Human Growth Factor–Enhanced Regeneration of Transplantable Human Hematopoietic Stem Cells in Nonobese Diabetic/Severe Combined Immunodeficient Mice

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 481-487 ◽  
Author(s):  
Johanne D. Cashman ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Human Growth ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Nonobese Diabetic

Abstract Self-renewal is considered to be the essential defining property of a stem cell. Retroviral marking, in vitro amplification, and serial transplantation of human cells that can sustain long-term lymphomyelopoiesis in vivo have provided evidence that human hematopoietic stem cell self-renewal occurs both in vitro and in vivo. To investigate whether this process can be manipulated by cytokines, we administered two different combinations of human growth factors to sublethally irradiated nonobese diabetic/severe combined immunodeficient (SCID) mice transplanted with 107 light-density human cord blood cells and then performed secondary transplants to compare the number of transplantable human lymphomyeloid reconstituting cells present 4 to 6 weeks post-transplant. A 2-week course of Steel factor + interleukin (IL)-3 + granulocyte-macrophage colony-stimulating factor + erythropoietin (3 times per week just before sacrifice) specifically and significantly enhanced the numbers of transplantable human lymphomyeloid stem cells detectable in the primary mice (by a factor of 10). Steel factor + Flt3-ligand + IL-6 (using either the same schedule or administered daily until sacrifice 4 weeks post-transplant) gave a threefold enhancement of this population. These effects were obtained at a time when the regenerating human progenitor populations in such primary mice are known to be maximally cycling even in the absence of growth factor administration suggesting that the underlying mechanism may reflect an ability of these growth factors to alter the probability of differentiation of stem cells stimulated to proliferate in vivo.

Human Growth Factor–Enhanced Regeneration of Transplantable Human Hematopoietic Stem Cells in Nonobese Diabetic/Severe Combined Immunodeficient Mice

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 481-487 ◽  
Author(s):  
Johanne D. Cashman ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Human Growth ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Nonobese Diabetic

Self-renewal is considered to be the essential defining property of a stem cell. Retroviral marking, in vitro amplification, and serial transplantation of human cells that can sustain long-term lymphomyelopoiesis in vivo have provided evidence that human hematopoietic stem cell self-renewal occurs both in vitro and in vivo. To investigate whether this process can be manipulated by cytokines, we administered two different combinations of human growth factors to sublethally irradiated nonobese diabetic/severe combined immunodeficient (SCID) mice transplanted with 107 light-density human cord blood cells and then performed secondary transplants to compare the number of transplantable human lymphomyeloid reconstituting cells present 4 to 6 weeks post-transplant. A 2-week course of Steel factor + interleukin (IL)-3 + granulocyte-macrophage colony-stimulating factor + erythropoietin (3 times per week just before sacrifice) specifically and significantly enhanced the numbers of transplantable human lymphomyeloid stem cells detectable in the primary mice (by a factor of 10). Steel factor + Flt3-ligand + IL-6 (using either the same schedule or administered daily until sacrifice 4 weeks post-transplant) gave a threefold enhancement of this population. These effects were obtained at a time when the regenerating human progenitor populations in such primary mice are known to be maximally cycling even in the absence of growth factor administration suggesting that the underlying mechanism may reflect an ability of these growth factors to alter the probability of differentiation of stem cells stimulated to proliferate in vivo.

scholarly journals Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3736-3749 ◽  
Author(s):  
Wanda Piacibello ◽  
Fiorella Sanavio ◽  
Antonella Severino ◽  
Alessandra Danè ◽  
Loretta Gammaitoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Hematopoietic Cells ◽  
Scid Mice ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

An RNA Interference Screen Identifies the Cell Fate Determinants Msi2 and Prox1 as Novel Regulators of Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 394-394
Author(s):  
Kristin J Hope ◽  
Sonia Cellot ◽  
Stephen Ting ◽  
Guy Sauvageau
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Fate ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Control Shrna ◽  
Fate Determinants ◽  
Cell Fate Determinants

