WO2018167317A1

WO2018167317A1 - Method for in-vitro stem cell expansion

Info

Publication number: WO2018167317A1
Application number: PCT/EP2018/056766
Authority: WO
Inventors: Sebastian Jessberger; Marlen Knobloch
Original assignee: Universität Zürich
Priority date: 2017-03-17
Filing date: 2018-03-16
Publication date: 2018-09-20

Abstract

The present invention relates to a method of hematopoietic stem cell generation, comprising the steps of providing a hematopoietic stem cell isolate from a donor and expanding said hematopoietic stem cells in vitro in the presence of a compound selected from malonic acid, malonyl-CoA, and a malonate salt. The invention further relates to the use of malonyl-CoA, malonic acid or a malonate salt in stem cell expansion and in the therapy of neuropsychiatric disorders and conditions requiring hematopoietic stem cell transplantation.

Description

Method for in-vitro Stem Cell Expansion

The present invention relates to a method of stem cell expansion, and to the use of malonate, malonyl-CoA and derivatives or conjugates thereof in the treatment of neuropsychiatric disorders and conditions requiring hematopoietic stem cell transplantation. Background

Hematopoietic stem cell transplantation is used in the therapy of many conditions including hematological malignancies, solid tumors, immunodeficiencies and hematologic diseases like anemias. In autologous transplantation, hematopoietic stem cells (HSCs) are obtained from the patient prior to myoablative chemotherapy. The HSCs are then administered back to the patient to replace the destroyed bone marrow. In allogenic transplantation, the HSCs are obtained from a compatible healthy donor.

HSCs can be extracted from bone marrow, peripheral blood, amniotic fluid or umbilical cord blood, wherein extraction from peripheral blood is clinically the most relevant method. HSCs are collected from the blood through a process called apheresis. Prior to apheresis, HSCs need to be recruited from the donor's bone marrow into the peripheral circulation through treatment with Granulocyte-colony stimulation factor (G-CSF). This treatment is expensive and has potential side effects.

In vitro expansion of HSCs would make treatment with G-CSF less necessary and would thus lower the threshold to recruit healthy volunteers for HSC donations. Previous approaches to expand hematopoietic stem cells in vitro yielded inconsistent results, led to a loss of cellular potency, or used overexpression of genes/gene therapy approaches, which by itself has potential side effects. A safe, reliable method for in vitro expansion of HSCs would be highly desirable.

New neurons are generated throughout life in the mammalian hippocampus (Spalding et al., 2013; van Praag et al., 2002). This process, called adult neurogenesis, is critically involved in a variety of hippocampus-dependent forms of learning and memory. In addition, failing or altered neurogenesis has been associated with a number of neuropsychiatric diseases such as major depression, epilepsy, and cognitive aging, suggesting adult hippocampal neurogenesis is relevant for human health and disease (Christian et al., 2014; Kempermann et al., 2008; Scharfman and Hen, 2007).

The neurogenic capacity of adult neural stem/progenitor cells (NSPCs) depends on a balance between quiescent and proliferative states. Large screens have been performed (e.g., by Roche) to identify ways to enhance neurogenic capacity of NSPCs in the aging brain or in disease states where NSPC activity is reduced. In these studies, proliferating NSPCs have been used as a starting cell type. Given that the vast majority of NSPCs within the adult brain is quiescent (or even dormant), these screens do not recapitulate the in vivo situation. A safe, reliable method for in vivo activation and expansion of quiescent NSCPs would be highly desirable. Description

Malonyl-CoA (CAS No. 524-14-1 ) is the coenzyme A derivative of malonic acid (propanedioic acid).

According to a first aspect of the invention, a method of hematopoietic stem cell generation is provided, comprising the steps of

a. providing a hematopoietic stem cell isolate from a donor and

b. expansion of said hematopoietic stem cells in vitro in the presence of a compound selected from malonic acid, malonat conjugates, particularly malonyl-CoA, and a malonate salt.

In certain embodiments, the malonate salt is a sodium or disodium, potassium or dipotassium or mixed sodium potassium salt of malonic acid. Using the method of the invention, a relatively small amount of hematopoietic stem cells can be expanded in vitro to generate an amount sufficient for subsequent stem cell transplantation.

In certain embodiments, the compound is present in a concentration from 50 to 500 μΜ. In certain embodiments, the compound is present in a concentration from 100 to 200 μΜ. In certain embodiments, the compound is present in a concentration from 150 to 350 μΜ. In certain embodiments, the compound is present in a concentration from 100 to 400 μΜ. In certain embodiments, the compound is present in a concentration of approximately (+- 20 μΜ) of 100 μΜ, 150 μΜ, 200 μΜ, 250 μΜ or 300 μΜ.

In certain embodiments, the expansion is performed for 3 to 10 days. In certain embodiments, the expansion is performed to effect an approximate 5-fold expansion of the starting cell number.

In certain embodiments, the hematopoietic stem cell isolate is a peripheral blood sample.

In certain embodiments, the hematopoietic stem cell isolate comprises 500 to 4000 CD34+ stem cells per ml, particularly 500 to 2000 CD34+ stem cells per ml, more particularly approx. 1000 CD34+ stem cells per ml. This cell concentration is usually achieved if the donor has not been treated with factors that lead to the mobilization of stem cells from the bone marrow into the peripheral blood (HSC mobilizing factors). Starting from this cell concentration, the obtained stem cells can be expanded according to the first aspect of the invention to levels similar to those obtained from donors that have been treated with HSC mobilizing factors. The method according to the first aspect of the invention thus allows abstaining from treatment of donors with HSC mobilizing factors, while still obtaining sufficient stem cell numbers for transplantation. It is expected that this will increase the willingness of healthy individuals to agree with a peripheral blood stem cell donation.

