scholarly journals Histone Deacetylase Inhibitors As Enhancers of Human Hematopoietic Stem Cell Activity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5044-5044
Author(s):  
Manja Wobus ◽  
Guruchandar Arulmozhivarman ◽  
Martin Kraeter ◽  
Jens Friedrichs ◽  
Martin Stoeter ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Hdac Inhibitors ◽  
Cell Activity ◽  
Adhesion Assay ◽  
Hematopoietic Stem ◽  
Notch 3 ◽  
Human Cd34 ◽  
Stem Cell Activity

Abstract Introduction The identification of compounds which increase the number but also keep or enhance the activity of hematopoietic stem and progenitor cells (HSPCs) could improve the clinical outcome after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). So far, most attempts to increase HSPC numbers ex vivo have been unsuccessful because of either inadequate cell numbers and/or loss of engraftment capacity and HSPC quality during expansion. Executing drug discovery screens in vertebrate systems is generally expensive, technically challenging and time consuming. Therefore, the zebrafish represents a versatile vertebrate model allowing HSPC regulation and development studies during embryogenesis and adulthood. Methods We used a semi-automated chemical screen to identify modulators of HSPC activity by transgenic (cmyb:EGFP) zebrafish embryos. Verification of identified histone deacetylase (HDAC) inhibitor candidates was carried out in vitro using human CD34+ HSPCs which were isolated from apharesis samples of healthy donors after mobilization with G-CSF by anti-CD34 coupled magnetic beads. The influence of HDAC inhibitors on HSPC phenotype, gene expression pattern as well as adhesion and migration capacity was analyzed after 5 days of treatment either in single or in co-culture with bone marrow-derived mesenchymal stromal cells (MSCs). Results The HDAC inhibitors valproic acid (VPA), resminostat and entinostat were shown to significantly amplify the number of hematopoietic precursors in a chemical in vivo zebrafish embryo screen (Arulmozhivarman et al. 2016). Treatment of human CD34+ HSPCs with these compounds in vitro resulted in a significantly increased percentage of CD34+CD90+ cells up to 60% compared to controls which showed only 2% of double positive cells as well as in 3-fold higher CD34+ and about 12-fold higher CD34+CD90+ absolute cell numbers. CD34 is a well-known surface marker for human immature HSPCs and in combination with CD90 it defines a potentially pluripotent subpopulation. In a co-culture setting, we found that VPA treated cells showed 2 to 3-fold higher attachment capacity on MSCs compared to the control cells. This finding led us to quantify the adhesive capacity of cells using static adhesion assay and atomic force microscopy based single-cell force spectroscopy (AFM-SCFS). Interestingly, detachment forces of VPA treated HSPCs were 3 times increased on MSCs compared to control cells and a similar phenotype was observed by static adhesion assay. Accordingly, the chemokine-mediated migration of VPA treated HSPCs towards SDF-1/CXCL12 was inhibited. To reveal underlying downstream molecules and mechanisms mediating the modified cellular characteristics, a whole genome expression array was carried out for HSPCs treated with VPA in comparison to untreated controls. Amongst a panel of regulated genes, the melanoma cell adhesion molecule (MCAM/CD146), Notch 3 and its downstream effector Hes-1 as well as the SDF-1 receptor CXCR-4 were found to be significantly changed. Whereas the decreased expression of CXCR4 correlates with the inhibited migration potential of VPA-treated HSPCs and Notch-3/Hes-1 have a known role in normal and malignant hematopoiesis (Gu et al. 2016), the induced expression of MCAM on HSPCs was not described so far. The result was confirmed by flow cytometry which revealed a 40% MCAM-positive cell population when treated with VPA, whereas the control showed only negative cells. Additionally, significant higher transcript levels were detected for MCAM by quantitative real-time PCR in VPA expanded cells. Recently, we described a role of MCAM in MSCs for the hematopoietic support (Stopp et al. 2013). The inducible expression in HSPCs may reflect homotypic interactions which preserve a more immature subpopulation with high stem cell activity. Conclusion We describe for the first time the ability of the HDAC inhibitors VPA, resminostat and entinostat to efficiently expand CD34+ HSPCs ex vivo especially supporting a CD34+CD90+ subpopulation with potentially high stem cell activity. Moreover, a potential role of MCAM in this context may offer new perspectives of the HSPC expansion ex vivo for the improvement of HSCT. Disclosures No relevant conflicts of interest to declare.