Abstract Abstract 394 Hematopoietic stem cells (HSC) can not yet be unambiguously prospectively identified, a fact which has made it difficult to determine whether a segregation of cell fate determinants underlies the asymmetric/symmetric self-renewal of these cells or whether deregulation of such determinants could contribute to the pathogenesis of hematopoietic malignancies by inducing constitutive symmetric self-renewal divisions. We have addressed these questions through a functional genetics approach taking advantage of systematic RNAi to evaluate the function of conserved polarity factors and cell fate determinants in HSCs. From a list of 72 of such factors identified in the literature, 30 murine homologues were chosen based on their differentially higher level of expression in HSC-enriched populations as measured by qRT-PCR. For each candidate we designed 3 unique short hairpin RNA (shRNA) encoding retroviral constructs also carrying EGFP for the purposes of following transduced cells. Primitive hematopoietic cells enriched for HSC were infected at high efficiency with the library in an arrayed 96-well format and their in vivo reconstituting potential was then evaluated through competitive repopulating unit assays. Genes for which shRNA vectors altered late transplant EGFP levels below or above thresholds as defined by a control shRNA to luciferase were considered as hits. Using this approach, we identified and comprehensively validated 4 genes, including the RNA binding protein Msi2, for which shRNA-mediated depletion dramatically impairs repopulation but does not induce cell death or a cell cycle block. Importantly, we show that the loss in the repopulating ability of these shRNA transduced cells is mediated at the stem cell level and is not due to progenitor or downstream cell toxicity or to any defect in the process of bone marrow homing. Subsequent expression profiling indicated that Msi2 is also upregulated in HOXB4-overexpressing symmetrically expanding HSC in line with our findings that it functions as a positive HSC regulator and further suggesting that it represents a potential novel HSC marker. As well as finding HSC agonists, the RNAi screen identified the homeodomain containing transcription factor Prox1 as a negative HSC regulator since its shRNA-mediated transcript loss consistently led to the dramatic in vivo accumulation of EGFP+ transduced cells. Grafts comprised of Prox1 shRNA-transduced cells did not exhibit any lineage skewing however, repeatedly contained an average of 10-fold more primitive Lin-Sca+CD150+48- cells as compared to non-transduced donor cells within the same recipient or to control shRNA-luciferase grafts indicating Prox1 knockdown leads to a significant in vivo expansion of phenotypic HSCs. Moreover, following a 7 day in vitro culture, cells infected with shRNAs to Prox1 were both morphologically and immunophenotypically more primitive than control cells and when transplanted at this time yielded a significantly enhanced engraftment level relative to control shRNAs (51+/-6% GFP vs 8+/-3% GFP). These results further suggest that Prox1 reduction by RNAi expands functional HSCs in vitro. Together these findings have identified conserved cell fate determinants as important and novel regulators of murine hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

Steel factor responsiveness regulates the high self-renewal phenotype of fetal hematopoietic stem cells

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 5043-5048 ◽  
Author(s):  
Michelle B. Bowie ◽  
David G. Kent ◽  
Michael R. Copley ◽  
Connie J. Eaves
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Wild Type ◽  
Developmentally Regulated ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Reduced Rate

Abstract Fetal hematopoietic stem cells (HSCs) regenerate daughter HSCs in irradiated recipients more rapidly than do adult HSCs. However, both types of HSCs divide in vitro with the same cell-cycle transit times, suggesting different intrinsically determined self-renewal activities. To investigate the mechanism(s) underlying these differences, we compared fetal and adult HSC responses to Steel factor (SF) stimulation in vitro and in vivo. These experiments were undertaken with both wild-type cells and W41/W41 cells, which have a functionally deficient c-kit kinase. In vitro, fetal HSC self-renewal divisions, like those of adult HSCs, were found to be strongly dependent on c-kit activation, but the fetal HSCs responded to much lower SF concentrations in spite of indistinguishable levels of c-kit expression. Fetal W41/W41 HSCs also mimicked adult wild-type HSCs in showing the same reduced rate of amplification in irradiated adult hosts (relative to fetal wild-type HSCs). Assessment of various proliferation and signaling gene transcripts in fetal and adult HSCs self-renewing in vitro revealed a singular difference in Ink4c expression. We conclude that the ability of fetal HSCs to execute symmetric self-renewal divisions more efficiently than adult HSCs in vivo may be dependent on specific developmentally regulated signals that act downstream of the c-kit kinase.

scholarly journals Evidence of both ontogeny and transplant dose-regulated expansion of hematopoietic stem cells in vivo

Blood ◽  
1996 ◽  
Vol 88 (8) ◽  
pp. 2852-2858 ◽  
Author(s):  
R Pawliuk ◽  
C Eaves ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fetal Liver ◽  
Complete Recovery ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Adult Mice

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

scholarly journals Engraftment in Nonobese Diabetic Severe Combined Immunodeficient Mice of Human CD34+ Cord Blood Cells After Ex Vivo Expansion: Evidence for the Amplification and Self-Renewal of Repopulating Stem Cells