In certain embodiments, the hematopoietic stem cell isolate comprises 4.000 to 40.000 CD34+ stem cells per ml, particularly 5.000 to 20.000 CD34+ stem cells per ml, more particularly approx. 10.000 to 20.000 CD34+ stem cells per ml. This cell concentration is usually achieved if the donor has been treated with HSC mobilizing factors. In some instances, the number of cells obtained from donors, even from donors treated with HSC mobilizing factors is not sufficient for stem cell transplantation, which requires approx. 2 x 10⁶ CD34+ stem cells per kg body weight. In these cases, several apheresis sessions are required. The method according to the first aspect of the invention thus allows to further expand the stem cell isolated from a donor, making several apheresis sessions unnecessary, while still obtaining sufficient stem cell numbers for transplantation. It is expected that this will increase the willingness of healthy individuals to agree with a peripheral blood stem cell donation.

In certain embodiments, the hematopoietic stem cell isolate is a peripheral blood sample comprising less than 10.000 CD34+ cells per ml, particularly less than 5.000 CD34+ cells per ml, more particularly less than 2.500 CD34+ cells per ml.

In certain embodiments, the hematopoietic stem cell isolate is a peripheral blood sample comprising less than 50.000 CD34+ cells per ml, particularly less than 20.000 CD34+ cells per ml, more particularly less than 10.000 CD34+ cells per ml.

According to a second aspect of the invention, malonyl-CoA, malonic acid or a malonate salt is provided for use in treatment / therapy of a condition requiring hematopoietic stem cell transplantation. The stem cell transplantation may be autologous or allogenic.

In certain embodiments of this aspect of the invention, the condition is selected from the group comprising hematological malignancies including leukemias, lymphomas and myelomas; solid tumors including neuroblastoma, Ewing sarcoma and choriocarcinoma; hematologic diseases including myelodysplastic syndromes, anemias and myeloproliferative disorders; amyloidoses; radiation poisoning; viral diseases including HTLV and HIV; lysosomal storage disorders; immunodeficiencies including ataxia teleangiectasia, DiGeorge syndrome, severe combined immunodeficiency and Wiskott-Aldrich syndrome.

Therapy of the abovementioned conditions includes chemotherapy or radiation therapy, leading to damage or ablation of the bone marrow. The patients rely on the transplantation of a sufficient amount of potent hematopoietic stem cells to rebuild their hematopoietic system. Thus, the method / use of malonyl-CoA or malonic acid or a malonate salt described herein is used in the context of a treatment or therapy of the above mentioned conditions, however the use is not directed towards a therapy of the underlying disease, but to the generation or expansion of the stem cells required to compensate the loss of cells during treatment.

In certain embodiments of this aspect of the invention, malonyl-CoA, malonic acid or a malonate salt is employed for in vitro expansion of hematopoietic stem cells obtained from a donor prior to transplantation.

According to a third aspect of the invention, malonyl-CoA, malonic acid or a malonate salt is provided for use in therapy or prevention of neuropsychiatric disorders including major depression, epilepsy, and cognitive aging.

The suitability of malonyl-CoA, malonic acid or a malonate salt for the abovementioned indications is demonstrated by the inventors in behavioral animal experiments.

According to another aspect of the invention, a dosage form is provided, comprising malonyl- CoA, malonic acid or a malonate salt for use in therapy or prevention of neuropsychiatric disorders.

According to yet another aspect of the invention, the use of malonyl-CoA, malonic acid or a malonate salt for stem cell expansion is provided.

In certain embodiments of this aspect of the invention, said stem cell is a hematopoietic stem cell and said expansion occurs in vitro.

The inventors present evidence that the regulation of lipid metabolism by malonyl-CoA affects HSC activity. Surprisingly, the inventors show that increasing levels of malonyl-CoA is instructive and sufficient to cause the expansion of murine and human HSCs.

In certain embodiments of this aspect of the invention, said stem cell is a neural stem/progenitor cell (NSPC) and said expansion occurs in vivo.

The inventors have used several complementary approaches to show that quiescent NSPCs in the embryonic and adult brain are in a distinct metabolic state that depends on high levels of fatty acid oxidation to maintain quiescence. This state is controlled by levels of malonyl-CoA, which in turn are regulated by the small regulatory protein Spot14. Surprisingly, the inventors show that manipulating levels of malonyl-CoA is instructive and sufficient to change quiescence behavior of NSPCs.

According to yet another aspect of the invention, a method of treating a condition requiring hematopoietic stem cell transplantation is provided, said method comprising the steps of a. obtaining hematopoietic stem cells from a donor;

b. expanding said hematopoietic stem cells in vitro using malonyl-CoA, , malonic acid or a malonate salt and c. administering said expanded hematopoietic stem cells to a patient in need thereof.

According to another aspect of the invention, a preparation of hematopoietic stem cells obtained by a method according to the first aspect of the invention is provided.

The preparation of hematopoietic stem cells according to this aspect of the invention is different from an untreated isolated preparation of hematopoietic stem cells with regard to distinct biochemical markers.