Ciclopirox Ethanolamine Is a Novel Modifier of Human Hematopoietic Stem Cell Ex Vivo Expansion

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 36-36
Author(s):  
Mehrnaz Safaee Talkhoncheh ◽  
Fredrik Ek ◽  
Aurelie Baudet ◽  
Christine Karlsson ◽  
Roger Olsson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Cell Activity ◽  
Cell Number ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Cells Cultured

Abstract Despite extensive studies over the last decades, little is known about the mechanisms governing human hematopoietic stem cell (HSC) fate decisions. In particular, it has been challenging to define culture conditions in which HSCs can be expanded for clinical benefit. Application of small molecule screening to modulate stem cells has emerged as a useful tool for identification of new compounds with ability to expand hematopoietic stem and progenitor cells (HSPCs). Such screens have mainly relied on the expression of CD34 as predictor of stem cell activity in cultured cells. However, CD34 defines a broad repertoire of progenitor cells and does not define stem cell function. We found that the long-term repopulation potential of cultured human HSPCs is exclusively contained within a discrete cell population co-expressing CD34 and CD90, while the vast majority of progenitor cells are found in the CD34+CD90- population. Tracking the CD34+ CD90+ population is therefore a sensitive and specific tool to predict stem cell activity in cultured hematopoietic cells and provides a good basis for a screen aimed at discovering modifiers of stem cell expansion. To search broadly for novel and potential modifiers of ex vivo HSCs expansion we next developed and optimized a small molecule screen in human cord blood (CB) derived CD34+ cells. We screened >500 small molecules from 8 different annotated chemical libraries for the phenotypic expansion of CD34+ CD90+ cells following a 6-day culture in serum-free medium supplemented with stem cell factor (SCF), thrombopoietin (TPO) and fms-like tyrosine kinase 3 ligand (FL). The numbers of CD34+ CD90+ cells for each molecule, tested at two different concentrations, was compared to DMSO treated controls. Following the initial screen, several candidate hits were selected and subjected to a dose response validation experiment from which we selected four top candidate molecules. Two of these molecules were histone deacetylase (HDAC) inhibitors, which recently have been reported to facilitate expansion of CB derived HSCs. One of the top candidates, Ciclopirox ethanolamine (CE), had previously not been implicated in HSC expansion. Ciclopirox ethanolamine is known as an antifungal agent and iron chelator. It has further been shown to suppress cancer cell survival through inhibition of Wnt/beta catenin signaling. We found that CB cells cultured with CE had a 4-fold increase in CD34+90+ cell number compared to DMSO treated controls following 6 days of culture. Interestingly, the total cell count was not different, suggesting a specific increase in CD34+ CD90+ cell number rather than an overall higher proliferation rate. When plated in methylcellulose, CE cultured cells generated increased numbers of myeloid colonies. Moreover, CE treated cells gave rise to multilineage colonies (CFU-GEMM) that could not be detected from the control cultures. To further test the functional capacity of cells cultured with CE, we transplanted cultured equivalents of 30,000 CB CD34+ cells (cultured with or without CE) into sub lethally irradiated NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Human hematopoietic reconstitution in peripheral blood was determined 16 weeks later. Mice transplanted with CE cultured cells showed higher human CD45 engraftment 16 weeks post transplant compared to control cells (33.2±6.7% vs 14.6±5% p=0.04). The engrafted cells contributed to both myeloid and lymphoid lineages. This shows that Ciclopirox ethanolamine enhances the long-term engraftment capacity of ex vivo cultured HSCs and suggests that it should be considered in stem cell expansion protocols, either alone or in combination with other molecules. We are currently addressing the basis for the increased stem cell activity mediated by Ciclopirox ethanolamine using parameters for differentiation, cell cycling and apoptosis. In addition, we are comparing Ciclopirox ethanolamine with other recently defined modifiers of HSC expansion. Disclosures No relevant conflicts of interest to declare.

scholarly journals In Vitro Maintenance of Highly Purified, Transplantable Hematopoietic Stem Cells

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4337-4347 ◽  
Author(s):  
Kateri A. Moore ◽  
Hideo Ema ◽  
Ihor R. Lemischka
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Lines ◽  
Progenitor Cell ◽  
Stromal Cell ◽  
Fetal Liver ◽  
Cell Activity ◽  
Hematopoietic Stem ◽  
Stem Cell Activity

