuries that may or may not cause brain damage. Common causes of head trauma include traffic accidents, home and workplace accidents, falls and attacks. Various types of problems can be caused by head trauma, including skull fractures, scalp lacerations, subdural hematoma (under subdural hemorrhage), epidural hematoma (bleeding between the subdural and skull), brain contusion (brain bruises), concussion (temporary loss of function due to trauma), coma or even death. The treatment of head trauma varies with the type of injury. If the brain is damaged, there is no quick device to fix the brain and the damage is often irreversible due to available treatment equipment. Lung disease is a broad term for respiratory diseases including the lungs, chest, bronchi, trachea, upper respiratory tract, and nerves and muscles for breathing. Examples of pulmonary diseases include obstructive pulmonary disease (where bronchial narrowing), localized or fibrotic lung disease (where lung loss is compliant and causes incomplete -43-201114895 lung expansion and increased lung stiffness), respiratory infections ( Can be caused by common colds or pneumonia), respiratory tumors (such as those caused by cancer), chest disease and pulmonary vascular disease (which affects the circulation of the lungs). Pulmonary disease Therapy varies depending on the type of disease, but can include agents (such as corticosteroids and antibiotics), oxygen, respirator ventilation, radiation therapy, and surgery. Depression is a psychiatric condition characterized by low emotions that accompany a low self-image and loss of interest or fun in general joyful activities. Depression is biologically related to changes in the activity of multiple parts of the brain, including the nucleus, which is a small nucleus in the serotonin-derived upper brainstem; the upper nucleus, which controls the biological rhythm, such as sleep And the wake-up cycle; the hypothalamus-cerebral gland-adrenal axis, which is the structural chain of activity during the body's response to various stressors: the midbrain ventral tegmental area, which is thought to be responsible for the brain> Nucleus accumbens, which we consider to play a role in compensation, laughter, fun, addiction, and fear; and the anterior cingulate cortex, which is active due to negative experiences. Treatment for depression includes an increase in the amount of extracellular serotonin in the brain for antidepressants, exercise, and psychotherapy. However, the efficacy of these treatments remains suspect. Non-obstructive azoospermia is a medical condition in males that does not have any measurable sperm concentration in its semen due to problems with sperm production. This is often caused by hormone imbalance and can be treated with an agent that repairs imbalance. Male menopause is a menopausal symptom experienced by middle-aged men, which includes a reduction in the production of hormone steroids and dehydroepiandrosterone. Treatment includes hormone replacement therapy and exercise. • 44- 201114895 Scleroderma is a chronic autoimmune disease that affects connective tissue. Hardening of the skin is the most common manifestation of the disease, although it can affect connective tissue throughout the body. There are no known direct cure treatments for scleroderma. Psoriasis is a chronic autoimmune disease that causes scaly erythema on the skin. The cause of psoriasis is related to overgrown skin cells. One hypothesis suggests that it is associated with T-cells that migrate to the dermis and trigger the release of cytokines that are rapidly produced by skin cells. Treatment of psoriasis includes drugs that target T-cells. Pigmented retinitis is a type of progressive retinal dystrophy in which photoreceptor or retinal pigment epithelial cells are abnormal and cause visual loss. There are limited medical treatments for the treatment of pigmented retinitis. The symptoms described herein can be treated with a particular type of target cell or a combination of such types. In a preferred embodiment, the symptoms described herein can be treated by infusion of the cell types recited in Table 2. -45- 201114895 Table 2 Treatment system using various types of symptoms that have been reprogrammed, that is, have been dedifferentiated or have been transdifferentiated or have been re-differentiated. The treatment of cell types (individual types or combinations thereof) Regeneration of anemia, hematopoietic cells - thalassemia hematopoietic cells, diabetic mesenchymal stem cells, pluripotent stem cells and/or pancreatic islet cell motoneuron diseases, pluripotent stem cells, alveolar epithelial cells, ectodermal cells and/or neuronal Parkinson's disease pluripotent stem cells and / or neuronal spinal cord injury pluripotent stem cells and / or neuronal muscle atrophy pluripotent stem cells, mesenchymal stem cells and / or skeletal muscle cells kidney disease kidney cells, mesenchymal stem cells and / or pluripotent stem cells multiple sclerosis Pluripotent stem cells, mesenchymal stem cells and/or neuronal congestive heart failure cardiomyocytes, mesenchymal stem cells, pluripotent stem cells and/or endothelial cells, hepatitis C virus hematopoietic cells, pluripotent stem cells, mesenchymal stem cells / or liver hepatocytes human immunodeficiency virus hematopoietic cell head trauma pluripotency Cyto and/or neuronal lung disease pluripotent stem cells, mesenchymal stem cells, alveolar epithelial cells and/or endothelial cell depression pluripotent stem cells and/or neuron non-obstructive azoospermia or male climacteric pluripotent stem cells, winter Capable bovine cells and / or sperm climacteric and infertility pluripotent stem cells 'oocyte, pluripotent bovine cells, rejuvenation pluripotent germ cells, pluripotent stem cells, keratinocytes, dermal papilla cells , neurons, sperm, oocytes, chondrocytes, cardiomyocytes, skeletal muscle cells, neurons, endothelial cells and/or melanocytes scleroderma ulcer pluripotent stem cells, endothelial cells, keratinocytes and/or mesenchymal cells Stem cells psoriasis mesenchymal stem cells and/or pluripotent stem cell wrinkles _ temporal stem cells, keratinocytes and/or pluripotent stem cells cirrhosis liver hepatocytes 'segmental stem cells, endothelial cells, pluripotent stem cells autoimmune Disease mesenchymal stem cells and / or winter stem cells alopecia dermal papilla cells and / or n color | | fine | ^ pigmented retinitis or horn Crystallization dystrophy/blindness neural crest and/or pluripotent stem cells-46- 201114895 Patients can be treated with the following steps in an example manner as described above: 1) Inserting a Fistula cannula into a patient's arm; 2) Using automation Systems such as the COB E® Spectra device (Gambro PCT) harvest white blood cells via plasmapheresis; 3) produce autologous, already differentiated stem cells from the patient's white blood cells; 4) wash the autologously differentiated stem cells and then Intravenous infusion into the patient; 5) Monitor patient progress, including taking blood tests and assessing the area of injury. The invention is further described by the following non-limiting examples, which are not to be construed as limiting the scope of the invention. [Examples] Example 1 Materials and Methods This clinical study evaluated infusion of a single amount of autologous re-calibrated cells to 4 patients with regenerative anemia after exposure to hematopoietic-induced culture conditions. Hours of security. This clinical study was approved by the King Edward Memorial (KEM) doctor and approved by the Immunohematology Institute (IIH). Patients must meet the criteria outlined in Table 3. RESULTS: Four patients with β-stomach and stomach anemia (3 males) and congenital aplastic anemia (1–47-201114895 females) were enrolled. These 4 patients were selected and monitored by IIH/KEM personnel. The clinical and therapeutic history of the patient is described in Table 4, and the infusion dose of CD 3 4 + cells is shown in Table 5. Table 3 Inclusion criteria Each criterion is necessary 1- Absolute neutrophil count <0.5x109/L 2-platelet count <20x109/L 3-corrected reticular red blood cell anemia < 1 % of the criteria only one is necessary 4-myeloid cell <25% 5-myeloid cell <50%, with less than 30% of hematopoietic cells 6 - Subjects evaluated within 3 months prior to diagnosis 7 - Subjects have not previously received immunosuppressive therapy and the patient was infused to 2 units for radiation exposure Red blood cell thick solution and 4 units of platelets to maintain a hemoglobin concentration greater than 8 grams per square inch and a platelet count greater than 50,000. The patients were separated by plasma using a Cobe Spectra blood separation machine and a white blood cell separation kit (both from Gambro BCT) to treat the total blood volume of the patient 2-3 times. Plasmapheresis includes cervical and anterior venous catheterization with a single lumen catheter for venous access. Cell fractionation was collected aseptically for CD34 analysis after collecting 150-200 ml skin color layers. The skin layer of the skin color is then reprogrammed under hematopoietic inducing conditions. Briefly, the reprogramming procedure consisted of 1 000 micrograms of purified CR3/43 (especially prepared by DakoCytomation for TriStem Corp.) diluted in 30 milliliters of ISK-modified medium and aseptically added to a white blood cell bag. The cell bag is then maintained at 3 7 . (: incubated for 3 hours in a sterile tissue culture incubator under 5% C02 " After completion of the reprogramming method at -48 - 201114895, the CD 3 4 + cell content of the transformed cells was analyzed. The cells were then utilized by the Cobe cell processor. 