The preparation of hematopoietic stem cells according to this aspect of the invention is different from preparation of hematopoietic stem cells by previously described methods with regard to stem cell potency, wherein the inventive stem cell preparation exhibits an increased potency. The potency can be determined by (serial) transplantation assays in which the ability of the cells to reconstitute the previously ablated bone marrow stem cell population of a mouse is determined (reconstitution assay). The preparation of hematopoietic stem cells according to this aspect of the invention is different from preparation of hematopoietic stem cells by previously described methods with regard to genetic modifications of the cells. The cells of the inventive hematopoietic stem cell preparation do not comprise any genetic manipulation, in particular no genetic manipulation that results increased proliferative activity of the cells, such as c-myc overexpression.

Wherever alternatives for single separable features are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Brief description of the figures

Fig. 1 shows that the cellular composition of quiescent NSPCs is set to allow optimal FAO. (A) mRNA levels of the key FAO enzyme Carnitine palmitoyl transferase 1 a (Cpt1 a) are highly and reversibly upregulated in quiescent (quie) NSPCs compared to proliferating (prol) and formerly quiescent (ex quie) NSPCs (mean ± SEM). ^***p < 0.001 (One-way ANOVA followed by Holm- Sidak's multiple comparisons test, n = 3 per condition). (B) The increase in Cpt1 a mRNA levels is also reflected on protein levels, as revealed by Western blot analysis. The bar graph shows quantification of Cptl a levels normalized to β-actin in proliferating and quiescent NSPCs (mean ± SEM). ^***p < 0.001 (unpaired t-test, n = 3 per condition). (C) Co-stainings against Cpt1 a and a mitochondrial marker (Mitotracker) reveals the mitochondrial localization of Cpt1 a in quiescent NSPCs. Shown is a representative confocal image. (D) mRNA levels of the previously described novel quiescence marker Spot14 are highly upregulated in quiescent (quie) NSPCs compared to proliferating (prol) NSPCs. This upregulation is reversible, as formerly quiescent (ex quie) NSPCs show highly reduced levels of Spot14 mRNA, however levels are still higher than in prol NSPCs. This suggests that the BMP4-induced in vitro quiescence system indeed reflects features of in vivo NSPC quiescence (mean ± SEM). ^***p < 0.001 , ^**p < 0.01 (One-way ANOVA followed by Holm-Sidak's multiple comparisons test, n = 3 per condition). (E) The endogenous Cptl a inhibitor Malonyl-CoA is lowered in quiescent NSPCs compared to proliferating NSPCs, as measured by mass spectrometry analysis (mean ± SEM). ^**p < 0.01 (unpaired t-test, n = 3 per condition). Scale bar represents 20 μηι.

Fig. 2 shows that quiescent NSPCs use FAO for energy purposes and as a carbon source. (A) Radioactive FAO measurements using ¹⁴C-labelled palmitic acid revealed a significant increase in ¹⁴C02 in quiescent NSPCs compared to proliferating NSPCs, suggesting that at least part of the fatty acids are fully oxidized for energy purposes (mean ± SEM). ^***p < 0.001 (unpaired t-test, n = 3 per condition). (B) Scheme outlining the path of ¹³C-labeled palmitic acid upon FAO. The oxidation of fatty acids results in acetyl-CoA, which can be fed into the tricarboxylic acid cycle (TCA). The metabolites measured with mass spectrometry in a ^Reincorporation assay are shown in bold. (C) Quiescent NSPCs show an increase in ^Reincorporation in amino acids derived from TCA intermediates compared to proliferating NSPCs (mean ± SD). ^***p < 0.001 , (unpaired t-test, n = 3 per condition). (D) Quiescent NSPCs show an increase in RC-incorporation in tricarboxylic acid cycle (TCA) intermediates compared to proliferating NSPCs. These RC-incorporation assay results suggest that fatty acids are also used as an alternative carbon source in quiescent NSPCs (mean ± SD). ^***p < 0.001 , ^**p < 0.01 (unpaired t-test, n = 3 per condition).

Fig. 3 shows that manipulating Malonyl-CoA levels is sufficient to prevent NSPC quiescence and to trigger cell cycle re-entry under quiescence condition. (A) Schematic outline of the experimental setup. (B) Addition of malonyl-CoA (100 μΜ or 200 μΜ) at the beginning of quiescence induction is sufficient to prevent quiescence entry in a dose dependent manner, as revealed with the cell cycle marker Ki67 and the mitotic marker phospho Histone 3 (pH3). Shown are representative images of indicated doses and the quantification of cycling and proliferating cells after 3 days of quiescence induction (mean ± SEM). ^***p < 0.001 , ^**p < 0.01 (One-way ANOVA followed by Holm-Sidak's multiple comparisons test, n = 3 per condition). (C) Schematic outline of the experimental setup. (D) Replating NSPCs after fully established quiescence in quiescence medium containing malonyl-CoA (100 μΜ or 200 μΜ) is sufficient to trigger cell cycle re-entry. This suggests that malonyl-CoA levels can overrule the present quiescence cues. Shown are representative images of indicated doses and the quantification of cycling and proliferating cells after 3 days of quiescence induction (mean ± SEM). ^**p < 0.01 , ⁺p = 0.06 (One-way ANOVA followed by Holm-Sidak's multiple comparisons test, n = 3 per condition). Scale bar represents 50 μηι.