Abstract The cellular and molecular mechanisms that regulate the most primitive hematopoietic stem cell are not well understood. We have undertaken a systematic dissection of the complex hematopoietic microenvironment to define some of these mechanisms. An extensive panel of immortalized stromal cell lines from murine fetal liver were established and characterized. Collectively, these cell lines display extensive heterogeneity in their in vitro hematopoietic supportive capacity. In the current studies, we describe a long-term in vitro culture system using a single stromal cell clone (AFT024) that qualitatively and quantitatively supports transplantable stem cell activity present in highly purified populations. We show multilineage reconstitution in mice that received the equivalent of as few as 100 purified bone marrow and fetal liver stem cells cultured for 4 to 7 weeks on AFT024. The cultured stem cells meet all functional criteria currently ascribed to the most primitive stem cell population. The levels of stem cell activity present after 5 weeks of coculture with AFT024 far exceed those present in short-term cytokine-supported cultures. In addition, maintenance of input levels of transplantable stem cell activity is accompanied by expansion of other classes of stem/progenitor cells. This suggests that the stem/progenitor cell population is actively proliferating in culture and that the AFT024 cell line provides a milieu that stimulates progenitor cell proliferation while maintaining in vivo repopulating activity.

Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1587-1587 ◽  
Author(s):  
Olivier Herault ◽  
Kristin J Hope ◽  
Eric Deneault ◽  
Matthias Trost ◽  
Nadine Mayotte ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Insertional Mutagenesis ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Cell Activity ◽  
Relative Reduction ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Abstract 1587 Although important efforts have been invested in the discovery of genes that regulate normal or leukemic hematopoietic stem cells (HSC) self-renewal, the number of validated candidates remains low, due largely to the unavailability of functionally pure stem cell populations. Moreover, it is often difficult to identify the normal counterpart cell from which leukemia originated, further complicating studies based on comparative gene expression. In this study, we used a series of recently characterized Hoxa9 + Meis1 acute myeloid leukemias (AML) derived from fetal liver (FL) cells (Wilhelm BT et al., submitted). These leukemias are remarkably similar in several aspects including their L-HSC frequency (between ∼1 in 100 to 350) except for one leukemia (FLA2) in which 70% of the cells show repopulation ability (i.e., L-HSC). We reasoned that comparative mRNA profiling of FLA2 to the phenotypically similar FLB1 (0.3% L-HSC) might identify genes uniquely associated with L-HSC self-renewal. We observed a 2–3-fold upregulation of Gpx3 in FLA2, which was confirmed by qRT-PCR. In accordance with this, all 14 of the tested Gpx3 promoter region CpG sequences were methylated in FLB1 and hypomethylated in FLA2 cells. The higher expression of GPx3 in FLA2 was confirmed at the protein level and reflected in elevated glutathione peroxidase activity in comparison to FLB1. Importantly, we also observed in FLA2 a relative reduction in reactive oxygen species (ROS) level (DCFDA) and a concomitant decrease in p38 MAPK activation (western blot and mass spectrometry). The correlation of Gpx3 levels with L-HSC frequency could be reflective of their functional dependence on this enzyme. FLA2 cells being difficult to manipulate ex vivo, to address this we utilized retroviruses encoding shRNAs and a GFP reporter to explore the in vivo function of FLA2 cells with downregulated Gpx3. The decrease in percentage of GFP+ donor cells when leukemia became apparent (∼19 days) from that of populations initially transplanted, was 4-fold higher following Gpx3 knockdown in comparison to shLuciferase transduction. Moreover, those shGpx3 infected FLA2 remaining at day 19 displayed a 3-fold decrease in GFP mean fluorescence intensity relative to their control counterparts. These results show that GFPhigh cells were selectively depleted, and suggest that Gpx3 is critical for the competitiveness of L-HSCs. Because redox metabolism has been implicated in HSC self-renewal, we also analyzed its expression and function in normal HSC to gain further insight into the role of GPx3 in stem cell activity. Interestingly, compared to FL-HSCs, isolated 3 and 4 week bone marrow (BM), HSCs exhibited a 39- and 6-fold decrease in Gpx3 expression, respectively. A correlation of Gpx3 levels with enhanced self-renewal was also observed in vitro as overexpression of several nuclear determinants of HSC expansion such as Hoxb4, NA10HD, Klf10 and Prdm16 promoted Gpx3 expression by 3.2 to 19.2-fold. We next infected BM cells enriched for HSCs with retroviruses carrying shRNAs to Gpx3. shRNA targeting of Gpx3 dramatically inhibited hematopoietic reconstitution. Transplantations of sublethally irradiated recipients indicated that Gpx3 knockdown significantly impaired both early and late donor-derived hematopoiesis. These results suggest that GPx3 is critical for repopulation mediated by both short and long-term repopulating cells. In reciprocal gain-of-function experiments, Lin-CD150+CD48- cells engineered to overexpress Gpx3, showed a marked competitive advantage over controls when transplanted following a 7-day ex vivo culture step. Insertional mutagenesis was ruled out as proviral integration analyses of six recipients confirmed polyclonal hematopoiesis. Moreover, some mice were in part reconstituted by the same clones, indicating that self-renewal occurred in vitro prior to transplantation. Phenotypic analysis of late-transplant hematopoietic tissues showed that Gpx3-transduced cells contributed to lymphoid and myeloid repopulation, confirming their multipotentiality. Together, these results indicate that Gpx3 enhances HSC expansion ex vivo possibly through modulation of self-renewal activity. In conclusion, a unique model of primary L-HSC was exploited to identify Gpx3 as a critical determinant for the competitiveness of L-HSCs and complementary experiments demonstrated a key role for this gene in normal HSC self-renewal. Disclosures: No relevant conflicts of interest to declare.