2991 was washed twice with saline solution. Once stirred and resuspended in saline solution, the cell suspension was infused into the patient via the jugular vein under gravity using an infusion device. Continuous monitoring before and after infusion of autologous reprogrammed cells The vital signs of the patient' included CBC counts. Table 4 Clinical history of patients with aplastic anemia until autologous human reprogrammed stem cells (HRSC) infusion and clinical history of patients until HRSC infusion __ Until RSC infusion Treated patients A: 25 years old male with severe aplastic anemia • diagnosed as SAA in 2002 • signs of weakness and difficulty breathing • frequent rectal and gum bleeding and vomiting • 4 and 2 units per month Blood and platelets • Eye congestion with severe jaundice • Accepted 8 months of anabolic steroid (T^bMeiwbol) test* No significant Improve patient B: 26-year-old woman with congenital aplastic anemia • diagnosed as congenital aplastic anemia in January 2004 • presented with signs of excessive menstrual discharge during the 6-month period. Patients were only received before RSC infusion Red blood cell concentrate and platelets in units 4 and 6 • Blood serum received from 3 months prior to infusion of RSC, no response to patients C: 19-year-old male with very severe aplastic anemia • was diagnosed very in 2003 Severe aplastic anemia • Dysphagia, weakness, and difficulty breathing; • 10-15 days of fever with chills due to severe neutropenia • Multiple episodes of infection, vomiting, and purpura • Hip abscess • Fainted twice due to brain hemorrhage • Received 5 and 2 units of blood and platelets every two weeks • Received cyclosporine therapy for 6 months, ending in March 2003, no effect D : 35 Very serious regenerative anemia in elderly men • anemia, weakness, difficulty breathing • fever with chills due to severe neutropenia and multiple times Attack and bleeding • diagnosed 3 years ago. Receive 4 and 2 units of blood and platelets per month. • Receive cyclosporine and anti-tuberculosis therapy. No immunosuppressive response for 6 months. -49- 201114895 All clinical monitoring All are performed by ΠΗ/KEM personnel. The blood transfusion requirements before or after the infusion are determined by the blood transfusion records obtained after receiving the blood samples from any unit. The transfusion unit is only used after obtaining the consent of the patient and the immediate relatives as witnesses. Before the patient's blood transfusion, all blood samples were irradiated with radiation at the Tata Memorial Hospital. The patient was also asked questions about his well-being before and after infusion of the reprogrammed cells. All patients carry a copy of their symptoms and all laboratory and clinical follow-ups or original records. The monitoring period for these patients was originally set to 2 years, but was later extended. The patient was left in a sterile positive pressure chamber during the first month after the infusion. Table 5 Patient, infusion date, weight and height, collected monocytes and transplanted CD34+ cell patient ID and infusion date Weight collected by mononuclear cells received CD34+/kg patient A 7/6/ 2004 48 152 lxlO9 11.7χ106 Patient B 7/14/2004 38 153 3.6x109 20χ106 Patient C 7/27/2004 52 166 3·9χ109 25x106 Patient D 8/17/2004 52.5 160 4·3χ109 23χ106 Will be 100 The 10,000 cells were stained with the following monoclonal antibody plates (all from DakoCytomation) according to the manufacturer's instructions: The first plate consisted of the same negative control group IgGl-FITC, IgGl-PE-Cy5 and IgGl-RPE conjugates. The disc is composed of anti-human CD45-FITC and CD34-RPE-Cy5-50-201114895. The third disc is composed of anti-human CD38-FITC and CD34-RPE-Cy5. The fourth disc is made up of CD61-FITC and CD34-RPE. -Cy5 consists of the fifth disc consisting of CD33/13 RPE and CD7-FITC. The sixth disc is composed of CD45 and Glycophorin-A-RPE. The seventh disc is made up of anti-human CD3-FITC and CD19- The cell analysis consisting of RPE was performed using the BD Cell Quest software in the FACSCalibur system (BD bioscience). For the single-strain assay, bone marrow mononuclear cells (MNC) of patients infused before and after reprogramming of cells were inoculated into meth〇Cult GFH4434 supplemented with recombinant growth factors according to the manufacturer's instructions (Stem Cell Technologies). Phase difference conversion microscopy was used to evaluate and differentiate into hematopoietic cell populations by time scoring. Patients were continuously monitored for CBC, liver enzyme and heme variants before and after the procedure. After discharge, patients with CBC, liver enzymes, heme variants, and karyotypes of peripheral blood karyotypes and G-chromosome strips were monitored by independent laboratories for reconfirmation. These tests are often performed after infusion of autologous reprogrammed cells. Analysis of peripheral blood samples before and after infusion of autologous reprogrammed cells. This trial was repeated on a yearly basis at the 6-month interval and 2 years after the first autologous reprogrammed stem cell therapy. In addition, the reprogrammed cells prior to infusion were analyzed to investigate cell stability. This analysis was also performed after the 3-51 transformation step of -51 - 201114895 and the long-term culture establishment of the transformed cells for up to 1 month. Karyotype identification and G-chromosome stripe lines were monitored by a third independent laboratory. Bone marrow smears and ring chisels are performed before and after infusion of autologous reprogrammed cells. This test was performed on an annual basis 1 to 20 days after autologous reprogramming of cells. All smear and ring chisel areas were scanned with a microscope attached to the camcorder before and after infusion of the reprogrammed stem cells. Results All patients were resistant to plasmapheresis and individual reprogrammed stem cell infusion procedures without any adverse events. Patient A and Patient D were independently transfused after individual infusion of reprogrammed hematopoietic stem cells (RHSC) (see Tables 6 and 7). The platelet, neutrophil and red blood cell implantation phases then occurred 3 and 6 days after infusion of patient A and patient D, respectively. The fetal hemoglobin switch is indicated in Patient A and Patient D (see Tables 4 and 5), but not in Patient B and Patient C (no data shown). Before the infusion, liver enzymes rose in patients A and D, although negative for HCV (as measured by ELISA). Liver enzyme was normalized after infusion of RHSC and reached normal concentration 4 years after infusion of RHSC. Patients B and C died 2 and 6 months after the infusion, respectively. Fetal Hb switching is indicated in patient 〇〇 1 and patient 〇〇 4 (see Tables 6 and 7). Both patients showed long-term engraftment after infusion of autologous HRSC. -52- 201114895 Table 6 Blood routine examination, heme variation and liver enzyme blood test in patients with severe regenerative anemia before and after autologous HRSC infusion. Nadir before infusion 07/19/ 2004 After infusion 05/26 / 2005 After infusion 03/07/ 2006 After infusion 01/05/ 2008 After infusion WBC [103///L] 1.3 3.4 3.1 3.7 5.4 HB[g/dLl 2.6 7.1 9 12 14 RBC[106//zL] 0.9 2 2.3 2.9 3.86 RETIC[%] 1 4 1.7 MCVrfLl 101.5 101.5 121.74 100 101.4 MCHiPsl 33.6 35.5 39.13 30.8 36.3 MCHC[g/dLl 33.8 35 32.14 30.8 35.8 Platelets [103//zL] 18 26 37 58 189 Neutrophils [103 /^L] 0.32 1.000 1.054 1.184 1.994 ESO[103//zL] 0.62 0.74 0.65 BASO [103///L] 0 0 0.054 LYMPH [103///L] 1.500 1.922 2.220 2.214 MONO [103///L] 0.500 0.062 0.222 0.324 HBA[g/dLl 5.1 6.7 9.2 12 HBA2[g/dll 0.13 0.14 0.24 0.39 HBF[g/dll ND 1.2 2.4 2.6 1.82 SGOT[U/Ll 150 127 74 32 30 SGPT[U/L1 145 181 49 37 44 WBC = white blood cells; HB = hemoglobin; RBC = red blood cells; RETIC = reticulocyte; MCV = mean hematocrit; MCH = mean hemoglobin; MCHC = flat Hemoglobin concentration; ESO = eosinophils; BASO = basophils; LYMPH = lymphocytes; MONO = single cells; HBA = heme A; HBA2 = heme A2; HBF = fetal hemoglobin; SGOT = Serum glutamate acetic acid transaminase; SGPT = serum glutamate pyruvate transaminase-53- 201114895 Table 7 Blood routine examination of patients with severe regenerative anemia before and after infusion of autologous HRSC, Blood _ variability and liver enzymes - ', blood test Nadir before infusion 09/28/ 2004 after infusion 05/30/ 2005 after infusion 03/07/ 2006 after infusion 01/05/ 2008 after infusion WBC [103//zL] 1.7 3 2.2 2.7 4 HB 3 11.1 7.7 11.5 13 RBC 3.6 2.4 3.2 3.45 RETIC [%] 2.4 3.4 1.7 MCV 90.3 108.33 113 110.3 MCH 30.9 32 35.9 37.8 MCHC 34.2 29.62 31.9 34.2 Platelets [103///L] 5 30 20 25 68 Neutrophils [103/^L] 0.1 0.72 0.50 1.0 1.24 ESO[103//iL] 0.90 0.22 0.54 0.64 BASO [103//zL] 0 0 0 0 LYMPH [l03/βL] 2.01 1.67 1.59 1.88 MONO [103 //zL] 0.18 0.44 0.54 0.12 HBA 10.6 7.13 10.33 12.1 HBA2 0.33 0.19 0.28 0.36 HBF 0.11 0.2 0.38 0.9 0.42 SGOT 160 46 46 69 34 SGPT 150 27 32 60 46 WBC = white blood cells; HB = hemoglobin; RBC = red blood cells: RETIC = reticulocyte; MCV = mean hematocrit; MCH = mean hemoglobin; MCHC = mean blood cells Red blood concentration; ESO = eosinophils; BASO basophils; LYMPH: lymphocytes; MONO single cells; HBA: heme A; HBA2: heme A2: HBF = fetal hemoglobin; SGOT = serum glutamic acid Oxalic acid transaminase; SGPT = serum glutamate pyruvate transaminase - 54 - 201114895 Flow cytometry of plasma isolated cells 3 hours before and after hematopoietic reprogramming was shown in Figure 1. The number of C D 3 4 positive cells produced after regenerating hematopoiesis is shown in Table 4. Representative flow cytometry of plasma isolated monocytes before and after nascent programming showed a significant increase in performance and in the absence of CD45, CD38 and CD34 positive cells (Figure 1). At the time of infusion, CD34 fine-ring peripheral blood is differentiated into osteoblasts for 3-6 days and then at sustained concentrations, as described by CD33 & 1 cells with significantly increased and without CD7, with high anterior and lateral Scattering (see Figure 2). This reprogramming pattern was observed in patients. There is simply no community formation of bone marrow extracts from patients with pre-autologous HRSC after infusion of methylcellulose cells (see figure. Cloning in patients B who suffer from congenital aplastic anemia only shows depression Hematopoiesis. However, all bone marrow extracts obtained from 14 to 20 days after infusion resulted in a variety of hematopoietic communities to the right circumference with a moderately elevated burst of red blood cells (BFU-red blood cells). After infusion of autologous HRSC 14 A -20 day bone marrow smear and ring chisel Figure 3) shows a significant increase in the number of bone marrow cells compared to baseline in mature and mature megakaryocytes in all patients at various stages of differentiation. Red blood cell proliferation in the differentiation stage is also indicated in all samples. In all patients, there was no karyotype and G-chromosome strip pattern in the peripheral blood or bone obtained before and after infusion of autologous HRSC (in patients D for more than 4 years). (Figure 4 ). Mononuclear programming of all blood cells of CD7 cytoskeleton fine 3) 3 days of fixed-day ф-常常区(未未在在 Α and myeloid changes -55- 201114895 in autologous HRSC infusion of incomplete avian anemia After the onset, the original (CD38 negative) and committed (CD38 positive) CD34 cells were cycled to the peripheral circulation for 3 days before reprogramming to bone marrow cells (Fig. 2) » after infusion in