Blood ◽  
1999 ◽  
Vol 93 (11) ◽  
pp. 3736-3749 ◽  
Author(s):  
Wanda Piacibello ◽  
Fiorella Sanavio ◽  
Antonella Severino ◽  
Alessandra Danè ◽  
Loretta Gammaitoni ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Growth Factors ◽  
Hematopoietic Cells ◽  
Scid Mice ◽  
Human Cd34 ◽  
Nonobese Diabetic ◽  
Cd34 Cells

Abstract Understanding the repopulating characteristics of human hematopoietic stem/progenitor cells is crucial for predicting their performance after transplant into patients receiving high-dose radiochemotherapy. We have previously reported that CD34+cord blood (CB) cells can be expanded in vitro for several months in serum containing culture conditions. The use of combinations of recombinant early acting growth factors and the absence of stroma was essential in determining this phenomenon. However, the effect of these manipulations on in vivo repopulating hematopoietic cells is not known. Recently, a new approach has been developed to establish an in vivo model for human primitive hematopoietic precursors by transplanting human hematopoietic cells into sublethally irradiated nonobese diabetic severe combined immunodeficient (NOD/SCID) mice. We have examined here the expansion of cells, CD34+ and CD34+38− subpopulations, colony-forming cells (CFC), long-term culture initiating cells (LTC-IC) and the maintenance or the expansion of SCID-repopulating cells (SRC) during stroma-free suspension cultures of human CD34+ CB cells for up to 12 weeks. Groups of sublethally irradiated NOD/SCID mice were injected with either 35,000, 20,000, and 10,000 unmanipulated CD34+ CB cells, which were cryopreserved at the start of cultures, or the cryopreserved cells expanded from 35,000, 20,000, or 10,000 CD34+ cells for 4, 8, and 12 weeks in the presence of a combination of early acting recombinant growth factors (flt 3/flk2 ligand [FL] + megakaryocyte growth and development factor [MGDF] ± stem cell factor [SCF] ± interleukin-6 [IL-6]). Mice that had been injected with ≥20,000 fresh or cryopreserved uncultured CD34+ cells did not show any sign or showed little engraftment in a limited number of animals. Conversely, cells that had been generated by the same number of initial CD34+ CB cells in 4 to 10 weeks of expansion cultures engrafted the vast majority of NOD/SCID mice. The level of engraftment, well above that usually observed when the same numbers of uncultured cells were injected in the same recipients (even in the presence of irradiated CD34− cells) suggested that primitive hematopoietic cells were maintained for up to 10 weeks of cultures. In addition, dilution experiments suggest that SRC are expanded more than 70-fold after 9 to 10 weeks of expansion. These results support and extend our previous findings that CD34+ CB stem cells (identified as LTC-IC) could indeed be grown and expanded in vitro for an extremely long period of time. Such information may be essential to design efficient stem cell expansion procedures for clinical use.

scholarly journals A Stemness Screen Reveals C3ORF54/INKA1 As a Gate-Keeper of Human Stem Cell Latency