A Forward RNAi Screen Identifies p38 MAP Kinase As a Druggable Target for Expansion of Human Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2352-2352
Author(s):  
Aurelie Baudet ◽  
Jonas Larsson
Keyword(s):  
Stem Cell ◽  
Next Generation Sequencing ◽  
Ex Vivo ◽  
Cell Activity ◽  
P38 Map Kinase ◽  
Next Generation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem Cell Activity ◽  
Generation Sequencing

Abstract Abstract 2352 While the mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood, intense efforts are being devoted to develop conditions that would enable ex vivo amplification of transplantable stem cells. We have developed a screening strategy to assess modulators of human HSCPs using pooled lentiviral shRNA libraries transduced into cord blood-derived stem/progenitor cells. We use the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs leading to prolonged maintenance or expansion of undifferentiated HSPCs. This approach enables complex, pooled screens in large numbers of cells. We further take advantage of next generation sequencing to track shRNA-transduced cells with high fidelity, allowing thousands of perturbations to be tested in parallel in a single pool of cells. Here we used a library of 2500 shRNAs targeting around 800 genes, mainly kinases and phosphatases, which include large numbers of “druggable” genes. The shRNAs composing the library were monitored by next generation sequencing in cord blood CD34+ cells sampled one day after transduction and following 20 days of culture, to determine their relative change in distribution during the culture period. The sequencing of all integrated proviruses containing shRNAs generated over 3 million sequences per sample. Analysis of the shRNA distribution before and after culture in 3 replicate screens revealed a dramatic enrichment of 3 independent shRNAs targeting MAPK14 (p38α). We could confirm that inhibition of MAPK14, mediated by RNA interference, leads to a proliferation advantage of CD34+ cells in culture, identifying p38 as a possible target for ex vivo stem cell expansion. We next used the chemical inhibitor SB203580 to inhibit p38 without genetic perturbation and in a non-permanent fashion. Culture of CD34+ cells under optimized conditions for expansion (serum-free medium supplemented with SCF, TPO and FLT3) with or without SB203580 showed a 3-fold increase of the stem cell enriched CD34+CD90+ cell population during 5 days of culture in SB203580 treated cells compared to control cells. Furthermore, when transplanted to immune-deficient NSG mice, SB203580 treated cells showed a dramatic increase in repopulating activity, as evidenced by the percentage of human engraftment 10 weeks after transplantation (SB203580: 30±6.4% vs control: 7.5±3.6%, p< 0,001). Thus, under otherwise optimized culture conditions for stem cell expansion, the addition of the p38 inhibitor leads to a significant increase in stem cell activity. To understand the basis for the increase in stem cell activity, we assayed SB203580 treated cells with respect to cell cycling and survival rate, but found a similar cell division history (shown by cytoplasmic dye dilution assays) and similar levels of apoptotic cells (shown by Annexin V staining) compared to control cells. Interestingly, however, when the cells were assayed for reactive oxygen species (ROS), we detected significantly reduced levels of ROS in SB203580 treated cells, implicating modulation of ROS as a possible mechanism behind the enhanced stem cell output. Taken together, using a functional forward genetic screen, we have been able to identify p38 MAP kinase as a highly promising target to enhance hematopoietic stem cell activity in ex vivo expansion settings. These results further support the feasibility of pooled RNAi screens in conjunction with next generation sequencing to identify genes and pathways that regulate primary human stem cell populations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RNAi screen identifies MAPK14 as a druggable suppressor of human hematopoietic stem cell expansion