Fig. 4 shows that Malonyl-CoA increases numbers of long-term (LT)-and short-term(ST)-HSCs in vitro. (A) Treatment of LT-HSCs with different doses of Malonyl-CoA (5 μΜ or 50 μΜ) leads to an increase in cell number compared to control treated LT-HSCs (LiCI) after 7d of in vitro expansion. After culturing LT-HSCs in the presence of Malonyl-CoA, there is a significant increase in the number of small cells (5-15 μηη diameter), corresponding to the initial cell size after isolation and suggestive of symmetric expansion. ^*p<0.05 (One-way ANOVA followed by Holm-Sidak's multiple comparisons test, n = 3-4 wells per condition). Shown are representative phase contrast images and masks of LT-HSCs at d7 (left panel), the quantification of the increase in cell numbers from d1 to d7 (middle panel) and the quantification of the number of small cells at d7. (B) Treatment of ST-HSCs with different doses of Malonyl-CoA (5μΜ or 50μΜ) leads to an increase in cell number compared to control treated ST-HSCs (LiCI) after 7d of in vitro expansion. After culturing ST-HSCs in the presence of Malonyl-CoA, there is a significant increase in the number of small cells (5-15μηι diameter), corresponding to the initial cell size after isolation and suggestive of symmetric expansion. ^**p<0.01 (One-way ANOVA followed by Holm-Sidak's multiple comparisons test, n = 3-4 wells per condition). Shown are representative phase contrast images and masks of ST-HSCs at d7 (left panel), the quantification of the increase in cell numbers from d1 to d7 (middle panel) and the quantification of the number of small cells at d7.

Fig. 5 shows that exogenously applied malonyl-CoA is incorporated into new lipids and that increased proliferation upon malonyl-CoA exposure in quiescent NSPCs is at least partially regulated by an increase in FASN-dependent de novo lipogenesis. (A) Scheme of the experimental procedure to detect whether exogenously applied malonyl-CoA can be used by NSPCs. Radioactively labeled malonyl-CoA (14C-malonyl-CoA) was applied together with non-labelled malonyl-CoA (100μΜ) to proliferating NSPCs for 48h. Intracellular lipids were isolated, separated by thin layer chromatography and their radioactivity was measured by scintillation counts. (B) Both in the polar lipid fraction (containing the phospholipids) and in the neutral lipid fraction (containing triacylglycerides), significantly higher radioactivity (decay per minute, dpm) was detected in the samples incubated with 14C-malonyl-CoA, indicating that exogenously applied malonyl-CoA is taken up and integrated into newly synthesized lipids in NSPCs. (C) NSPCs were replated after fully established quiescence in quiescence medium containing either vehicle, the FASN inhibitor Xenical (Orlistat, 20 μΜ), malonyl-CoA (200 μΜ) or both Xenical and malonyl-CoA (20 μΜ and 200 μΜ). (D) The number of mitotic cells was analyzed 3 days later by pH3 staining. Inhibition of FASN significantly reduced the remaining small percentage of mitotic cells, whereas malonyl-CoA significantly increased proliferation. Xenical abolished the pro-proliferative effect of malonyl-CoA when applied together with malonyl-CoA. This suggests that increased proliferation of quiescent NSPCs is at least partially dependent on FASN-driven de novo lipogenesis. Shown are representative images of indicated doses (A) and the quantification of proliferating cells (B) (mean ± SEM). Scale bar represents 50μηι. ^***p < 0.001 , ^**p < 0.01 , ^* p < 0.05.

Fig. 6 shows a surface marker analysis of LKS cells expanded in the presence of malonyl- CoA. (A) depicts the experimental procedure, (B) shows the flow cytometry analysis of control and malonyl-CoA treated cells. The surface markers LKS (lin-, c-kit+, sca1 +) (left graph), CD150+/CD48- (middle graph) and CD34+/CD34- (right graph) were measured in both treatment groups. (C) depicts the absolute numbers of LKS, CD150+ and CD34- cells.

Fig. 7 shows the experimental setup for the primary LKS transplant.

Fig. 8 shows the outcome of the blood cell measurement in the time-course experiment of the primary LKS transplant. Chimerism is a measure of the origin of the blood cells. The higher the percentage indicated the more blood cells of this type originated from the indicated treatment group. (A) depicts the chimerism of all blood cells, (B) depicts the chimerism of the myeloid lineage of blood cells. (C) depicts the chimerism of the lymphoid lineage of blood cells. The chimerism of all blood cells and of the lymphoid lineage in the malonyl-CoA treated group differed significantly from the control group (no treatment).

Fig. 9 shows the experimental setup of the secondary LKS transplant.

Fig. 10 shows the outcome of the blood cell measurement in the time-course experiment of the secondary LKS transplant. Chimerism is a measure of the origin of the blood cells. The higher the percentage indicated the more blood cells of this type originated from the indicated treatment group. (A) depicts the chimerism of all blood cells, (B) depicts the chimerism of the myeloid lineage of blood cells. (C) depicts the chimerism of the lymphoid lineage of blood cells. The chimerism of the malonyl-CoA treated group tended to be higher than the control group, but the difference was not statistically different.