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 325-325
Author(s):  
Kerstin B. Kaufmann ◽  
Laura Garcia Prat ◽  
Shin-Ichiro Takayanagi ◽  
Jessica McLeod ◽  
Olga I. Gan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Fold Increase ◽  
Steady Increase ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The controversy generated from recent murine studies as to whether hematopoietic stem cells (HSC) contribute to steady-state hematopoiesis emphasizes how limited our knowledge is of the mechanisms governing HSC self-renewal, activation and latency; a problem most acute in the study of human HSC and leukemia stem cells (LSC). Many hallmark stem cell properties are shared by HSC and LSC and therefore a better understanding of stemness regulation is crucial to improved HSC therapies and leukemia treatments targeting LSC. Our previous work on LSC subsets from >80 AML patient samples revealed that HSC and LSC share a transcriptional network that represent the core elements of stemness (Eppert, Nature Med 2011; Ng, Nature 2016). Hence, to identify the key regulators of LSC/HSC self-renewal and persistence we selected 64 candidate genes based on expression in functionally validated LSC vs. non-LSC fractions and assessed their potential to enhance self-renewal in a competitive in vivo screen. Here, we transduced cord blood CD34+CD38- cells with 64 barcoded lentiviral vectors to assemble 16 pools, each consisting of 8 individual gene-transduced populations, for transplantation into NSG mice. Strikingly, individual overexpression (OE) of 5 high scoring candidates revealed delayed repopulation kinetics of human HSC/progenitor cells (HSPC): gene-marking of human CD45+ and lin-CD34+ cells was reduced relative to input and control at 4w post transplantation, whereas by 20w engraftment of marked cells reached or exceeded input levels. For one of these candidates, C3ORF54/INKA1, we found that OE did not alter lineage composition neither in in vitro nor in vivo assays but increased the proportion of primitive CD34+ cells at 20w in vivo; moreover, secondary transplantation revealed a 4.5-fold increase in HSC frequency. Of note, serial transplantation from earlier time points (2w, 4w) revealed superior engraftment and hence greater self-renewal capacity upon INKA1-OE. Since we observed a 4-fold increase of phenotypic multipotent progenitors (MPP) relative to HSC within the CD34+ compartment (20w) we assessed whether INKA1-OE acts selectively on either cell population. The observation of latency in engraftment was recapitulated with sorted INKA1-OE HSC but not MPP. Likewise, liquid culture of HSPC and CFU-C assays on sorted HSC showed an initial delay in activation and colony formation upon INKA1-OE that was completely restored by extended culture and secondary CFU-C, respectively. INKA1-OE MPP showed a slight increase in total colony count in primary CFU-C and increased CDK6 levels in contrast to reduced CDK6 levels in INKA1-OE HSC emphasizing opposing effects of INKA1 on cell cycle entry and progression in either population. Taken together, this suggests that INKA1-OE preserves self-renewal capacity by retaining HSC preferentially in a latent state, however, upon transition to MPP leads to enhanced activation. Whilst INKA1 has been described as an inhibitor of p21(Cdc42/Rac)-activated kinase 4 (PAK4), no role for PAK4 is described in hematopoiesis. Nonetheless, its regulator Cdc42 is implicated in aging of murine HSPC by affecting H4K16 acetylation (H4K16ac) levels and polarity and has recently been described to regulate AML cell polarity and division symmetry. In our experiments immunostaining of HSPC subsets cultured in vitro and from xenografts indicates that INKA1-OE differentially affects epigenetics of these subsets linking H4K16ac to the regulation of stem cell latency. In AML, transcriptional upregulation of INKA1 in LSC vs. non-LSC fractions and at relapse in paired diagnosis-relapse analysis (Shlush, Nature 2017) implicates INKA1 as a regulator of LSC self-renewal and persistence. Indeed, INKA1-OE in cells derived from a primary human AML sample (8227) with a phenotypic and functional hierarchy (Lechman, Cancer Cell 2016) revealed a strong latency phenotype: In vitro and in vivo we observed label retention along with a steady increase in percentage of CD34+ cells, transient differentiation block, reduced growth rate, G0 accumulation and global reduction of H4K16ac. In summary, our data implicates INKA1 as a gate-keeper of stem cell latency in normal human hematopoiesis and leukemia. Studying the detailed pathways involved will shed light upon the mechanisms involved in HSC activation and latency induction and will help to harness these for novel therapeutic approaches. Disclosures Takayanagi: Kyowa Hakko Kirin Co., Ltd.: Employment.

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 Clonal-level lineage commitment pathways of hematopoietic stem cells in vivo

2019 ◽  
Vol 116 (4) ◽  
pp. 1447-1456 ◽  
Author(s):  
Rong Lu ◽  
Agnieszka Czechowicz ◽  
Jun Seita ◽  
Du Jiang ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Different Levels

While the aggregate differentiation of the hematopoietic stem cell (HSC) population has been extensively studied, little is known about the lineage commitment process of individual HSC clones. Here, we provide lineage commitment maps of HSC clones under homeostasis and after perturbations of the endogenous hematopoietic system. Under homeostasis, all donor-derived HSC clones regenerate blood homogeneously throughout all measured stages and lineages of hematopoiesis. In contrast, after the hematopoietic system has been perturbed by irradiation or by an antagonistic anti-ckit antibody, only a small fraction of donor-derived HSC clones differentiate. Some of these clones dominantly expand and exhibit lineage bias. We identified the cellular origins of clonal dominance and lineage bias and uncovered the lineage commitment pathways that lead HSC clones to different levels of self-renewal and blood production under various transplantation conditions. This study reveals surprising alterations in HSC fate decisions directed by conditioning and identifies the key hematopoiesis stages that may be manipulated to control blood production and balance.

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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