disease replacement A, patient B, and patient D. A 3-day bone marrow implantation period occurred. On the other hand, when analyzed by flow cytometry, patient 003 then developed a bone marrow implantation period on day 20. Individual infusions of HRSC without any preconditioning system resulted in 4 Two of the patients with aplastic anemia had a long implantation period. Patients A and B showed a long implantation period without any blood transfusion after infusion of HRSC. Neutrophils in this patient, The implantation period of red blood cells and platelets was accompanied by switching or increased Hb F heme concentration (Tables 6 and 7). This was not indicated in the other 2 patients who died. Hb F switching confirmation in these two patients Infusion of HRSCs towards reprogramming of juvenile Hb genotypes Thus, the implantation period and reconstitution are observed as umbilical cord blood stem cell transplants (Elhasid et al. Leukemia 2000, 14: 93 1 -934; Locatelli et al., Bone Marrow Transplant 1996, 18: 1095-101). The number of chromosomes and the long implants of the strips clearly reflect the safety of infusion of HRSC in hematopoietic conditions, where clonal evolution is not a rare event in traditional therapy. It is important that autologous HRSC can be used without any immunosuppression. The system of severe regenerative anemia patients has a long implantation period and survival rate, just as those seen with isogenic stem cells. Summary, 14 days after the infusion, bone marrow analysis of patients who have been infused shows with lipids Cells and stromal cells (which are severely regenerative anemia-56-201114895 dominant occupants of the bone marrow) decrease and increase bone marrow cellularity and bone marrow cells, red blood cells and megakaryocyte lineage at various stages of differentiation. Hemoglobin concentration and reticular red blood cell concentration significantly increase the red blood cell mass in the bone marrow. It also steadily increases fetal hemoglobin. In order to improve the important components of sickle cell anemia and /3 thalassemia. In addition, significantly improve the red blood cell index 'as measured by red blood cell size, heme content and concentration. Reprogrammed cells show normal chromosomal nucleus after infusion Type and genetic stability. Finally, implanted and long-term reconstituted populations were observed in more than 3 patients with aplastic anemia. Example 2 Materials and Methods Autologous reprogrammed hematopoietic cells (target cells) were involved Tested in 21 patients with no thalassemia. 1 9 patients with severe / 3 - ground sea anemia and 2 patients with moderate thalassemia. One of the cases of moderate thalassemia is thalassemia/Hb E mutation (commonly found in Eastern and Indian patients) and another suffering from thalassemia/sickle cell anemia. The patient was separated by plasma by treating the total blood volume of the patient 2-3 times. Autologous reprogrammed cell lines are generated by reprogramming white blood cells until the target cells are obtained, as indicated by their distinguishing features as described above. The patient is administered autologous reprogrammed cells by intravenous infusion into the jugular vein or into the veins of the arms or thighs. Results -57- 201114895 No toxicity or adverse side effects were observed after reprogramming of cells transfused to patients with beta thalassemia when compared to baselines, such as monitoring of vital signs, ultrasound imaging of the heart, Bone density, liver and kidney enzymes (including karyotype and G-chromosome strips) were measured. There was a satisfactory and significant mean reduction (5 〇 %) in transfusion requirements in patients with severe/3 thalassemia for nearly 9 months after infusion of reprogrammed cells when compared to baseline. Thalassemia patients with moderate/3 thalassemia (one with thalassemia/sputum and another thalassemia/sickle cell anemia) for nearly 9 months after infusion of reprogrammed cells are not dependent Blood transfusion. When compared to the baseline, the average weight and height after infusion of the reprogrammed cells were significantly greater, and the organ size of the spleen and/or hepatic thalassemia was normalized. When compared to the baseline, the absolute mean fetal hemoglobin concentration was significantly increased in patients with severe and moderate thalassemia after infusion of reprogrammed cells (Figure 5). The mean red blood cell index, as reflected by improved red blood cell size, heme content (Figure 6), and concentration (Figure 7), was also significantly improved when compared to baseline. Finally, the mean serum ferritin (a biomarker for iron overload) was significantly reduced in patients with thalassemia after infusion of reprogrammed cells (Figure 8). Iron overload is the leading cause of mortality and morbidity in patients with thalassemia, sickle cell anemia, and any disease induced by transfusion iron overload. Example 3 -58- 201114895 Materials and Methods Two patients with diabetes were treated by plasma separation 2-3 times. Autologous reprogrammed mesenchymal stem cells, pluripotent and pancreatic islet cells (target cells) are obtained by reprogramming the blood cells until they develop the target cells, as indicated by their distinguishing features. The patient is administered autologous reprogrammed cells via intravenous infusion into the veins of the neck or thigh. Results After infusion of autologous reprogrammed cells, the patient synthesized tt insulin concentrations, such as fasting and 90 minutes of feeding stimulation. This normal c-peptide concentration has been reprogrammed for up to 3 months in the infusion autologous (Figure 9). In addition, the Hb degree representing glycemic control was normalized after infusion of autologous reprogrammed cells (Figure 1 < For example, a patient with greater than 10% Hb A1C prior to infusion now has 5.8% Hb A1C after receiving the programmed cells. In addition, when compared, the blood glucose levels of these patients appeared to be normal concentrations after infusion. In addition, insulin intake/injection by diabetic patients is indicated after the infusion has been reprogrammed. Example 4 Materials and Methods Four patients with amyotrophic lateral sclerosis (ALS) were self-programmed by autologous cells. The diagnosis of this disease does not include the total amount of blood stem cells isolated from the above or the arm 丨 normal - peptide measured ί after maintaining A1C concentration)). Example 丨 已 〗 〖Baseline = reach positive: large reduction Patient biotics -59- 201114895 mark. This disease is diagnosed by clinical exclusion of other similar conditions. The patient was separated by plasma by treating 2-3 times the total blood volume. Autologous Reprogrammed pluripotent stem cells, alveolar epithelial cells, and neurons (target cells) are obtained by reprogramming plasma-separated white blood cells until development of the labeled cells, as indicated by their distinguishing features as described above. The patient is administered autologous reprogrammed cells via intravenous infusion into the jugular vein or veins of the arms or thighs. Results In patients suffering from ALS and treated with autologous reprogrammed cells, in a pulmonary function test ( There is a significant improvement in PFT). The obstacles in this lung function test are one of the causes of early mortality. Most patients experience less rigid limbs and neck, and note that some patients have improved spleen function. Other patients showed improvement in their ability to walk and raise their head. EXAMPLE 5 Materials and Methods Four patients with Parkinson's disease were separated by plasma by treating 2-3 times their total blood volume. Autologous reprogrammed pluripotent stem cells and neurons (target cells) are obtained by reprogramming plasma-separated white blood cells until development of the target cells, as indicated by their distinguishing features as described above. The patient is administered autologous reprogrammed cells via intravenous infusion into the jugular vein or veins of the arms or thighs. -60- 201114895 Results Patients with significant tremor effects of the disease experienced significantly reduced shaking and conventional agents used to control their condition. The first patient took 4 capsules of Sinemet (dopamine modifier) daily, but only took 1 capsule a day after 4 months of infusion. Example 6 Materials and Methods Two patients with spinal cord injury were plasma separated by treating 2-3 total blood volumes. Autologous reprogrammed pluripotent stem cells and neurons (target cells) are obtained by reprogramming plasma-separated white blood cells until the target cells are developed, as indicated by their distinguishing features as described above. The patient is administered autologous reprogrammed cells via intravenous infusion into the jugular vein or veins of the arms or thighs. Results One patient lost his grief. Another patient was a paralyzed limb with C5-C6 spinal cord injury. The patient cannot sit up or spin his torso on the bed. After treatment, it is able to sit for a very long time and be able to turn its body on the bed. It can also stand alone after supporting its body against the wall. It is further capable of twisting its toes and describing the sensitivity of its bladder. M RI analysis before and after infusion of reprogrammed cells showed a slight reduction in lesion size after transfusion of cells that had been reprogrammed. It began to take the initiative to physical therapy and usually feels better than before. -61 - 201114895 Example 7 Materials and Methods Two patients with muscular dystrophy (MD) were treated with autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, and skeletal muscle cells (target cells). This is achieved by reprogramming the plasma-separated white blood cells until the target cells are developed, as indicated by their distinguishing features as described above. The first patient suffered from limb-type MD, which means that the most severely affected muscles are usually the MD category of the buttocks and shoulders, while the second patient suffers from the nemelin MD. The patient was separated by plasma by treating 2-3 times the total blood volume. Autologous Reprogrammed cell lines are generated via reprogramming in accordance with the present invention. The patient is administered autologous reprogrammed cells via intravenous infusion into the jugular vein or veins of the arms or thighs. Results Muscular atrophy associated with MD can be measured by monitoring the concentration of the muscle enzyme creatinine phosphotransferase (CPK). This enzyme is reduced by the infusion of reprogrammed cells (Figure 11). Lactate dehydrogenase (the enzyme that rises during tissue destruction) is also reduced (Figure 11). Patients also experience reduced liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), both of which are associated with inflammation and injury to hepatocytes and skeletal muscle. Moreover, when compared to baseline, patients showed improved patient morbidity, such as patients with infusion of reprogrammed cells before and after the patient's cam record and -62- 201114895 before and after infusion of reprogrammed cells Examples of pulmonary function 8 Materials and methods Patients with kidney disease are obtained by self-heavy, mesenchymal stem cells and kidney cells (target cells) obtained by reprogramming plasma-separated white blood cells, as described above. The difference is characterized by 2-3 times the total blood volume and plasma separation. Produced by reprogramming in accordance with the present invention. Inject into the jugular vein or the veins of the arms or thighs and the cells. Results The improvement in the concentration of autologous reprogrammed cell infusion was obtained, which was the renal function of the Jiankang, serum creatinine and BUN or urea. For example, 24 functions after autologous reprogrammed cell therapy (see Table 8). The patient showed a protein molecule in the red blood cells that increased oxygen and a concentration of IGF-1) (the growth factor). Pathogen (an organic compound), creatinine (decomposition product of its acid salt), uric acid (which is urine and phosphorus (which is a mineral found in bones); as determined in the test. Newly programmed pluripotent stem cell therapy The cell lines are indicated by development of the target cells. The patient is reprogrammed by intravenously transfecting the patient's line through intravenous administration to the patient who has been reprogrammed to experience various fluid targets, such as Urinary volume of 175 years of age in men with diabetes showed improved hemoglobin in the kidney (which is an organic compound that carries elegans growth factor-1 (i, also showing reduced urine for creatinine phosphate secretion in muscle) A large number of these markers are renal function indicators of poor-63-201114895. Patients further show reduced glycated hemoglobin (HbAlC), which is in the form of heme and is often used as an indicator of plasma glucose concentration in humans suffering from diabetes. Similarly, 53-year-old men with diabetes showed similar improvement at 12 months after treatment (see Table 8). The patient showed increased hemoglobin concentration and decreased The concentration of creatinine, HbAlC, and blood urea nitrogen (BUN), which is a measure of the amount of nitrogen in the form of urea in the blood. Table 8 Renal function markers in two diabetic male patients 75 1 Sugar g years old male suffering 51 Hemoglobin [g/dl] 8 12.9 12 14.5 Urea [g/dl] 190 38 n/an/a Creatine 酉 dry [mg/dl] 24 months after baseline treatment for aged men with diabetes ] 12 1.9 15 4.5 Uric acid [mg/dl] 8.5 4 n/an/a Phosphorus [mg/dl] 6.1 3.8 n/an/a HbAlC[%] 14.4 6.1 12 6 IGF-1"_11 45 112 n/an/ a BUNfmg/dll n/an/a 79 19

HbAlC=醣化血紅素；IGF-1=似胰島素生長因子1 ; BUN=血尿素氮 具有Π型尿病且以自體已重新編程細胞治療的12位 病患的分析揭露出降低的微量白蛋白濃度（圖3)，其與 洩漏至尿中的白蛋白有關聯且爲腎疾病與血管內皮細胞功 能障礙和心血管疾病的指標。1 2位病患亦經歷降低的 HbAlC 濃度（圖 14 )。 -64- 201114895 另外，遭受自體免疫腎絲球性腎炎之45歲女性病患 在以自體已重新編程細胞治療後18個月內展現出改善的 腎功能（參閱表9 )。 表9 在遭受自體免疫腎絲球性腎炎之45歲女性病患中的腎功能標誌濃度 基準線 治療後18個月 血紅素_ 9 11.5 肌酸酐[mg/dl] 9.3 2.1 BUN「mg/dll 89 15 BUN=血尿素氮 此外，遭受末期腎疾病之5 9歲女性病患在以已重新 編程細胞治療後顯示出改善的肌酸酐濃度、血紅素濃度及 副甲狀腺激素（PTH)(參閱表10)。在遭受自體免疫之 45歲女性病患中的腎功能標誌濃度。病患亦降低每月前 往的血液透析次數，從12次降至約8次。 -65- 201114895 表10 在以已重新編程細胞醫療前及後遭受末期腎疾病之59歲女性病患的腎功能標誌濃度 腎功能標誌 基準線 醫療後 03/02/ 2009 04/29/ 2009 05/23/ 2009 08/05/ 2009 08/11/ 2009 血紅素[g/dl] 9.8 9.2 11.5 TLCAVBC (每微升） 3800 6800 5100 PLT (每微升） 361000 273000 348000 HbAlC[%] 5.6 ASTrU/1] 25 16 14 ALT『U/1] 49 11 12 尿素[mg/dl] 90 70 22.13 肌酸酐 [mg/dll 14.5 11.2 11.86 8.86 9.58 血清白蛋白 Γ洲 3 3.4 3.3 3 PT[秒] 31.2 INR 2.71 鈣[mg/dl] 8.4 9.5 9.4 8.7 9.5 磷[mg/dl] 8 9.2 5.9 押[mmol/dl] 5.9 5.2 6.9 4.1 納[mmol/1] 133 136 尿酸[mg/dl] 7.9 6.1 7.5 4.8 PTH『pg/ml] 195.6 108.2 TLC=總白血球數量；WBO白血細胞；PLT=血小板；HbAlC=醣化血紅素；AST=天 冬胺酸轉胺酶；ALT=丙胺酸轉胺酶；PT=凝血酶原時間；INR=(用於凝血）國際正 規化比値；PTH=副甲狀腺激素 由於糖尿病而遭受慢性腎衰竭之46歲病患在以已重 新編程細胞治療時亦顯示出改進的肌酸酐濃度、尿素及血 紅素濃度（參閱表11)。病患降低每個月前往的血液透 -66 - 201114895 析次數，從1 2次降至約5次，且降低經由皮下輸送宜保 利（epoetin al fa ) ( EP REX® )的治療，從每週兩次4000 單元降至每兩周一次4000單元。 表11 在以已重新編程細胞醫療前及後遭受慢性腎衰竭之46歲病患的腎功能標誌濃度 腎功能標誌 基準線 醫療後 09/21/2008 02/24/2009 04/20/2009 血紅素[g/dl] 6.5 10.3 9.4 TLC/WBC (每微升） 5500 8200 10100 PLT (每微升） 159000 246000 300000 FBSrmg/dll 133 120 121 2小時PP [mg/dll 170 172 169 ASTrU/L] 9 34 32 ALTrU/L] 5 22 15 尿素[mg/dl] 109 51 65 肌酸酐[mg/dl] 7.5 4.9 4 血清白蛋白 _11 3.5 3.7 4 PT[秒] 12.7 12 11 INR 1 1 1 I5[mg/dl] 10 9.9 11 憐[mg/dl] 6 5.3 5 紳[mmol/L] 6.2 5.1 4.7 鎂[mg/dl] 2.3 2.4 2.8 TLC=總白血球數量；WBC=白血細胞；PP=膳食後；PLT=血小板；FBS=禁食血糖； AST=天冬胺酸轉胺酶；ALT=丙胺酸轉胺酶；PT=凝血酶原時間；INR=(用於凝血 )國際正規化比値 實例9 -67- 201114895 材料及方法 患有多發性硬化症之病患係以自體已重新編程多能性 幹細胞、間葉幹細胞及神經元（標的細胞）治療。標的細 胞係經由重新編程已血漿分離之白血細胞直到發育出標的 細胞爲止，如以彼等如上述之區別特徵所指明。病患係藉 由處理2_3次彼之總血量而血漿分離。自體已重新編程細 胞係經由根據本發明的重新編程而產生。病患係經由靜脈 內輸注於頸靜脈或手臂或大腿的靜脈中而投予自體已重新 編程細胞。 結果 以自體已重新編程細胞治療之病患顯示出減輕腦部及 脊髓中的損害。減輕損害可發生在接受治療3個月之內（ 圖1 5 a-b )。減輕腦組織損傷係發生在6個月之內（圖 1 6a-b )。 以自體已重新編程細胞治療之病患亦顯示出移除脊髓 損害（圖17a-b ) »這些病患進一步展現改善的Kurtzke 擴展殘疾狀況（EDSS)評分、顯示出緩解且不參與至多4 年的慣例療法，因爲接受個別輸注。 實例1 0 材料及方法 患有Η IV之女性病患係以自體已重新編程造血幹細 胞治療。病患係藉由處理2-3次彼之總血量而血漿分離。 -68- 201114895 標的細胞係經由重新編程已血漿分離之白血細胞直到發育 出標的細胞而獲得，如以彼等如上述之區別特徵所指明。 病患係經由靜脈內輸注於頸靜脈或手臂或大腿的靜脈中而 投予自體已重新編程細胞。 結果 在治療前，HIV-1及HIV-2抗體之篩選試驗顯示3.68 之試驗値。以1 或更大之値被認爲陽性。 在以自體已重新編程細胞治療後兩個月，Η IV -1及 HIV-2抗體篩選之試驗値爲0.46，其表示病患未顯示出 HIV-1及HIV-2的陽性結果。在治療後6個月，HIV-1及 HIV-2抗體篩選之試驗値爲0.48，進一明證明病患未顯示 出HIV的陽性結果。 實例1 1 以自體已重新編程細胞治療的效果在遭受其他症狀及 疾病的病患中予以證明。本文所列之標的細胞係經由重新 編程已血漿分離之白血細胞直到發育出標的細胞而獲得， 如以彼等如上述之區別特徵所指明》 充血性心臟衰竭 將遭受充血性心臟衰竭之6 1歲男性病患以自體已重 新編程心肌細胞、多能性幹細胞、間葉幹細胞及內皮細胞 輸注。治療導致改善的射出分率（EF )、腦部利鈉肽前驅 -69- 201114895 物濃度（Pro BNP )(其爲與冠狀動脈疾病有關聯的預測 因子）、血液葡萄糖禁食濃度及HbA 1 c濃度（參閱表1 2 )。此外，先前已擴大之心臟係藉由縮小如心臟超音波影 像所測量之舒張末期左心室內部尺寸（LVID/D )、收縮 末期左心室內部尺寸（LVID/S)及舒張末期心室間隔厚 度（IVSD )而恢復成如表12中所證明之正常尺寸。 表12 在以已重新編程細胞醫療前及後遭受充血性心臟衰竭之61歲病患的冠狀 動脈疾病標誌 _ 冠狀動脈 治療前 治療後 治療後 治療後 治療後 治療後 疾病標誌 1個月 1個月 2個月 4個月 6個月 10個月 LVID/Dfcml 6.5 6.3 5.9 6.9 6.5 5.7 LVID/S[cml 6.1 5.6 5.4 5.9 5.7 4.9 EF 15 18 22 29 33 45 IVSD[cml 1.3 1.3 1.2 1.2 0.6 1.0 LVPWDrcml 0.7 0.6 0.6 0.8 0.6 1.0 左心室重量指數 283 251 209.5 167 151.5 igm/ml 141.5 133.5 111.4 88.8 81 Pro BNP [pg/ml] 2969 800 600 600 600 497 禁食血液葡萄糖 [mg/dl] 244 118 145 87 95 80 HbAlC[%] 9.2 nd nd 7.2 6.5 6 LVID/EN舒張末期左心室內部尺寸；LVID/S=收縮末期左心室內部尺寸；EF=射出分 率；IVSD=舒張末期心室間隔厚度；LVPWD=舒張末期心室後壁厚度；Pr〇-BNP=腦 部利鈉肽前驅物：HbAlC=醣化血紅素 C型肝炎 將感染C型肝炎病毒（HCV)且具有地中海貧血 的1 3位病患以自體已重新編程造血細胞、多能性幹細胞 -70- 201114895 、間葉幹細胞及肝細胞輸注。治療效果揭露較低或清除之 HCV載量（參閱表1 3 )與改善的血液標誌（參閱表1 4 ) ，諸如肝酵素、膽紅素、白蛋白、凝血酶原時間及（用於 凝血）國際正規化比値。病患特別經歷正常化之肝酵素丙 胺酸轉胺酶（ALT )及天冬胺酸轉胺酶（AST )，二者皆 與肝細胞發炎及傷害有關聯（圖18a-b)。 表13 在13位感染C型肝 F炎病毒且以自體已重新編程幹細胞治療之病患中的病毒載量 病患ID 基準線 病毒載量xl 000 輸注後3個月 病毒載量xl000 1 0 0 2 164 82 3 3 0 4 123 16 5 16 0 6 2 0 7 0 0 8 7,700,000 7,700.000 9 152 106 10 63 27 11 387 97 12 0 0 13 17 0 -71 - 201114895 表14 在以已重新編程細胞醫療前及後感染C型肝炎病毒且遭受肝硬化之病患中 的血液標誌 血液標誌 基準線 輸注後3個月 血紅素[g/dl] 9.8 12.9 血小板（每微升） 65000 120000 WBC (每微升） 4300 7200 血糖（禁食）[mg/dl] 210 110 血液2小時（PP) [mg/dl] 190 134 ALT[U/L1 50 24 AST[U/L] 32 31 膽紅素（總）[mg/dl] 6.9 3.2 膽紅素（直接）[Mg/dl] 3.1 1.8 血清白蛋白[g/1] 1.9 3.1 尿素[mg/dl] 60 36 肌酸酐[mg/dl] 1.6 1.1 INR 1.8 1.0 PT[秒] 18 12.8 WBC=白血細胞；PP=膳食後；ALT=丙胺酸轉胺酶；AST=天冬胺酸轉胺酶；INR=( 用於凝血）國際正規化比値；PT=凝血酶原時間 例如，由於C型肝炎病毒感染而遭受肝硬化之44歲 男性病患亦顯示出改善的肝酵素丙胺酸轉胺酶及天冬胺酸 轉胺酶、（用於凝血）國際正規化比値、膽紅素及白蛋白 與隨機血糖（參閱表15)。事實上，此病患在以已重新 編程細胞醫療則亦以白蛋白治療’但在醫療後不接受白蛋 白。 -72- 201114895 表15 由於C型肝炎病毒感染而遭受肝硬化之44歲男性病患在基準線上及以已重新編 程紐 胞醫療後的血液標誌 血液標誌 基準線 醫療後 03/30/ 2009 04/04/ 2009 04/23/ 2009 05/06/ 2009 06/22/ 2009 血紅素[g/dl] 10.9 10.4 11.6 13.4 10.2 TLC (每微升） 2500 2700 6100 4700 2800 PLT (每微升） 20000 17000 43000 50000 27000 ASTiU/Ll 212 170 131 141 120 ALTrU/Ll 62 59 48 51 53 鹼性磷酸酶 TU/L1 196 147 58 GGT[U/L1 49 70 63 35 膽紅素τ Tnig/Ll 12.7 10.9 11.2 8.1 8.8 膽紅素D 『mg/Dll 6.8 5.4 4.71 4.9 4 尿素[mg/dl] 13 18 20 16 25 肌酸酐 [mg/dll 0.7 0.7 0.8 0.7 0.7 血清白蛋白 rs/Li 2.8 2.5 2.9 2.4 1.9 RBS[mg/dl] 212 154 158 188 174 INR 1.9 1.8 1.72 1.69 1.72 鉀[mmol/L] 3.2 3.8 4.4 鈉[mmol/L] 134 141 137 尿酸[Mg/dl] 3.5 2.4 3 胎兒蛋白 正常 冷凝球蛋白 陰性 TLC=總白血球計數；PLT=血小板；AST=天冬胺酸轉胺酶；ALT=丙胺酸轉胺酶; GGT=r-麩胺醯轉移酶；RBS=隨機血糖；INR=(用於凝血）國際正規化比値 頭部創傷 -73- 201114895 從運動意外遭受頭部創傷之病患係以輸注自體已重新 編程多能性幹細胞及神經元治療。治療導致損傷之腦組織 修補（圖1 9a-b )，改善的射出分率（EF )、腦部利鈉肽 前驅物濃度（Pro BNP )(其爲與冠狀動脈疾病有關聯的 預測因子）、血液葡萄糖禁食濃度及HbAlC濃度（參閱 表 10 )。 肺疾病 遭受與運動神經元疾病有關聯的侷限性肺疾病之病患 係以輸注自體已重新編程多能性幹細胞、間葉幹細胞、肺 泡上皮細胞及內皮細胞治療。在治療後6個月，用力肺活 量（FVC )(其爲在完全吸氣後可用力吹出之空氣容積） 從50%增加到71%，且1秒內用力呼氣容積（FEV,)( 其爲在1秒內可用力吹出之空氣容積）從64%增加到68 %。FEV,S FVC之比値進一步從100%降低至82%，在 健康的成人中爲約75-80%。肺的X-射線掃描顯示出在治 療後較不混濁的肺腔（圖20)。 更年期 以輸注自體已重新編程多能性幹細胞、多能性生殖細 胞及卵母細胞投予51歲更年期病患。在治療後，病患經 歷各種激素及蛋白質濃度的增加，包括似胰島素生長因子 (IGF-1 )、***及低密度脂蛋白（LDL )(參閱表16 -74- 201114895 表16 以自體已重新編程幹細胞投予更年期病患的效果 血液標誌 醫療前 醫療後 11/23/ 2006 12/09/ 2006 01/01/ 2007 02/17/ 2009 IGF-l[ng/ml] 91 53 100 143 IGF-1-結合[mg/L] 5.3 4 4.5 5.9 GH[^g/L] 2.4 3.5 3.5 1.5 助孕酮[ng/ml] 1.2 1.5 17.5 2.25 ***[pg/ml] 26 82 162 168.82 皮質醇Ug/dl] 17.8 24 4.3 24.5 DHEA-SrNmol/11 2.2 3.6 0.4 5.3 adrenocort Cpmol/l] 21.1 10 10 甲狀腺球蛋白[ng/ml] 47 18 31 1.4 抑鈣素[ng/ml] 28 40 50 FSH[IU/11 41.2 5.4 3.88 LHriU/11 15.1 1.8 6.76 催乳激素[/Zg/l] 16.2 11.1 10.5 0.6 游離試驗[pmol/1] 8 6.4 7.5 膽固醇[mg/dl] 242 258 280 268 HDL[mg/dl] 70 76 83 63.22 LDL[mg/dl] 22 162 173 179.9 triglycer[mg/dl] 111 102 122 125 Hbalc[%] 6.5 5.3 5.5 5.4 Fbs[Mg/dll 94 90 102 103 抑制素[u] 40 30 骨鈣化素[ng/mL] 6.9 TSH[//U/ml] 0.84 0.7 0.5 FT3 [pg/ml] 3.1 2.7 2.7 1.54 抑鈣素[ng/l] 28 40 50 FT4[pg/ml] 1.4 0.94 0.71 0.82 IGF-1=似胰島素生長因子1 ; IGF-1-結合=似胰島素生長因子結合；GH=生長激素； DHEA-S=去氫表雄固酮硫酸酯；adrenocort^腎上腺皮質激素；FSH=促卵泡激素； LH=黃體激素；HDL=高密度脂蛋白；LDL=低密度脂蛋白；triglyCer=三酸甘油酯； HBA1C=醣化血紅素；FBS=胎兒血液取樣；TSH=促甲狀腺激素；FT3=游離三碘甲 狀腺胺酸；FT4=游離甲狀腺素 -75- 201114895 抑鬱症 遭受抑鬱症之病患係以輸注自體已重新編程多能性幹 細胞及神經元治療。在治療後，病患經歷各種激素及蛋白 質濃度的增加，包括似胰島素生長因子-1 ( IGF-1 )、皮 質醇及睾固酮（參閱表17)。 表17 以自體已重新編程細胞投予抑鬱症病患的效果 血液標誌 醫療前 醫療後 06/11/2007 07/23/2007 <0.05 0.06 IGF-l[ng/mll 93.4 140 IGF-bp[mg/l] 4.4 5.6 皮質醇["g/dl] 4 20.9 adrenocort 『pg/mll <10 17.1 Sh-bg[nmol/l] 37.9 39.8 FSHriU/Ll 4.94 4.98 LH[IU/L1 10.1 3.72 e2『pg/ml] 29 <28 睪固酮[pmol/1] 28.4 57.8 催乳激素["g/1] 15.7 1.6 DHEA-S["mol/l] 2.3 2.9 TSH["U/ml] 1.707 3.318 FT3[pg/ml] 1.69 1.87 FT4[ng/dl] 0.81 0.89 GH=生長激素；IGF-1=似胰島素生長因子1 ; IGF-l-bp=似胰島素生長因子結合蛋白 ;adrenocort=腎上腺皮質激素；Sh-bg=性激素結合球蛋白；FSH=促據泡激素；LH= 黃體激素；e2=***：DHEA-S=去氫表雄固酮硫酸酯；TSH=促甲狀腺激素；FT3= 游離三碘甲狀腺胺酸；FT4=游離甲狀腺素 •76- 201114895 非阻塞性無精症_ 遭受非阻塞性無精症之病患係以輸注自體已重新編程 多能性幹細胞、多能性生殖細胞及***治療。治療後，病 患經歷8個月期間的睪固酮增加（圖2 1 )。 視覺障礙 由於已經移除之良性腫瘤而遭受視覺喪失之病患係以 輸注自體已重新編程多能性幹細胞及神經元治療。