Examples

Quiescent NSPCs have high levels of FAO

To characterize FAO in quiescent NSPCs, the inventors analyzed the expression of Cptl a, a rate-limiting enzyme of FAO we found to be upregulated in quiescent NSPCs using unbiased proteomics. The inventors confirmed the upregulation of Cptl a using quantitative reverse transcriptase PCR and Western blot analyses, showing a substantial increase in the expression of Cptl a in quiescent compared to proliferating NSPCs (Figures 1A-B). In line with the radioactive FAO measurements, the increase in Cptl a expression in quiescent NSPCs was also reversible, as formerly quiescent NSPCs re-exposed to proliferation conditions had similar Cptl a levels as proliferating NSPCs (Figure 1A). As expected by the previously described role of Cptl a to shuttle fatty acids to mitochondria for FAO, the inventors found co- labeling of Cptl a with the mitochondrial dye Mitotracker in both quiescent and proliferating NSPCs (Figure 1 C). In addition, Cptl a was highly expressed in NSPCs compared to its neuronal progeny when directly isolated from the adult DG (3.5-fold upregulated in SOX2+ cells vs. DCX+ cells), as described previously (Bracko et al., 2012;). Collectively, these data indicate that in contrast to proliferating NSPCs that are highly lipogenic (Knobloch et al., 2013, Nature 493, 226-230), quiescent NSPCs strongly express Cptl a and show high levels of functional FAO.

High levels of FAO are required to sustain cellular quiescence

The inventors next aimed to understand the molecular mechanism underlying high levels of FAO in quiescent NSPCs. The inventors have previously shown that Spot14 is selectively expressed in quiescent NSPCs in vivo (Knobloch et al., 2013, Nature 493, 226-230; Knobloch et al., 2014, Stem Cell Reports 3, 735-742). In analogy, quantitative reverse transcriptase PCR showed more than 30-fold upregulation of Spot14 mRNA upon quiescence induction in vitro, again reversed upon re-exposure to proliferation conditions (Figure 1 D). Given that Spot14 negatively regulates malonyl-CoA levels we expected that high levels of Spot14 in quiescent NSPCs leads to low levels of malonyl-CoA. Indeed, the increase in Spot14 expression in quiescent NSPCs was associated with a substantial decrease in malonyl-CoA, as measured with mass spectrometry (Figure 1 E). The levels of acetyl-CoA were comparable between proliferating and quiescent NSCPs. Because malonyl-CoA is an endogenous inhibitor of Cptl a, its levels determine the rate of FAO (Houten and Wanders, 2010, Journal of inherited metabolic disease 33, 469-477). Thus, high levels of Spot14 accompanied by low levels of malonyl-CoA in quiescent NSPCs allows for optimal FAO. Furthermore, these data suggest that the BMP4-induced quiescence in vitro reflects features of in vivo NSPC quiescence (Knobloch et al., 2013, Nature 493, 226-230; Shin et al., 2015, Cell Stem Cell 17, 360-372).

Quiescent NSPCs use FAO for energy purposes and as a carbon source

Given the importance of FAO for NSPC behavior in vitro and in vivo, the inventors next sought to understand why NSPCs require FAO to maintain quiescence. We used two complementary approaches to analyze the fate of beta-oxidized fatty acids in NSPCs. First, using radioactively labeled ¹⁴C-palmitic acid, the inventors determined the amount of ¹⁴C02 that is produced upon full oxidation of fatty acids. This measurement serves as readout for the amount of energy produced by FAO. The inventors found significantly higher levels of ¹⁴C02 in quiescent NSPCs compared to proliferating NSPCs, suggesting that quiescent NSPCs use fatty acids as an alternative fuel source (Figure 2A). The inventors next used ¹³C-labeled palmitic acid in combination with mass spectrometry to trace the incorporation of the labeled carbon atoms in quiescent vs. proliferative NSPCs (Figure 2B). The inventors found highly significant increases in the incorporation of ¹³C into TCA intermediates as well as into proteins derived from TCA intermediates in quiescent NSPCs (Figure 2C-D). Taken together, these data indicate that quiescent NSPCs use FAO for energy purposes as well as an alternative carbon source for amino acid biosynthesis.

Malonyl-CoA levels regulate NSPC proliferation

After showing that adult NSPCs require high levels of FAO for quiescence, the inventors tested if manipulating FAO is sufficient to change quiescence behavior. We reasoned that elevating levels of malonyl-CoA might lead to a block of Cptl a-dependent FAO and provide sufficient substrate (together with acetyl-CoA) to fuel FASN-dependent de novo lipogenesis that is required for NSPC proliferation. The inventors first tested if exogenously applied malonyl-CoA could prevent BMP4-mediated induction of NSPC quiescence. Adult NSPCs were exposed to BMP4-containing medium in the presence of different concentrations of malonyl-CoA (Figure 3A). Indeed, elevated levels of malonyl-CoA dose-dependently prevented the induction of quiescence, as measured using the cell cycle markers Ki67 and phosphorylated histone H3 (phosphoH3) (Figure 3B). These data show that manipulating FAO through malonyl-CoA levels is sufficient to override BMP4-induced quiescence and keeps NSPCs in a proliferating state.

The inventors next analyzed if manipulation of FAO may be instructive for NSPC behavior. We induced BMP4-mediated quiescence over 3 days, after which proliferation is almost completely inhibited, followed by re-plating the cells in quiescence medium together with malonyl-CoA (Figure 3C). Strikingly, the inventors found that malonyl-CoA triggered NSPCs to enter into cell cycle in a dose-dependent manner, despite the continuous presence of quiescence cues and despite a fully established quiescence state. In contrast, control cells without malonyl-CoA remained largely quiescent (Figure 3D). These data reveal that differential metabolic states are not a mere consequence of a given stem cell state but that manipulating levels of FAO through malonyl-CoA is instructive to regulate the behavior of adult NSPCs.