Blood ◽  
2012 ◽  
Vol 119 (26) ◽  
pp. 6255-6258 ◽  
Author(s):  
Aurélie Baudet ◽  
Christine Karlsson ◽  
Mehrnaz Safaee Talkhoncheh ◽  
Roman Galeev ◽  
Mattias Magnusson ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ex Vivo ◽  
Cell Activity ◽  
Rnai Screen ◽  
Hematopoietic Stem ◽  
Stem Cell Expansion ◽  
Stem Cell Activity ◽  
Stem And Progenitor Cells ◽  
Hematopoietic Stem Cell Expansion ◽  
Pharmacologic Inhibition

We report on a forward RNAi screen in primary human hematopoietic stem and progenitor cells, using pooled lentiviral shRNA libraries deconvoluted by next generation sequencing. We identify MAPK14/p38α as a modulator of ex vivo stem cell proliferation and show that pharmacologic inhibition of p38 dramatically enhances the stem cell activity of cultured umbilical cord blood derived hematopoietic cells. p38 inhibitors should thus be considered in strategies aiming at expanding stem cells for clinical benefit.

Ex Vivo Expansion of Human SCID-Repopulating Cells Using Recombinant TAT-HOXB4 Protein.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3159-3159 ◽  
Author(s):  
Gorazd Krosl ◽  
Marie-Pier Giard ◽  
Jana Krosl ◽  
Stephanie Beauchemin ◽  
Keith Humphries ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Ex Vivo ◽  
Day Treatment ◽  
Fold Increase ◽  
Limited Information ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Short Term Exposure

Abstract One of the major obstacles to the successful clinical application of hematopoietic stem cell (HSC) transplantation, particularly in the context of related haplotype-mismatched transplantation, unrelated cord blood transplants for adults, and grafts that are processed ex vivo to remove malignant, or alloreactive T cells, is the number of available long-term repopulating HSCs. The addition of soluble recombinant TAT-HOXB4 protein was recently reported to enable rapid in vitro expansion of murine HSCs that retain their in vivo proliferation and differentiation capacity. However, the ability of this recombinant protein to effectively expand human hematopoietic stem cells remains hypothetical. In addition, limited information is available on underlying mechanisms of HOXB4 HSC expansion. First, to determine the capacity of recombinant TAT-HOXB4 protein to promote human HSC expansion, we treated human CD34+ cells for 4 and 8 days with 40 nM, or 80 nM TAT-HOXB4 protein in X-Vivo 15 medium supplemented with Stem Cell Factor, TPO, IL-6 and Flt3-ligand. Cultures exposed to TAT-HOXB4 treatment for 8 days had no pronounced effect on the total cell yield. During this period, a 2-fold net loss of CFU-GEMM was observed for controls, in comparison to ~8-fold and ~5-fold expansions in response to 40 nM and 80 nM TAT-HOXB4 (p&lt;0.05), respectively. Recombinant TAT-HOXB4 also induced ~10–15-fold expansion of large CFU-GM, compared to only ~2.5-fold increase for controls (p&lt;0.05). HSC numbers were enumerated at the beginning and at the end of the treatment using the principle of limiting dilution in a 4-month NOD/SCID repopulation assay. Culture for 8 days in cytokines devoid of TAT-HOXB4 resulted in ~2-fold loss of SCID Repopulating Cells (SRCs), while cultures supplemented with 40 and 80 nM TAT-HOXB4 protein showed a 2.5-fold (95% CI 1.7 – 3.3 fold) and 5.5-fold (95% CI 3.6 – 7.4 fold) increase, respectively. Then, to determine whether the increase in HSC numbers resulted from HOXB4-enhanced proliferation of HSCs, we examined the cell cycle profile of control and HOXB4-treated cell populations using Hoechst 33342 and pyronin Y dyes. After 4-day treatment with 80 nM TAT-HOXB4, 44%±12% of CD34+CD38+ cells were in Go, compared to only 19%±6% of the controls (p&lt;0.05). In contrast, similar proportions (89–91%) of quiescent CD34+CD38− cells were observed for both conditions. Tracking cell divisions using CFSE also showed that during this period, HOXB4-treated CD34+CD38+ cells underwent ~2 population doublings less than controls (p&lt;0.05). In conclusion, short-term exposure of human CD34+ populations to recombinant TAT-HOXB4 protein has the potential to achieve clinically relevant HSC expansion levels. At the HSC level, these observations suggest that TAT-HOXB4 preferentially affects cell fate (self-renewal?) rather than cell proliferation.