治療後 ，病患經歷增加的視網膜敏感度及改善的視覺（圖22) 〇 由此已詳細敘述本發明較佳的具體例，應瞭解由以上 段落所定義之本發明不限於以上發明內容所陳述之特殊細 節，因爲許多其顯而易見的變異有可能不違背本發明的精 神及範疇。 【圖式簡單說明】 以實例方式所提供但不意欲使本發明僅限於所述之特 殊具體例的下列詳細敘述可結合所附之圖形予以最完整的 瞭解，其中： 圖1顯示已血漿分離單核細胞在誘發重新編程前（上 格板）及後（下格板）的免疫表現型分析。將細胞以與 R-藻紅素（RPE) Cy-5或藻紅素（PE)結合之單株抗體標 記（垂直圖例），分別用於免疫球蛋白G1 ( IgGl )及 CD3 4或CD19同型控制。亦將細胞以與螢光異硫氰酸鹽 -77- 201114895 (FITC)結合之單株抗體染色（濃度圖例）’用於CD45 ' CD38 ' CD61及IgGl同型控制。下格板顯示出增加的 造血幹細胞，如以降低白血球成熟標誌（諸如CD45與 CD19)伴隨增加的CD34及CD34CD38 -細胞之相對數量 所描述。 圖2 a顯示患有嚴重的再生不全性貧血之病患在輸注 自體HRSC後（事後第1天、第2天、第3天、第6天及 第1 4天）的末梢血液樣品之序列免疫表現型分析。將細 胞以對抗CD34與CD45(第二濃度格板）及CD34與 C D 3 8 (第三濃度格板）的單株抗體標記。上濃度格板顯 示在自體HRSC的流動式細胞測量術、骨髓抹片及環鏨區 的前及側散射。第1天至第.14天顯示具有大的前及側散 射之細胞（其爲顆粒性細胞之指標）增加’且第1天至第 3天顯示循環CD34之造血幹細胞的相對數量增加。圖2b 顯示患有嚴重的再生不全性貧血之病患在輸注自體人類已 重新編程幹細胞（HRSC )後（事後第1天、第2天、第 3天、第6天及第14天）的末梢血液樣品的序列免疫表 現型分析。將細胞以對抗CD34與CD61 (第一濃度格板 )、CD19與CD3(第二濃度格板）及CD33 & 13與CD7 (第三濃度格板）的單株抗體標記。FAC掃描圖顯示增加 的骨髓細胞數量，如以逐漸增加表現CD33 & 13之細胞（ 包括祖細胞）所描述，其係藉由增加共同表現CD3 3 & 1 3 與CD7之細胞的相對數量所描述。亦逐漸增加淋巴細胞 的相對數量，如以增加CD1 9及CD 3淋巴細胞之相對數量 -78- 201114895 所顯示^ 圖3顯示患有嚴重的再生不全性貧血之病患在輸注自 體HRSC後的骨髓分析。在醫療前及後（a及b)的骨髓 抹片顯示出增加的紅血細胞；在治療前及後（c及d)的 環鑿區顯示出增加的骨髓細胞性；在輸注HRSC後的骨髓 克隆分析顯示出增加的巨核細胞群落形成單位（e)、單 細胞群落形成單位（f)、顆粒性細胞與巨噬細胞群落形 成單位（g)、骨髓細胞與紅血球群落形成單位（h)及紅 血球爆發群落形成單位（i)的生長。 圖4顯示患有嚴重的再生不全性貧血之病患在以自體 HRSC輸注4年後的末梢血液樣品的染色體核型鑑定及g-條紋帶，顯示沒有任何基因異常。 圖5顯示患有地中海貧血之病患在以自體已重新編程 細胞治療後增加的絕對平均胎兒血紅素濃度。 圖6顯示增加的平均血球容積，其代表患有地中海貧 血之病患在以自體已重新編程細胞治療後的紅血細胞平均 尺寸，以毫微微升表示。 圖7顯示增加的平均細胞血紅素，其爲患有地中海貧 血之病患在以自體已重新編程細胞治療後的每一細胞血紅 素重量，以微微克表示。 圖8顯示患有地中海貧血之病患在以自體已重新編程 細胞治療後降低的血清中鐵蛋白濃度，以每毫升毫微克表 不° 圖9顯示患有糖尿病之病患在以自體已重新編程細胞 -79- 201114895 治療後於禁食及攝取混合膳食後增加的C-肽濃度。 圖1 0顯示患有糖尿病之病患在以自體已重新編程細 胞治療後降低的醣化血紅素（Hb A 1 C )濃度。 圖11顯示患有肌肉萎縮症之病患在以自體已重新編 程細胞治療後降低的肌胺酸磷酸轉移酶（CPK)及乳酸鹽 脫氫酶（LDH )濃度。 圖12顯示患有肌肉萎縮症之病患在以自體已重新編 程細胞治療後降低的肝酵素丙胺酸轉胺酶（ALT)及天冬 胺酸轉胺酶（AST)濃度。 圖1 3顯示1 2位患有腎疾病之病患在以自體已重新編 程細胞治療後降低的微量白蛋白尿素濃度。 圖14顯示12位由於糖尿病而患有腎疾病之病患在以 自體已重新編程細胞治療後降低的醣化血紅素（Hb A 1 C ) 濃度。 圖15a顯示患有多發性硬化症之病患在以自體已重新 編程細胞治療前（左掃描）及後三個月（右掃描）的腦部 磁共振影像（MRI)掃描，其描述以幹細胞醫療後降低的 病變提升。圖15b顯示病患在以自體已重新編程細胞治療 前（左掃描）及後三個月（右掃描）於不同的腦部位之 MRI掃描。在描述治療前的掃描中，箭頭指向腦中的病變 ，而在描述治療後的掃描中，箭頭指向改善的病變。 圖1 6 a顯示患有多發性硬化症之病患在以自體已重新 編程細胞治療前（上掃描）及後六個月（下掃描）的腦部 磁共振影像（MRI)掃描。圖16b顯示病患在以自體已重 -80- 201114895 新編程細胞治療前（上掃描）及後六個月（ 外腦部MRI掃描。在描述治療前的掃描中 中的病變，而在描述治療後的掃描中，箭頭 縮的病變改善，如以減輕腦室及腦渠擴張所 圖1 7 a顯示患有多發性硬化症之病患在 編程細胞治療前（左掃描）及後六個月（右 磁共振影像（MRI)掃描。圖17b顯示病患 新編程細胞治療前（左掃描）及後六個月（ 髓橫向MRI掃描在描述治療前的掃描中 髓上的病變，而在描述治療後的掃描中，箭 病變。 圖18a顯示以C型肝炎感染之病患在以 程細胞治療後降低的肝酵素丙胺酸轉胺酶（ 圖18b顯示降低的天冬胺酸轉胺酶（AST); 圖19a顯示由於運動意外而有頭部創傷 磁共振影像（M RI )掃描。在治療前（上掃 示擴張及以寬的穿刺血腫置換。在以自體已 治療後（下掃描），腦室顯示出降低的腦萎 以改善血腫而減輕腦室及腦渠擴張。圖1 9b 療前（上掃描）及治療後（下掃描）的額外 描。 圖2 0顯示患有肺疾病之病患在以自體 胞治療前（左X-射線）及後（右x-射線）| 。在治療後，病患顯示改善的肺容積及減少 下掃描）的額 ，箭頭指向腦 指向減輕腦萎 描述。 以自體已重新 掃描）的矢狀 在以自體已重 右掃描）的脊 ，箭頭指向脊 頭指向改善的 自體已重新編 ALT)濃度及 1度。 之病患的腦部 描），腦室顯 重新編程細胞 縮參數，諸如 顯示病患在治 腦部MRI掃 已重新編程細 匀胸部X-射線 的病變尺寸， -81 - 201114895 如以降低的低密度區域所描述。 圖2 1顯示在患有非阻塞性無精症且以自體已重新編 程細胞治療之病患中的濾泡刺激激素（fsh )、黃體激素 (lh )、助孕酮（pro )及睪固酮（test )之性激素濃度。 濃度顯示隨超聲波所測定增加之睪九尺寸（未顯示數據） 而顯著增加的游離睪固酮。 圖2 2顯示遭受視覺障礙之病患在以自體已重新編程 細胞治療前（上格板）及後（下格板）的視網膜敏感度及 視覺障礙。在視網膜敏感度的結果中’橘色區域指出降低 的視網膜敏感度。在視覺障礙的結果中’白色區域指出正 常視覺，而粉紅色 '橘色及黑色區域指出增加的視覺障礙 。在治療後，病患經歷改善其視野’因爲在 '治療前^^視_ 膜敏感度中的橘色區域在治療後轉變成綠色及白色’且在 治療前的視覺障礙中的黑色區域在治療後轉變成白色。 -82-HbAlC = glycated hemoglobin; IGF-1 = insulin-like growth factor 1; BUN = blood urea nitrogen has sputum type urine disease and analysis of 12 patients treated with autologous reprogrammed cells revealed reduced microalbumin concentration (Fig. 3), which is associated with albumin leaking into the urine and is an indicator of renal disease and vascular endothelial cell dysfunction and cardiovascular disease. One or two patients also experienced reduced HbAlC concentrations (Figure 14). -64- 201114895 In addition, 45-year-old female patients suffering from autoimmune renal glomerulonephritis exhibited improved renal function within 18 months of autologous reprogrammed cell therapy (see Table 9). Table 9 Renal function marker concentration in 45-year-old female patients suffering from autoimmune renal glomerulonephritis 18 months after baseline treatment Hemoglobin _ 9 11.5 Creatinine [mg/dl] 9.3 2.1 BUN "mg/dll 89 15 BUN=blood urea nitrogen In addition, 59-year-old female patients suffering from end-stage renal disease showed improved creatinine concentration, heme concentration, and parathyroid hormone (PTH) after treatment with reprogrammed cells (see Table 10). The concentration of renal function markers in 45-year-old female patients who were autoimmune. The patient also reduced the number of hemodialysis trips per month, from 12 to about 8. -65- 201114895 Table 10 Reprogramming of cells in the 59-year-old female patient with end-stage renal disease before and after medical treatment. Renal function markers baseline medical care 03/02/ 2009 04/29/ 2009 05/23/ 2009 08/05/ 2009 08 /11/ 2009 Heme [g/dl] 9.8 9.2 11.5 TLCAVBC (per microliter) 3800 6800 5100 PLT (per microliter) 361000 273000 348000 HbAlC[%] 5.6 ASTrU/1] 25 16 14 ALT『U/1】 49 11 12 Urea [mg/dl] 90 70 22.13 Creatinine [mg/dll 14.5 11.2 11.86 8.86 9.58 Serum albumin Pazhou 3 3.4 3.3 3 PT [sec] 31.2 INR 2.71 Calcium [mg/dl] 8.4 9.5 9.4 8.7 9.5 Phosphorus [mg/dl] 8 9.2 5.9 Pend [mmol/dl] 5.9 5.2 6.9 4.1 Na [ Mmmol/1] 133 136 uric acid [mg/dl] 7.9 6.1 7.5 4.8 PTH "pg/ml] 195.6 108.2 TLC = total white blood cell count; WBO white blood cells; PLT = platelets; HbAlC = glycated hemoglobin; AST = aspartic acid Transaminase; ALT = alanine transaminase; PT = prothrombin time; INR = (for coagulation) international normalization ratio 値; PTH = parathyroid hormone due to diabetes, chronic kidney failure, 46-year-old patient Improved creatinine concentration, urea and heme concentration were also shown when reprogrammed cells were treated (see Table 11). Patients reduced the number of blood permeation per month to -66 - 201114895, down from 12 times About 5 times, and reduced treatment via subcutaneous delivery of epoetin al fa (EP REX®), from 4000 units twice a week to 4000 units once every two weeks. Table 11 Renal function markers in 46-year-old patients suffering from chronic renal failure before and after reprogrammed cell therapy Renal function markers baseline medical treatment 09/21/2008 02/24/2009 04/20/2009 Heme [g/dl] 6.5 10.3 9.4 TLC/WBC (per microliter) 5500 8200 10100 PLT (per microliter) 159000 246000 300000 FBSrmg/dll 133 120 121 2 hours PP [mg/dll 170 172 169 ASTrU/L] 9 34 32 ALTrU/L] 5 22 15 Urea [mg/dl] 109 51 65 Creatinine [mg/dl] 7.5 4.9 4 Serum albumin_11 3.5 3.7 4 PT [seconds] 12.7 12 11 INR 1 1 1 I5[mg/ Dl] 10 9.9 11 pity [mg/dl] 6 5.3 5 绅 [mmol/L] 6.2 5.1 4.7 Magnesium [mg/dl] 2.3 2.4 2.8 TLC = total white blood cell count; WBC = white blood cells; PP = post-meal; PLT = Platelets; FBS = fasting blood glucose; AST = aspartate transaminase; ALT = alanine transaminase; PT = prothrombin time; INR = (for coagulation) international normalization ratio 値 Example 9 -67- 201114895 Materials and Methods Patients with multiple sclerosis were treated with autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, and neurons (target cells). The target cell line is reprogrammed by plasma-separated white blood cells until the target cells are developed, as indicated by their distinguishing characteristics as described above. The patient was separated by plasma by treating 2 to 3 times the total blood volume. Autologous reprogrammed cells are generated via reprogramming in accordance with the present invention. The patient is administered autologous reprogrammed cells via intravenous infusion into the jugular vein or veins of the arms or thighs. Results Patients treated with autologous reprogrammed cells showed reduced damage in the brain and spinal cord. Mitigating damage can occur within 3 months of treatment (Figure 15 a-b). The reduction of brain tissue damage occurred within 6 months (Fig. 1 6a-b). Patients treated with autologous reprogrammed cells also showed removal of spinal cord lesions (Fig. 17a-b) » These patients further demonstrated improved Kurtzke Extended Disability (EDSS) scores, showed remission and did not participate for up to 4 years Conventional therapy because of the individual infusions received. EXAMPLE 1 Materials and Methods Female patients with sputum IV were treated with autologous reprogrammed hematopoietic stem cells. The patient was separated by plasma by treating 2-3 times the total blood volume. -68- 201114895 The target cell line is obtained by reprogramming the plasma-separated white blood cells until the development of the target cells, as indicated by their distinguishing characteristics as described above. The patient is administered autologous reprogrammed cells via intravenous infusion into the jugular vein or veins of the arms or thighs. Results Screening tests for HIV-1 and HIV-2 antibodies showed a test sputum of 3.68 prior to treatment. It is considered positive after 1 or more. Two months after treatment with autologous reprogrammed cells, the test for ΗIV-1 and HIV-2 antibody screening was 0.46, indicating that the patient did not show positive results for HIV-1 and HIV-2. At 6 months after treatment, the test for HIV-1 and HIV-2 antibody screening was 0.48, and it was confirmed that the patient did not show positive results for HIV. Example 1 1 The effect of autologous reprogrammed cell therapy was demonstrated in patients suffering from other symptoms and diseases. The target cell lines listed herein are obtained by reprogramming the plasma-separated white blood cells until the development of the target cells, as indicated by their distinctive features as described above. Congestive heart failure will be affected by congestive heart failure. Male patients have reprogrammed cardiomyocytes, pluripotent stem cells, mesenchymal stem cells, and endothelial cells for autologous infusion. Treatment results in improved ejection fraction (EF), brain natriuretic peptide precursor-69-201114895 concentration (Pro BNP) (which is a predictor associated with coronary artery disease), blood glucose fasting concentration, and HbA 1 c Concentration (see Table 1 2). In addition, the previously expanded heart system reduces the end diastolic left ventricular dimension (LVID/D), end-systolic left ventricular endoscopic size (LVID/S), and end-diastolic ventricular septal thickness (IVSD) as measured by cardiac ultrasound images. ) and restored to the normal size as demonstrated in Table 12. Table 12 Coronary artery disease markers in 61-year-old patients suffering from congestive heart failure before and after reprogrammed cell therapy _ Coronary treatment before and after treatment After treatment After treatment, disease markers 1 month and 1 month after treatment 2 months 4 months 6 months 10 months LVID/Dfcml 6.5 6.3 5.9 6.9 6.5 5.7 LVID/S[cml 6.1 5.6 5.4 5.9 5.7 4.9 EF 15 18 22 29 33 45 IVSD[cml 1.3 1.3 1.2 1.2 0.6 1.0 LVPWDrcml 0.7 0.6 0.6 0.8 