Malonyl-CoA increases numbers of LT-and ST-HSCs in vitro

Given a report that FAO also plays an important role in HSCs (Ito and Suda, 2014, Nat Rev Mol Cell Biol 15, 243), the inventors next asked whether there is a more universal mechanism of stem cell regulation and tested whether treatment with malonyl-CoA can lead to expansion of HSCs in vitro. Mouse LT-HSCs and ST-HSCs were isolated and kept under basal conditions with or without malonyl-CoA. Analysis of cell size and number after seven days in vitro suggested that malonyl-CoA indeed lead to an increase in the number of cells, specifically of small size similar to the cells initially isolated, indicative of an expansion of HSCs (Fig. 4).

Surface marker analysis of malonyl-CoA induced HSC

The surface marker expression of malonyl-CoA induced HSCs was investigated. Therefore, LKS cells were isolated from CD45.1 donor mice and cultured in the presence of malonyl-CoA (1 ΟΟμΜ) for 7 days (Fig. 6 A). After 7 days the surface markers of ST-HSC (lin-, c-kit+, Seal +, CD48-, CD150+, CD34+) and LT-HSC (lin-, c-kit+, Sca1 +, CD48-, CD150+, CD34-) were analysed by flow cytometry (Fig. 6 B). The number of LKS cells, CD150+ and CD34- cells was significantly increased in the group of malonyl-CoA treated cells as compared to untreated controls (Fig. 6 C).

In vivo potency of malonyl-CoA induced HSC

Potency of the cells expanded according to the method of the present invention was tested in vivo, to assess if the method is associated with a loss in potency.

In a first step, LKS cells were isolated from CD45.1 donor mice and cultured in the presence of malonyl-CoA (100μΜ) for 7 days (Fig. 7). Cultured LKS cells (20.000 cells/mice) were combined with 350.000 total bone marrow cells from CD45.1/2 competitor mice and injected into irradiated CD45.2 recipient mice (850Gy, split dose). Blood samples were analysed for myeloid and lymphoid blood cells as well as total blood cell count in a time course experiment after 4, 8, 12, 16, 20 and 24 weeks (Fig. 8). Total blood cells and lymphoid blood cells were significantly elevated in the group of malonyl-CoA treated LKS cells (Fig. 8 A, C) whereas myeloid cells showed only a slight increase in the malonyl-CoA treated group (Fig. 8 B). This demonstrates the usability of the malonyl-CoA treated cells in a primary transplant in vivo.

In a second step, the longterm effect of malonyl-CoA treatment was evaluated in a secondary LKS transplant. Towards these ends total bone marrow cells of the CD45.2 recipient mice from the primary LKS transplant described above were isolated after the end of the time course experiment and injected into irradiated CD45.2 mice (850Gy, split dose) (Fig. 9). Blood samples were analysed for myeloid and lymphoid blood cells as well as total blood cell count in a time course experiment after 4, 8, 12, 16, 20 and 24 weeks (Fig. 10). Total blood cell count, as well as the myeloid and lymphoid blood cells were slightly elevated, although not statistically significantly, in the mice receiving bone marrow cells from the malonyl-CoA treated group as compared to the untreated control animals. This result demonstrates that the malonyl-CoA treated cells maintain their pluripotency in a longterm in vivo setting without any negative effects on stem cell exhaustion. Experimental procedures

Animals

Mice were kept with littermates under a 12h dark/light cycle in single ventilated cages and with ad libitum access to food and water. The Cpt1a-EGFP reporter mouse line (STOCK Tg(Cpt1 a- EGFP)IP41 Gsat/Mmucd) was generated by the Mutant Mouse Regional Resource Centers (MMRRC). Time-mated C57/BI6 female mice were obtained from Janvier Labs (France). All animal experiments were performed according to Swiss regulatory standards and approved by the Veterinary office of the Canton of Zurich.

Plasmids

Cptl a shRNA sequences were designed using the RNAi Consortium hairpin candidate sequences selection (www.broadinstitute.org/rnai/trc) against mouse Cptl a. The shRNA knockdown constructs (derived from Lentil_ox3.7) were cloned to express mCherry under the CMV promoter and shRNAs under the U6 promoter. Knockdown efficiency was tested in transfected mouse liver hepatoma cells.

Cell culture

Adult mouse DG NSPCs were obtained and cultured as previously described (Knobloch et al., 2013, Nature 493, 226-230; Ray and Gage, 2006, Molecular and cellular neurosciences 31 , 560-573). In brief, cells were kept as monolayers in DMEM/F12 medium supplemented with N2, FGF2, EGF and Heparin as well as an antibiotic/antimycotic. For all experiments involving quiescence induction and/or immunocytochemistry, cells were plated on laminin-coated glass coverslips/plastic dishes. All experiments were done with a minimum of 3 replicates per condition. Quiescence was induced as previously described (Martynoga et al., 2013, Genes & development 27, 1769-1786; Mira et al., 2010, Cell Stem Cell 7, 78-89). In brief, cells were kept as described above, but EGF was exchanged with recombinant mouse BMP4, which resulted in a significant drop in proliferation over the course of 3 days. Proliferating NSPCs were grown in parallel without BMP4. For Cptl a inhibition, various doses of Etomoxir or malonyl-CoA were added to the medium as outlined in the figures.