A Functional Screen to Identify Novel Effectors of Hematopoietic Stem Cell Activity

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2459-2459
Author(s):  
Eric Deneault ◽  
Sonia Cellot ◽  
Amélie Faubert ◽  
Jean-Philippe Laverdure ◽  
Mélanie Fréchette ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Functional Screen ◽  
Nuclear Factors ◽  
Stem Cell Activity

Abstract The maintenance of blood homeostasis depends on hematopoietic stem cells (HSCs), which rely on two critical properties, namely multipotency and self-renewal. The former enables differentiation into multiple lineages, the latter ensures preservation of fate upon cellular division. By definition, a self-renewal division implies that a HSC is permissive to cell cycle entry, while restrained from engaging in differentiation, apoptosis or senescence pathways. Despite the tremendous progress made towards the identification of the molecular circuitry that governs ESC fate, genes controlling this process in adult HSCs have proven more difficult to unmask. This is principally due to our inability to maintain or expand HSC ex vivo as homogenous populations, to the absence of a stringent surrogate marker to follow the HSC multipotent state and to changes in cell phenotype observed shortly upon facing the selective pressures of in vitro culture conditions, impeding HSC tracking in this context. We now report the results of a novel in vitro to in vivo functional screen, which identified a series of nuclear factors that induced high levels of HSC activity similar to that previously achieved with Hoxb4. We created a database consisting of 689 nuclear factors considered as potential candidate regulators of HSC activity. This list was mostly derived from microarray gene expression profiling of normal and leukemia stem cells including our recently generated FLA2 leukemia (1 in 1.5 cells are leukemia stem cells, G.S. et coll., in preparation). It was also enriched by genes obtained following a review of the literature on stem cell self-renewal. Genes in this database were next ranked from 1 (lowest priority) to 10 (highest priority) based on 3 factors: differential expression between primitive and more mature cellular fractions (e.g., LT-HSC-enriched: 3 points), expression levels (high, highest priority: max 3 points) and the consistency of findings between datasets (max 4 points). Genes with a score of 6 and above (n=139) were selected for functional studies, of which 104 were tested in HSCs, using a high-throughput overexpression in vitro to in vivo assay tailored to circumvent current limitations imposed by the biology of HSCs. In total, 18 new determinants have emerged, 11 of which act in a cell autonomous manner, namely Ski, Smarcc1, Vps72, Trim27, Sox4, Klf10, Prdm16, Erdr1, Cnbp, Xbp1 and Hnrpdl, while the remaining provide a non-autonomous influence on HSC activity, i.e, Fos, Hmgb1, Tcfec, Sfpi1, Zfp472, Hdac1 and Pml. Clonal and phenotypic analyses of hematopoietic tissues derived from selected recipients confirmed that the majority of these factors induced HSC expansion in vitro without perturbing their differentiation in vivo. Epistatic analyses further reveals that 3 of the most potent candidates, namely Ski, Prdm16 and Klf10 may exploit both mechanisms, i.e., cell and non-cell autonomous. The utilization of this novel screening method together with the creation of a database enriched for potential determinants and candidate regulators of adult stem cell activity can now be exploited to devise regulatory networks in these cells.

scholarly journals In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells

Cell Reports ◽  
2016 ◽  
Vol 17 (10) ◽  
pp. 2789-2804 ◽  
Author(s):  
Yukiko Ishikura ◽  
Yukihiro Yabuta ◽  
Hiroshi Ohta ◽  
Katsuhiko Hayashi ◽  
Tomonori Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Cell Activity ◽  
Spermatogonial Stem Cell ◽  
Stem Cell Activity

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

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