0.6 1.0 Left ventricular mass index 283 251 209.5 167 151.5 igm/ml 141.5 133.5 111.4 88.8 81 Pro BNP [pg/ml] 2969 800 600 600 600 497 Fasting blood glucose [mg/dl] 244 118 145 87 95 80 HbAlC[%] 9.2 nd nd 7.2 6.5 6 LVID/EN end diastolic left ventricular size; LVID/S = end systolic left ventricular size; EF = ejection fraction; IVSD = end diastolic ventricular septal thickness; LVPWD = end diastolic ventricle Posterior wall thickness; Pr〇-BNP = brain natriuretic peptide precursor: HbAlC = glycosylated hemoglobin hepatitis C will infect hepatitis C virus (HCV) and 13 patients with thalassemia have been reprogrammed by themselves Hematopoietic cells, pluripotency -70-201114895 cells, mesenchymal stem cells and liver cell infusion. The therapeutic effect reveals a lower or cleared HCV load (see Table 13) and an improved blood marker (see Table 14), such as liver enzymes, bilirubin, albumin, prothrombin time and (for coagulation) International formalization is more than awkward. The patients experienced normalized liver enzymes such as alanine transaminase (ALT) and aspartate transaminase (AST), both of which were associated with hepatocyte inflammation and injury (Fig. 18a-b). Table 13 Viral load in 13 patients infected with type C hepatic F virus and treated with autologous reprogrammed stem cells. Patient ID baseline viral load xl 000 3 months after infusion viral load xl000 1 0 0 2 164 82 3 3 0 4 123 16 5 16 0 6 2 0 7 0 0 8 7,700,000 7,700.000 9 152 106 10 63 27 11 387 97 12 0 0 13 17 0 -71 - 201114895 Table 14 in the reprogrammed cell medical Blood markers in patients infected with hepatitis C virus before and after liver cirrhosis blood markers 3 months after baseline infusion of hemoglobin [g/dl] 9.8 12.9 platelets (per microliter) 65000 120000 WBC (per microliter 4300 7200 Blood glucose (fasting) [mg/dl] 210 110 Blood 2 hours (PP) [mg/dl] 190 134 ALT[U/L1 50 24 AST[U/L] 32 31 Bilirubin (total) [ Mg/dl] 6.9 3.2 bilirubin (direct) [Mg/dl] 3.1 1.8 serum albumin [g/1] 1.9 3.1 urea [mg/dl] 60 36 creatinine [mg/dl] 1.6 1.1 INR 1.8 1.0 PT [sec] 18 12.8 WBC = white blood cells; PP = post-meal; ALT = alanine transaminase; AST = aspartate transaminase; INR = (for coagulation) international normalization ratio 値; PT = Blood zymogen time For example, a 44-year-old male patient suffering from cirrhosis due to hepatitis C virus infection also showed improved liver enzyme alanine transaminase and aspartate transaminase, (for blood coagulation) international regular Comparison of sputum, bilirubin and albumin with random blood glucose (see Table 15). In fact, the patient was treated with albumin when he was reprogrammed, but he did not receive white protein after medical treatment. -72- 201114895 Table 15 44-year-old male patient suffering from cirrhosis due to hepatitis C virus infection on the baseline and after reprogramming the vaccination with the blood mark blood marker baseline medical care 03/30/ 2009 04/ 04/2009 04/23/ 2009 05/06/ 2009 06/22/ 2009 Heme [g/dl] 10.9 10.4 11.6 13.4 10.2 TLC (per microliter) 2500 2700 6100 4700 2800 PLT (per microliter) 20000 17000 43000 50000 27000 ASTiU/Ll 212 170 131 141 120 ALTrU/Ll 62 59 48 51 53 Alkaline phosphatase TU/L1 196 147 58 GGT[U/L1 49 70 63 35 bilirubin τ Tnig/Ll 12.7 10.9 11.2 8.1 8.8 Bile Erythroline D 『mg/Dll 6.8 5.4 4.71 4.9 4 Urea [mg/dl] 13 18 20 16 25 Creatinine [mg/dll 0.7 0.7 0.8 0.7 0.7 serum albumin rs/Li 2.8 2.5 2.9 2.4 1.9 RBS[mg/dl 212 154 158 188 174 INR 1.9 1.8 1.72 1.69 1.72 Potassium [mmol/L] 3.2 3.8 4.4 Sodium [mmol/L] 134 141 137 Uric acid [Mg/dl] 3.5 2.4 3 Fetal protein normal condensed globulin-negative TLC = total white blood cells Count; PLT = platelet; AST = aspartate transaminase; ALT = alanine transaminase; GGT = r-glutamine Enzyme; RBS = random blood glucose; INR = (for coagulation) international normalization than sputum head trauma -73- 201114895 From the accident of head trauma caused by sports accidents, infusion of autologous reprogrammed pluripotent stem cells and nerves Yuan treatment. Treatment of brain tissue repair leading to injury (Figure 19a-b), improved ejection fraction (EF), brain natriuretic peptide precursor concentration (Pro BNP), which is a predictor associated with coronary artery disease, Blood glucose fasting concentration and HbAlC concentration (see Table 10). Pulmonary disease Patients with localized lung disease associated with motor neuron disease are treated with infusion of autologous reprogrammed pluripotent stem cells, mesenchymal stem cells, alveolar epithelial cells, and endothelial cells. At 6 months after treatment, forced vital capacity (FVC), which is the volume of air that can be blown out after full inhalation, increased from 50% to 71%, and forced expiratory volume (FEV,) within 1 second (which is The volume of air that can be blown out in 1 second increases from 64% to 68%. The ratio of FEV, S FVC is further reduced from 100% to 82%, and is about 75-80% in healthy adults. X-ray scans of the lungs showed a less turbid lung cavity after treatment (Figure 20). Menopause Infusion of autologous reprogrammed pluripotent stem cells, pluripotent germ cells, and oocytes were administered to 51-year-old menopausal patients. After treatment, the patient experienced an increase in various hormone and protein concentrations, including insulin-like growth factor (IGF-1), estradiol, and low-density lipoprotein (LDL) (see Table 16-74-201114895, Table 16 for autologous The effect of reprogramming stem cells on menopausal patients has been shown blood labeling before medical treatment 11/23/ 2006 12/09/ 2006 01/01/ 2007 02/17/ 2009 IGF-l[ng/ml] 91 53 100 143 IGF -1-binding [mg/L] 5.3 4 4.5 5.9 GH [^g/L] 2.4 3.5 3.5 1.5 Progesterone [ng/ml] 1.2 1.5 17.5 2.25 Estradiol [pg/ml] 26 82 162 168.82 Cortisol Ug/dl] 17.8 24 4.3 24.5 DHEA-SrNmol/11 2.2 3.6 0.4 5.3 adrenocort Cpmol/l] 21.1 10 10 thyroglobulin [ng/ml] 47 18 31 1.4 Calcitonin [ng/ml] 28 40 50 FSH[ IU/11 41.2 5.4 3.88 LHriU/11 15.1 1.8 6.76 Prolactin [/Zg/l] 16.2 11.1 10.5 0.6 Free test [pmol/1] 8 6.4 7.5 Cholesterol [mg/dl] 242 258 280 268 HDL[mg/dl] 70 76 83 63.22 LDL[mg/dl] 22 162 173 179.9 triglyceride [mg/dl] 111 102 122 125 Hbalc[%] 6.5 5.3 5.5 5.4 Fbs[Mg/dll 94 90 102 103 Inhibin [u] 40 30 Bone calcification Prime [ng/mL] 6.9 TSH[//U/ml] 0.84 0.7 0.5 FT3 [pg/ml] 3.1 2.7 2.7 1.54 Calcitonin [ng/l] 28 40 50 FT4[pg/ml] 1.4 0.94 0.71 0.82 IGF-1 = insulin-like growth Factor 1; IGF-1-binding = insulin-like growth factor binding; GH = growth hormone; DHEA-S = dehydroepiandrosterone sulfate; adrenocort^ adrenocortical hormone; FSH = follicle stimulating hormone; LH = luteinizing hormone; HDL=high-density lipoprotein; LDL=low-density lipoprotein; triglyCer=triglyceride; HBA1C=glycated hemoglobin; FBS=fetal blood sampling; TSH=thyroid stimulating hormone; FT3=free triiodothyronine; FT4= Free thyroxine-75- 201114895 Depression patients suffering from depression are treated with infusion of autologous reprogrammed pluripotent stem cells and neurons. After treatment, the patient experienced an increase in various hormone and protein concentrations, including insulin-like growth factor-1 (IGF-1), cortisol and testosterone (see Table 17). Table 17 Effect of autologous reprogrammed cells on patients with depression. Blood markers before medical treatment 06/11/2007 07/23/2007 <0.05 0.06 IGF-l[ng/mll 93.4 140 IGF-bp[ Mg/l] 4.4 5.6 Cortisol ["g/dl] 4 20.9 adrenocort 『pg/mll <10 17.1 Sh-bg[nmol/l] 37.9 39.8 FSHriU/Ll 4.94 4.98 LH[IU/L1 10.1 3.72 e2『 Pg/ml] 29 <28 steroid [pmol/1] 28.4 57.8 prolactin ["g/1] 15.7 1.6 DHEA-S["mol/l] 2.3 2.9 TSH["U/ml] 1.707 3.318 FT3 [pg/ml] 1.69 1.87 FT4 [ng/dl] 0.81 0.89 GH = growth hormone; IGF-1 = insulin-like growth factor 1; IGF-l-bp = insulin-like growth factor binding protein; adrenocort = adrenocortical hormone; Sh -bg=sex hormone binding globulin; FSH=promoting vesicle hormone; LH=progesterone; e2=estradiol: DHEA-S=dehydroepiandrosterone sulfate; TSH=thyroid stimulating hormone; FT3=free triiodo Thyroin; FT4 = free thyroxine • 76- 201114895 Non-obstructive azoospermia _ Patients suffering from non-obstructive azoospermia have been reprogrammed with pluripotent stem cells, pluripotent germ cells, and Sperm treatment. After treatment, the patient experienced an increase in testosterone during the 8 months (Figure 21). Visual Disorders Patients suffering from visual loss due to the removal of benign tumors have been reprogrammed with pluripotent stem cells and neuronal therapy. After treatment, the patient experiences increased retinal sensitivity and improved vision (Fig. 22). Thus, a preferred embodiment of the invention has been described in detail, and it should be understood that the invention as defined by the above paragraph is not limited to the above description. The details are specific, as many of the obvious variations are possible without departing from the spirit and scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of the preferred embodiments of the invention, Immunophenotypic analysis of nucleated cells before induction of reprogramming (upper plate) and after (lower plate). Monoclonal antibody labeling (vertical legend) with R-phycoerythrin (RPE) Cy-5 or phycoerythrin (PE) for immunoglobulin G1 (IgGl) and CD3 4 or CD19 isotype control . The cells were also stained with monoclonal antibody (concentration legend) in combination with fluorescein isothiocyanate-77-201114895 (FITC) for CD45 'CD38' CD61 and IgGl isotype control. The lower plate shows increased hematopoietic stem cells, as described by decreasing the relative number of CD34 and CD34CD38- cells associated with increased white blood cell maturation markers (such as CD45 and CD19). Figure 2a shows the sequence of peripheral blood samples from patients with severe aplastic anemia after infusion of autologous HRSC (days 1st, 2nd, 3rd, 6th, and 14th afterwards) Immunophenotypic analysis. The cells were labeled with monoclonal antibodies against CD34 and CD45 (second concentration plates) and CD34 and C D 3 8 (third concentration plates). The upper concentration plate shows the flow cytometry, bone marrow smear and anterior and lateral scatter of the autologous HRSC. Days 1 to 14 showed an increase in cells with large front and side scatters, which are indicators of granulocyte growth, and the relative number of hematopoietic stem cells showing circulating CD34 increased from day 1 to day 3. Figure 2b shows patients with severe aplastic anemia after infusion of autologous human reprogrammed stem cells (HRSC) (1st, 2nd, 3rd, 6th and 14th day afterwards) Sequence immunophenotypic analysis of peripheral blood samples. Cells were labeled with monoclonal antibodies against CD34 and CD61 (first concentration plates), CD19 and CD3 (second concentration plates), and CD33 & 13 and CD7 (third concentration plates). The FAC scan shows an increase in the number of bone marrow cells, as described by the progressive increase in the expression of CD33 & 13 cells (including progenitor cells) by increasing the relative number of cells that collectively express CD3 3 & 1 3 and CD7. description. The relative number of lymphocytes is also gradually increased, as shown by increasing the relative number of CD1 9 and CD 3 lymphocytes -78-201114895. Figure 3 shows the patients with severe aplastic anemia after infusion of autologous HRSC. Bone marrow analysis. Bone marrow smears showed increased red blood cells before and after treatment (a and b); increased bone marrow cellularity in the chisel region before and after treatment (c and d); bone marrow clones after infusion of HRSC Analysis showed increased megakaryocyte community forming units (e), single cell community forming units (f), granulocyte and macrophage colony forming units (g), bone marrow cells and red blood cell community forming units (h) and red blood cell outbreaks The growth of the community forming unit (i). Figure 4 shows karyotype identification and g-striping of peripheral blood samples from patients with severe aplastic anemia after 4 years of autologous HRSC infusion, showing no genetic abnormalities. Figure 5 shows the absolute mean fetal hemoglobin concentration increased after treatment with autologous reprogrammed cells in patients with thalassemia. Figure 6 shows an increased mean hematocrit representing the average red blood cell size after treatment with autologous reprogrammed cells in patients with thalassemia, expressed in femtolite. Figure 7 shows the increased mean cellular hemoglobin, expressed as picograms of the hemoglobin weight per cell after treatment with autologous reprogrammed cells in patients with thalassemia. Figure 8 shows the concentration of ferritin in serum reduced in patients with thalassemia after treatment with autologous reprogrammed cells, expressed in nanograms per milliliter. Figure 9 shows that patients with diabetes have been autologous Reprogramming Cells - 79 - 201114895 Increased C-peptide concentration after fasting and ingestion of mixed diet after treatment. Figure 10 shows the reduced glycated hemoglobin (Hb A 1 C ) concentration in patients with diabetes after treatment with autologous reprogrammed cells. Figure 11 shows the concentration of creatinine phosphotransferase (CPK) and lactate dehydrogenase (LDH) decreased in patients with muscular dystrophy after treatment with autologous reprogrammed cells. Figure 12 shows liver enzyme alanine transaminase (ALT) and aspartate transaminase (AST) concentrations decreased in patients with muscular dystrophy after treatment with autologous reprogrammed cells. Figure 13 shows the microalbumin urea concentration that was reduced in 12 patients with kidney disease after treatment with autologous reprogrammed cells. Figure 14 shows the reduced glycated hemoglobin (Hb A 1 C ) concentration in 12 patients with kidney disease due to diabetes after treatment with autologous reprogrammed cells. Figure 15a shows a brain magnetic resonance imaging (MRI) scan of a patient with multiple sclerosis before treatment with autologous reprogrammed cells (left scan) and after three months (right scan), depicting stem cells Reduced lesions after medical treatment. Figure 15b shows an MRI scan of the patient at different brain sites before (left scan) and after three months (right scan) with autologous reprogrammed cells. In describing the pre-treatment scan, the arrow points to the lesion in the brain, while in the post-treatment scan, the arrow points to the improved lesion. Figure 167 shows a brain magnetic resonance imaging (MRI) scan of patients with multiple sclerosis before (auto-scanning) and after six-month (lower scan) treatment with autologous reprogrammed cells. Figure 16b shows patients undergoing autologous weight-80-201114895 new programmed cell therapy (upper scan) and after six months (extra-brain MRI scan. Describe the lesions in the pre-treatment scan, while describing In the post-treatment scan, the lesions of the arrow contraction were improved, such as to reduce the dilatation of the ventricles and cerebral canal. Figure 17 7 shows that patients with multiple sclerosis before the programmed cell treatment (left scan) and the last six months ( Right magnetic resonance imaging (MRI) scan. Figure 17b shows the patient's newly programmed cell before treatment (left scan) and the last six months (medullary lateral MRI scan in describing the lesion on the medullary scan before treatment, and after describing the treatment In the scan, the arrow lesions. Figure 18a shows the liver enzyme alanine transaminase reduced in patients infected with hepatitis C after treatment with Cheng Cheng (Figure 18b shows reduced aspartate transaminase (AST); Figure 19a shows a magnetic resonance imaging (MRI) scan of the head due to an accidental motion. Before treatment (upward dilatation and wide puncture hematoma replacement. After autologous treatment (lower scan), ventricle display Reduced brain stagnation to improve blood Swelling reduces the dilatation of the ventricles and cerebral canal. Figure 1 9b Extra treatment before (upper scan) and after treatment (lower scan) Figure 20 shows that patients with lung disease before treatment with autologous cells (left X -ray) and after (right x-ray)|. After treatment, the patient showed an improved lung volume and reduced the amount of the next scan), the arrow points to the brain pointing to alleviate the brain suffocation. The self-rescanned vector The ridges in which the autologous weight has been right-scanned, the arrow pointing to the horn pointing to the improved autologous re-programmed ALT) and the 1 degree. The brain of the patient), the ventricle reprogramming the cytoskeletal parameters, such as The patient's MRI scan in the brain has been reprogrammed to fine-tune the chest X-ray lesion size, -81 - 201114895 as described in the reduced low-density area. Figure 21 shows follicle stimulating hormone (fsh), luteinizing hormone (lh), progesterone (pro) and testosterone in patients with non-obstructive azoospermia and autologous reprogrammed cells (test) ) sex hormone concentration. The concentration showed a significant increase in free testosterone as a function of the increase in the size of the ninth (data not shown). Figure 2 2 shows the retinal sensitivity and visual impairment of patients suffering from visual impairment before and after autologous reprogramming of cells (upper plate) and posterior (lower plate). In the results of retinal sensitivity, the 'orange area' indicates reduced retinal sensitivity. In the results of visual impairments, the white area indicates normal vision, while the pink 'orange and black areas indicate increased visual impairment. After treatment, the patient experienced an improvement in his or her vision 'because the orange area in the 'film sensitivity' was converted to green and white after treatment and the black area in the visual disorder before treatment was treated After turning into white. -82-

Claims (1)

201114895 七、申請專利範圍： 1 · 一種重新補充病患中的組織或細胞系譜之細胞的方 法，其包含（i)獲得第一細胞系譜之定型細胞；（ii)重 新編程該定型細胞以獲得已重新編程細胞；及（iii )以該 已重新編程細胞投予病患。 2 .根據申請專利範圍第1項之方法，其中該病患患有 選自骨髓衰竭、血液學症狀、再生不全性貧血、A -地中 海貧血、運動神經元疾病、帕金森氏病、脊髓傷害 '肌肉 萎縮症、腎疾病、多發性硬化症、充血性心臟衰竭、C型 肝炎病毒、人類免疫缺乏病毒、頭部創傷、脊髓傷害、肺 疾病、抑鬱症、非阻塞性無精症、男性更年期、更年期與 ***症、回春作用、硬皮病潰瘍、牛皮癖、皴紋、肝硬化 、自體免疫疾病、禿髮、色素沉著性視網膜炎、角膜結晶 失養症/失明、糖尿病、肝硬化及***症之疾病或病症。 3.根據申請專利範圍第1項之方法，其中該已重新編 程細胞係選自多能性幹細胞、造血幹細胞、神經元幹細胞 、上皮幹細胞、間葉幹細胞、內胚層與神經外胚層幹細胞 、生殖細胞、胚外細胞、胚胎幹細胞、腎細胞、肺泡上皮 細胞、肺泡內胚層細胞、神經元、外胚層細胞、胰小島細 胞、腺泡細胞、卵母細胞、***、肝細胞、角質化細胞、 黑色素細胞、骨細胞、真皮乳頭細胞、軟骨細胞、脂肪細 胞、內皮細胞、心肌細胞及滋養層細胞。 4 .根據申請專利範圍第1項之方法，其中該定型細胞 係從全血、骨髓、神經元組織、肌肉組織、表皮或真皮獲 -83 - 201114895 得。 5 .根據申請專利範圍第4項之方法，其中該定型細胞 係從全血獲得。 6.根據申請專利範圍第5項之方法，其中該定型細胞 係經由血漿分離術獲得。 7 ·根據申請專利範圍第5項之方法，其中該定型細胞 係從流動的（mobilized)或未流動的（unm〇bUized)血 液獲得。 8. 根據申請專利範圍第5項之方法，其中該定型細胞 係選自T細胞、B細胞 '嗜酸性細胞、嗜鹼性、嗜中性細 胞、巨核細胞、單核細胞、紅血球（erythrocyte)、顆粒 性細胞、肥胖細胞、淋巴細胞、白血球、血小板及紅血細 胞（red blood cell)。 9. 根據申請專利範圍第1項之方法，其中該重新編程 包含定型細胞之反分化、轉分化、重新分化或其組合。 1〇·根據申請專利範圍第9項之方法，其中該重新編 程包含定型細胞之反分化以獲得已反分化細胞。 Π.根據申請專利範圍第10項之方法，其中該定型細 胞係藉由定型細胞與劑（agent )接觸而反分化。 12. 根據申請專利範圍第11項之方法，其中將該定型 細胞以劑培育。 13. 根據申請專利範圍第12項之方法’其中該劑接合 介導在定型細胞表面上的抗原補獲、識別或表現之受體。 根據申請專利範圍第11項之方法’其中該受體爲 ，84 - 201114895 第I型MHC抗原或第II型MHC抗原。 1 5 ·根據申請專利範圍第1 1項之方法，其中該劑爲受 體之抗體。 1 6 ·根據申請專利範圍第1 3項之方法，其中該劑爲受 體之單株抗體。 1 7.根據申請專利範圍第1 6項之方法，其中該抗體係 選自單株抗體CR3/4 3及單株抗體TAL 1B5。 18. 根據申請專利範圍第1項之方法，其中該已反分 化細胞係經由注射或植入而投予。 19. 根據申請專利範圍第18項之方法，其中該已反分 化細胞係藉由非經腸、肌肉內、靜脈內 '皮下、眼內、口 服、穿透皮膚注射或注入脊髓液中而投予。 20. 根據申請專利範圍第9項之方法，其中該重新編 程包含定型細胞之轉分化以獲得已轉分化細胞。 21. 根據申請專利範圍第20項之方法，其中該定型細 胞係藉由在包含一或多種反分化劑及一或多種分化促進劑 的組織培養基中培養定型細胞而轉分化。 22. 根據申請專利範圍第21項之方法，其中該組織培 養基係選自伊思可夫（Iscove’s )改良之杜爾貝克（ Dulbecco’s)培養基（IMDM)、杜爾貝克改良之伊格爾（ Eagle )培養基（DMEM )、伊格爾最低必需（ΕΜΕ )培養 基、最低必需培養基（α-ΜΕΜ)、洛斯維（Roswell) 公園紀念所（RPMI ;發展出培養基的場所）1 640、1^111-F-12、E199、MCDB、Leibovitz L-15 及威廉斯（Williams -85- 201114895 )培養基E或任何商業上可取得的培養基。 23·根據申請專利範圍第21項之方法，其中該分化促 進劑爲抗凝血劑、僞裝劑或抗生素。 24. 根據申請專利範圍第21項之方法，其中該分化促 進劑爲維生素、礦物質或其衍生物。 25. 根據申請專利範圍第24項之方法，其中該維生素 、礦物質或其衍生物係選自維生素A、維生素b3'維生素 C、維生素D3、維生素κ、視黃酸、薛鹼醯胺、鋅或鋅化 合物及鈣或鈣化合物。 26. 根據申請專利範圍第21項之方法，其中該分化促 進劑爲天然或合成激素。 27. 根據申請專利範圍第26項之方法，其中該天然或 合成激素爲氫皮質酮或地塞松。 2 8 .根據申請專利範圍第21項之方法，其中該分化促 進劑爲胺基酸或其衍生物。 29.根據申請專利範圍第28項之方法，其中該胺基酸 或其衍生物係選自L-麩醯胺酸（L-glu )、麥角硫醇（ EGT )、脯胺酸及非必需胺基酸（NEAA )。 3 0.根據申請專利範圍第21項之方法，其中該分化促 進劑爲化學化合物或其衍生物。 31.根據申請專利範圍第30項之方法，其中該化學化 合物或其衍生物係選自锍基十六烷醇、二丁基環腺苷 單磷酸鹽（db-cAMP )、單硫代甘油（MTG )、腐肉鹼、 二甲亞颯（DMSO)、次黃嘌呤、腺嘌呤、弗斯可林（ -86 - 201114895 forskolin)、西洛他醯胺（cil〇stamide)及 3-異丁基-1-甲 基黃嘌呤。 3 2 ·根據申請專利範圍第21項之方法，其中該分化促 進劑爲核苷或其類似物。 33.根據申請專利範圍第32項之方法，其中該核苷或 其類似物爲5 -氮雜胞嘧啶核苷。 3 4 ·根據申請專利範圍第21項之方法，其中該分化促 進劑爲酸或其鹽。 35·根據申請專利範圍第34項之方法，其中該酸或其 鹽係選自抗壞血酸、丙酮酸鹽、黑海綿酸（okadic acid ) 、亞麻油酸' 乙二胺四乙酸（EDTA)、二鈉EDTA、乙二 醇四乙酸（EGTA)、抗凝血檸檬酸鹽葡萄糖式A ( ACDA )、丁酸鈉及甘油磷酸鹽。 3 6.根據申請專利範圍第21項之方法，其中該分化促 進劑爲抗生素或藥物。 37.根據申請專利範圍第36項之方法，其中該抗生素 或藥物係選自 G418、健大黴素（gentamycine)、本西法 林（pentoxifylline ) ( 1- ( 5-酮己基）-3,7-二甲基黃嘌呤 )及美洒辛（indomethacin)。 3 8.根據申請專利範圍第21項之方法，其中該分化促 進劑爲蛋白質。 39.根據申請專利範圍第38項之方法，其中該蛋白質 爲組織血纖維蛋白溶酶原活化劑（TP A )。 4〇.根據申請專利範圍第21項之方法，其中該組織培 -87- 201114895 養基含有自體血漿、血小板、血清或哺乳動物源之血清。 41. 根據申請專利範圍第21項之方法，其中該細胞係 在血液袋、支架、組織培養袋或塑膠組織培養容器中培養 〇 42. 根據申請專利範圍第41項之方法，其中該組織培 養容器爲附著性或未附著性之組織培養容器。 4 3 .根據申請專利範圍第41項之方法，其中該組織培 養容器經塗佈或未經塗佈。 44. 根據申請專利範圍第43項之方法，其中該組織培 養容器係以選自明膠、膠原、基底膜基質（matrigel)或 細胞外基質之劑塗佈。 45. 根據申請專利範圍第21項之方法，其中該細胞係 在介於約1 8與約4 0 °C之間的溫度下培養。 46. 根據申請專利範圍第21項之方法，其中該細胞係 在介於約4與約1 0 %之間的二氧化碳濃度下培養。 47. 根據申請專利範圍第21項之方法，其中該細胞係 在介於約1 〇與約3 5 %之間的氧濃度下培養。 48. —種治療在需要此治療之病患中的疾病或組織傷 害之方法，其包含（Ο獲得定型細胞；（ii)重新編程該 定型細胞以獲得已重新編程標的細胞；及（iii)將該已重 新編程'標的細胞投予病患。 