Time-lapse imaging and analysis

NSPCs were plated as described above. 4-6 adjacent areas per well were imaged every 4h on a heated and C02-controlled inverted microscope over the indicated time. Stitched phase contrast images were analysed using ImageJ. Several processing steps (bandpass filtering, Gaussian blurs, thresholding) were used to automatically analyze the area covered by cells. Proteomic analysis

Proteins were extracted from proliferating and quiescent NSPCs and samples were processed using a modified protocol by filter aided sample preparation (FASP) (Wisniewski et al., 2009, Nature methods 6, 359-362), followed by Solid Phase Extraction (SPE) C18 clean up. All data was acquired on an Orbitrap Fusion Tribrid mass spectrometer (Thermo Scientific), which was connected to an Easy-nLC 1000 HPLC system (Thermo Scientific). Raw files were processed with Progenesis Ql for proteomics (Nonlinear Dynamics). Mascot (2.4.1 ) was used for searching a target-decoy mouse database downloaded from uniprot (03/01/2015). Resulting Mascot dat-files were imported into Scaffold 4 (Proteome Software) and the false discovery rate (FDR) for peptides was set to 0.01 , for proteins to 0.05 respectively. The Scaffold Spectrum Report was re-imported into Progenesis and relative quantitation using non- conflicting peptides was performed. Gene names, p-values and normalized protein abundance were exported to Metacore 6.24 build 67895 (Thomson Reuters) for gene ontology analysis.

Radioactive FAO measurements

Radioactively labeled ³H-palmitic acid and ¹⁴C-palmitic acid were purchased from Perkin Elmer. FAO was assessed by the production and release of tritiated water or ¹⁴C02 as previously described (Djouadi et al., 2003, Mol Genet Metab 78, 1 12-1 18; Huynh et al., 2014, Methods in enzymology 542, 391 -405). In brief, proliferating and quiescent NSCPs were incubated with labeled palmitic acid for 2.5h or 4h and medium was collected for subsequent processing. The amount of radioactive ³H20 or ¹⁴C02 generated was measured by scintillation counting. All measurements were normalized to protein content.

In utero electroporation and tissue preparation

ln-utero electroporation of mouse embryos (embryonic day 13, E13) was carried out as described previously (Asami et al., 201 1 ). In brief the shRNA plasmid DNA (Cpt1 a-shRNA1 , Cpt1 a-shRNA2 or non-targeting shRNA) was targeted into the ventricular wall by electroporation and 24h later, transfected brains were processed for immunohistochemical analysis.

Immunostainings

Mice were transcardially perfused with 0.9% saline solution followed by 4% PFA solution. Brains were processed as previously described for immunohistology (Knobloch et al., 2013, Nature 493, 226-230). Staining was performed on 40 μηη thick free-floating sections. Primary antibodies were incubated overnight at 4 °C, followed by secondary antibody incubation for several hours at room temperature (RT). Cells were fixed with 4% PFA and antibody stainings were done overnight at 4 °C, secondary antibodies were incubated 2-4 h at RT. Antibody details are available in the supplemental information. Image acquisition and analysis

Images of cell and tissue stainings were acquired using epifluorescent and confocal microscopy. Images were analysed using Imaris and ImageJ, with customized macros for automated detection. Image acquisition and analysis were performed in a blinded manner. RT-PCR

RNA of proliferating, quiescent, replated proliferating and formerly quiescent NSPCs (grown as described above) was isolated and processed as described before (Knobloch et al., 2013, Nature 493, 226-230). Taqman probes against Cptl a, Spot14 and β-actin and RT-PCR master-mix were used according to the manufacturer's protocol. Real time PCR and data analyses were performed on an Applied Biosystems 7900HT System. Fold changes were calculated using the deltadelta Ct methods.

Western Blots

Protein lysates of proliferating and quiescent NSPCs were separated by SDS-PAGE electrophoresis followed by transfer to PVDF membrane. Membranes were incubated with primary antibodies overnight at 4°C, followed by HRP-conjugated secondary antibodies for several hours at RT. The signal was revealed by enhanced chemiluminescence substrate and quantification was done with ImageJ.

Malonyl-CoA and Acetyl-CoA measurements

Metabolites of proliferating and quiescent NSPCs were extracted with cold acetonitrile:methanol:water solvent (40:40:20). Extracts were dried, re-suspended in water and analyzed by LC-MS/MS on a Thermo Quantum Ultra instrument equipped with a Waters Acquity UPLC (Buescher et al., 2010, Anal Chem 82, 4403-4412). Data analysis was performed using own software written in Matlab (The Mathworks). Measured malonyl-and acetyl-CoA values were normalized to the average cell number.

C13-incorporation measurements

Proliferating and quiescent NSPCs were incubated for 24h with the corresponding medium containing 100 μΜ ¹³C-labelled palmitate (Cambridge Isotope Laboratories Inc.). Metabolites were extracted with 80% methanol. Samples were processed as described in the supplemental information and GC-MS analyses were performed using an Agilent 7890A GC equipped with a HP-5 ms 5% Phenyl Methyl Silox capillary column, interfaced with a triple quadruple tandem mass spectrometer (Agilent 7000B, Agilent Technologies). The GC-MS analyses were performed in Single Ion Monitoring (SIM) scanning for the isotopic pattern of metabolites.