49. 根據申請專利範圍第1-48項中任一項之方法，其 中該標的細胞係以醫藥組成物形式投予。 5 0.—種申請專利範圍第49項之醫藥組成物。 -88- 201114895 51.—種一或多種已重新編程標的細胞之用途，其 製備用於修復或重新補充病患中的組織或細胞系譜之細 ，或用於治療疾病或組織傷害的藥劑或醫藥組成物。 5 2·—種獲得用於投予需要此投予之病患的已重新 程標的細胞之方法，其中該方法包含（i)獲得第一細 系譜之定型細胞；及（ii)重新編程該定型細胞以獲得 重新編程標的細胞。 53. —種醫藥組成物，其包含藉由申請專利範圍第 項之方法而獲得的已重新編程標的細胞及至少一種醫藥 可接受之賦形劑。 54. —種製備用於投予需要此投予之病患的藥劑或 藥組成物之方法，其中該方法包含（i)獲得第一細胞 譜之定型細胞；（Π)重新編程該定型細胞，獲得已重 編程標的細胞：及（iii)可隨意地組合該已重新編程標 細胞與一或多種醫藥賦形劑。 55. —種醫藥組成物，其係藉由申請專利範圍第54 之方法而製備。 係 胞 編 胞 已 52 上 盤 系 新 的 項 89 -201114895 VII. Patent Application Range: 1 · A method for replenishing cells of a tissue or cell lineage in a patient, comprising: (i) obtaining a committed cell of the first cell lineage; (ii) reprogramming the committed cell to obtain Reprogramming the cells; and (iii) administering the cells to the patient with the reprogrammed cells. 2. The method of claim 1, wherein the patient has a disease selected from the group consisting of bone marrow failure, hematological symptoms, aplastic anemia, A-thalassemia, motor neuron disease, Parkinson's disease, spinal cord injury Muscular atrophy, kidney disease, multiple sclerosis, congestive heart failure, hepatitis C virus, human immunodeficiency virus, head trauma, spinal cord injury, lung disease, depression, non-obstructive azoospermia, male menopause, menopause With infertility, rejuvenation, scleroderma ulcer, psoriasis, striate, cirrhosis, autoimmune disease, alopecia, pigmented retinitis, corneal dystrophies/blindness, diabetes, cirrhosis and no A disease or condition of pregnancy. 3. The method of claim 1, wherein the reprogrammed cell line is selected from the group consisting of pluripotent stem cells, hematopoietic stem cells, neuronal stem cells, epithelial stem cells, mesenchymal stem cells, endoderm and neuroectodermal stem cells, germ cells , extraembryonic cells, embryonic stem cells, renal cells, alveolar epithelial cells, alveolar endoderm cells, neurons, ectoderm cells, pancreatic islet cells, acinar cells, oocytes, sperm, hepatocytes, keratinocytes, melanocytes , bone cells, dermal papilla cells, chondrocytes, adipocytes, endothelial cells, cardiomyocytes and trophoblast cells. 4. The method according to claim 1, wherein the committed cell line is obtained from whole blood, bone marrow, neuronal tissue, muscle tissue, epidermis or dermis - 83 - 201114895. 5. The method of claim 4, wherein the committed cell line is obtained from whole blood. 6. The method of claim 5, wherein the committed cell line is obtained via plasmapheresis. 7. The method of claim 5, wherein the committed cell line is obtained from mobilized or unflowed (unm〇bUized) blood. 8. The method of claim 5, wherein the committed cell line is selected from the group consisting of a T cell, a B cell 'eosinophil, an basophilic, a neutrophil, a megakaryocyte, a monocyte, an erythrocyte, Granular cells, obese cells, lymphocytes, white blood cells, platelets, and red blood cells. 9. The method of claim 1, wherein the reprogramming comprises reverse differentiation, transdifferentiation, re-differentiation, or a combination thereof of the committed cells. The method according to claim 9, wherein the reprogramming comprises reverse differentiation of the committed cells to obtain the dedifferentiated cells. The method of claim 10, wherein the committed cell is dedifferentiated by contact of the committed cell with an agent. 12. The method of claim 11, wherein the committed cells are incubated with a dose. 13. The method according to claim 12, wherein the agent binds to a receptor that mediates antigen recruitment, recognition or expression on the surface of the committed cell. According to the method of claim 11, wherein the receptor is , 84 - 201114895 type I MHC antigen or type II MHC antigen. 1 5 . The method of claim 11, wherein the agent is a receptor antibody. The method according to claim 13 of the patent application, wherein the agent is a monoclonal antibody of the recipient. 1 7. The method according to claim 16 wherein the anti-system is selected from the group consisting of monoclonal antibody CR3/43 and monoclonal antibody TAL 1B5. 18. The method of claim 1, wherein the re-differentiated cell line is administered via injection or implantation. 19. The method according to claim 18, wherein the dedifferentiated cell line is administered by parenteral, intramuscular, intravenous subcutaneous, intraocular, oral, penetrating dermal injection or injection into spinal fluid. . 20. The method of claim 9, wherein the reprogramming comprises transdifferentiation of committed cells to obtain transdifferentiated cells. 21. The method according to claim 20, wherein the committed cell is transdifferentiated by culturing the committed cell in a tissue culture medium comprising one or more anti-differentiators and one or more differentiation promoters. 22. The method according to claim 21, wherein the tissue culture medium is selected from Iscove's modified Dulbecco's medium (IMDM), Durbeck's modified Eagle (Eagle) Medium (DMEM), Eagle's minimum essential (ΕΜΕ) medium, minimum essential medium (α-ΜΕΜ), Roswell Park Memorial (RPMI; site where the medium was developed) 1 640, 1^111-F- 12. E199, MCDB, Leibovitz L-15 and Williams (Williams -85-201114895) medium E or any commercially available medium. The method according to claim 21, wherein the differentiation promoting agent is an anticoagulant, a pretending agent or an antibiotic. 24. The method according to claim 21, wherein the differentiation promoting agent is a vitamin, a mineral or a derivative thereof. 25. The method according to claim 24, wherein the vitamin, mineral or derivative thereof is selected from the group consisting of vitamin A, vitamin b3 'vitamin C, vitamin D3, vitamin κ, retinoic acid, sulphuric acid amide, zinc Or zinc compounds and calcium or calcium compounds. 26. The method according to claim 21, wherein the differentiation promoting agent is a natural or synthetic hormone. 27. The method according to claim 26, wherein the natural or synthetic hormone is hydrocorticosterone or dexamethasone. The method of claim 21, wherein the differentiation promoting agent is an amino acid or a derivative thereof. 29. The method of claim 28, wherein the amino acid or derivative thereof is selected from the group consisting of L-glutamic acid (L-glu), ergothiol (EGT), proline and non-essential Amino acid (NEAA). The method of claim 21, wherein the differentiation promoting agent is a chemical compound or a derivative thereof. The method according to claim 30, wherein the chemical compound or a derivative thereof is selected from the group consisting of mercapto cetyl alcohol, dibutyl cyclic adenosine monophosphate (db-cAMP), and monothioglycerol ( MTG), carnitine, dimethyl hydrazine (DMSO), hypoxanthine, adenine, forskolin (-86 - 201114895 forskolin), cilostamide and 3-isobutyl- 1-methylxanthine. The method according to claim 21, wherein the differentiation promoting agent is a nucleoside or an analog thereof. 33. The method of claim 32, wherein the nucleoside or analog thereof is a 5-azacytidine nucleoside. The method according to claim 21, wherein the differentiation promoting agent is an acid or a salt thereof. The method according to claim 34, wherein the acid or a salt thereof is selected from the group consisting of ascorbic acid, pyruvate, okadic acid, linoleic acid, ethylenediaminetetraacetic acid (EDTA), disodium EDTA, ethylene glycol tetraacetic acid (EGTA), anticoagulant citrate glucose A (ACDA), sodium butyrate and glycerol phosphate. 3. The method according to claim 21, wherein the differentiation promoting agent is an antibiotic or a drug. 37. The method according to claim 36, wherein the antibiotic or drug is selected from the group consisting of G418, gentamycine, pentoxifylline (1-(5-ketohexyl)-3,7- Dimethylxanthine) and indomethacin. 3. The method of claim 21, wherein the differentiation promoting agent is a protein. 39. The method according to claim 38, wherein the protein is a tissue plasminogen activator (TP A ). 4. The method according to claim 21, wherein the tissue culture-containing serum of autologous plasma, platelets, serum or mammalian sources. 41. The method of claim 21, wherein the cell line is cultured in a blood bag, a stent, a tissue culture bag, or a plastic tissue culture container. 42. The method of claim 41, wherein the tissue culture container Culture the container for adherent or non-adherent tissue. The method according to claim 41, wherein the tissue culture container is coated or uncoated. 44. The method according to claim 43, wherein the tissue culture container is coated with a agent selected from the group consisting of gelatin, collagen, matrigel or extracellular matrix. 45. The method of claim 21, wherein the cell line is cultured at a temperature between about 18 and about 40 °C. 46. The method of claim 21, wherein the cell line is cultured at a concentration of carbon dioxide between about 4 and about 10%. 47. The method according to claim 21, wherein the cell line is cultured at an oxygen concentration of between about 1 Torr and about 35 %. 48. A method of treating a disease or tissue injury in a patient in need of such treatment, comprising: obtaining a committed cell; (ii) reprogramming the committed cell to obtain a reprogrammed target cell; and (iii) The method of re-programming the target cell is administered to the patient. The method according to any one of claims 1 to 48, wherein the target cell line is administered as a pharmaceutical composition. Pharmaceutical composition of the scope 49. -88- 201114895 51. - Use of one or more reprogrammed cells prepared for repairing or replenishing tissue or cell lineage in a patient, or for An agent or pharmaceutical composition for treating a disease or tissue injury. 5 2. A method for obtaining a re-targeted cell for administration to a patient in need of such administration, wherein the method comprises (i) obtaining a first pedigree a committed cell; and (ii) reprogramming the committed cell to obtain a reprogrammed target cell. 53. A pharmaceutical composition comprising a reprogrammed target obtained by the method of claim 2 a cell and at least one pharmaceutically acceptable excipient. 54. A method of preparing a medicament or a pharmaceutical composition for administration to a patient in need of such administration, wherein the method comprises (i) obtaining a first cell spectrum a committed cell; (Π) reprogramming the committed cell to obtain a reprogrammed cell: and (iii) optionally combining the reprogrammed target cell with one or more pharmaceutical excipients. , which was prepared by applying the method of Patent No. 54. The cell line has been 52 and the new item 89 -