HSC experiments Cells were isolated from hindlegs and hips of 12-week old BI6 mice according to a previously described protocol (Roch et al, Stem Cells 2015). In brief, bones were crushed in PBS-EDTA, red blood cells were lysed and lineage depletion was performed using a biotin-mouse lineage cocktail (BD Biosciences) combined with magnetic bead sorting (autoMACS, BD Biosciences). Cells were stained with Strep-PO (Life Technologies), cKit-PECY7 (BioLegend), Sca1 -APC (BioLegend), CD150 PECY5 (BioLegend), CD48-PB (BioLegend), CD34-FITC (eBioscience) and PI (Fluka). The LT-HSC population was defined as lineage depleted (lin neg), C-Kit+, Sca1 +, CD48-, CD150+, CD34-. The ST-HSC population was defined as lineage depleted (lin neg), C-Kit+, Sca1 +, CD48-, CD150+, CD34+. 80 cells per well and condition were sorted on a FACS Aria II flow cytometer (BD Biosciences) into round bottom well plates containing 200μΙ basal medium supplemented with Stemline II (Sigma-Aldrich), 100 ng/ml stem cell factor (SCF) and 2 ng/ml Flt-3 ligand as previously described (Roch et al, Stem Cells 2015). Cells were treated 24h after isolation with either 50μΜ Lithium chloride (LiCI, Sigma-Aldrich), or 5 μΜ or 50 μΜ Malonyl-CoA lithium salt (Sigma-Aldrich). Phase contrast images were taken with a 10x objective every day using an inverted microscope equipped with a heated and CO2 controlled chamber and analyzed with ImageJ. Circles were drawn manually over each cell and cell number and diameters were determined using the analyze particle function. d1 =24h after treatment (48h after isolation), d7=192h after treatment.

Statistical analyses

Statistical analysis was performed with the software Prism 6 (GraphPad). Unpaired t-tests, paired t-test and One-way-ANOVAs or Two-way-ANOVAs followed by Holm-Sidak's multiple comparisons tests were used as indicated in the figure legends. Significance levels were set at < 0.05.

Claims

1 . A method of hematopoietic stem cell generation comprising the steps of

a. providing a hematopoietic stem cell isolate obtained from a donor, particularly as a peripheral blood sample, and

b. expansion of said hematopoietic stem cells in vitro in in the presence of a compound selected from malonic acid, malonyl-CoA and a physiologically acceptable malonate salt, wherein the malonate salt is selected from a sodium or disodium, a potassium or dipotassium or a mixed sodium potassium salt of malonic acid.

2. The method according to claim 1 , wherein said compound is present in a concentration from 50 to 500 μΜ.

3. The method according to any one of the above claims, wherein said expansion is performed for 3 to 10 days.

4. The method according to any one of the above claims, wherein the expansion is performed to effect a 4- to 10-fold expansion of the starting cell number, in particular an approximate 5-fold expansion of the starting cell number.

5. The method according to any one of the above claims, wherein said hematopoietic stem cell isolate comprises 500 to 4000 CD34+ stem cells per ml, particularly 500 to 2000 CD34+ stem cells per ml, more particularly approx. 1000 CD34+ stem cells per ml.

6. The method according to any one of the above claims, wherein said hematopoietic stem cell isolate comprises less than 50.000 CD34+ cells per ml, particularly less than 20.000 CD34+ cells per ml, more particularly less than 10.000 CD34+ cells per ml

7. Malonyl-CoA, malonic acid or a malonate salt, wherein the malonate salt is selected from a sodium or disodium, a potassium or dipotassium or a mixed sodium potassium salt of malonic acid, for use in therapy of a condition requiring hematopoietic stem cell transplantation.

8. Malonyl-CoA, malonic acid or a malonate salt for use in therapy of a condition requiring hematopoietic stem cell transplantation according to claim 7, wherein said condition is selected from the group comprising hematological malignancies including leukemias, lymphomas and myelomas; solid tumors including neuroblastoma, Ewing sarcoma and choriocarcinoma; hematologic diseases including myelodysplastic syndromes, anemias and myeloproliferative disorders; amyloidoses; radiation poisoning; viral diseases including HTLV and HIV; lysosomal storage disorders; immunodeficiencies including ataxia teleangiectasia, DiGeorge syndrome, severe combined immunodeficiency and Wiskott-Aldrich syndrome.

9. Malonyl-CoA, malonic acid or a malonate salt for use in therapy of a condition requiring hematopoietic stem cell transplantation according to any one of claims 7 or 8, wherein said malonyl-CoA, malonic acid or malonate salt is employed for in vitro expansion of hematopoietic stem cells obtained from a donor prior to transplantation.

10. Malonyl-CoA, malonic acid or a malonate salt, wherein the malonate salt is selected from sodium or disodium, potassium or dipotassium or mixed sodium potassium salt of malonic acid, for use in therapy or prevention of neuropsychiatric disorders including major depression, epilepsy, and cognitive aging.

1 1 . A dosage form comprising malonyl-CoA, malonic acid or a malonate salt, wherein the malonate salt is selected from a sodium or disodium, a potassium or dipotassium or a mixed sodium potassium salt of malonic acid, for use in therapy or prevention of neuropsychiatric disorders.

12. Use of malonyl-CoA, malonic acid or a malonate salt, wherein the malonate salt is selected from a sodium or disodium, a potassium or dipotassium or a mixed sodium potassium salt of malonic acid, for stem cell expansion.

13. The use of malonyl-CoA, malonic acid or a malonate salt according to claim 12, wherein said stem cell is a hematopoietic stem cell and said expansion occurs in vitro.

14. The use of malonyl-CoA, malonic acid or a malonate salt according to claim 12, wherein said stem cell is a neural stem/progenitor cell and said expansion occurs in vivo.

15. A preparation of hematopoietic stem cells obtained by a method according to any one of claims 1 to 6.