Impaired Reconstitution Potential of TGFβ-Hypersensitive Human Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 394-394
Author(s):  
Emma Rörby ◽  
Matilda Nifelt Hägerström ◽  
Ulrika Blank ◽  
Göran Karlsson ◽  
Stefan Karlsson
Keyword(s):  
Lentiviral Vector ◽  
Control Vector ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Smad Pathway ◽  
Human Hscs ◽  
And Control

Abstract Abstract 394 Hematopoietic stem cells (HSCs) are primitive, tissue-specific cells that can self-renew and differentiate along all lineages of the blood system. These properties make the HSCs critical for tissue regeneration and clinical applications in cell therapy. Cord blood (CB) is an accessible source for HSCs. However, the yield of HSCs from one cord is too low in order to successfully transplant adult patients. The expansion of HSCs in vitro has met with limited success due to incomplete knowledge regarding the mechanisms regulating self-renewal. Members of the transforming growth factor-β (TGF-β) superfamily have been shown to regulate HSCs through the downstream Smad signaling pathway. TGF-β1 potently inhibits HSC growth in vitro, and overexpression of the inhibitory Smad7 has been demonstrated to increase in vivo self-renewal of murine HSC, indicating that the Smad pathway negatively regulates self-renewal (Blank et al. Blood, 2006). However, disruption of the entire Smad pathway in HSCs through conditional deletion of the common Smad4 resulted in reduced repopulative capacity (Karlsson et al. JEM, 2007). These findings demonstrate the complexity of Smad signaling and highlight the importance to investigate it further. Therefore, we asked whether enforced expression of Smad4 could reveal a role for TGF-β in human HSCs regulation in vivo or affect self-renewal and regenerative ability of HSCs in vitro. To investigate the effect of Smad4 overexpression in hematopoiesis, full-length cDNA of human Smad4 was cloned in to a lentiviral vector carrying a GFP reporter gene, referred to as Smad4 vector. As control, a lentiviral vector carrying GFP only, referred to as control vector, was generated. Human CB HSCs overexpressing Smad4 displayed increased sensitivity to TGF-β in colony assays (TGF-β treated-/untreated growth: 0.22 ±0.04 vs. 0.32 ±0.04 for Smad4 vector and control vector, respectively P=.0197). Importantly, the addition of a TGF-β inhibitor targeting ALK4, 5 and 7 receptors (SB431542) rescued the colony forming capacity (TGF-β treated-/untreated growth: 0.6 ±0.046 vs. 0.72 ±0.078 for Smad4 vector and control vector, respectively) demonstrating the functional overactivity of the TGF-β pathway in Smad4 overexpressing cells. Since TGF-β is a well-known growth inhibitor of hematopoietic progenitors (Batard et al. JCS, 2000; Cashman et al. Blood, 1990; Sitnicka et al. Blood, 1996) we further analyzed cell cycle status of transduced cells. Cells with enforced expression of Smad4 and increased TGF-β sensitivity were to a larger extent in the quiescent state of the cell cycle (G0) compared to control cells when cultured for six days (16.54 ±5.70% vs. 7.84 ±0.51% for Smad4 vector and control vector, respectively P=.0286) but could be released from G0 when treated with the inhibitor SB431542. Moreover, as TGF-β also is known to induce apoptosis (Jacobsen et al. Blood, 1995) we further investigated if enforced expression of Smad4 would affect apoptosis in cultured CB cells. After six days of culture Smad4 overexpressing cells had significantly higher AnnexinV expression compared to control cells (25.74 ±3.81% vs. 15.45 ±4.44% for Smad4 vector and control vector, respectively P=.0281), an effect that also was decreased when adding the inhibitor SB431542 to the culture (20.38 ±5.96% vs. 16.25 ±6.35% for Smad4 vector and control vector, respectively). Furthermore, we transplanted transduced CB cells into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Interestingly, despite having similar transduction efficiency as the empty vector control (30 ±16% vs. 29 ±13% for Smad4 vector and control vector, respectively) CD34+ CB HSCs transduced with the Smad4 vector had impaired engraftment as measured by FACS analysis of peripheral blood (PB) (Smad4 vector 1.03 ±1.3% GFP vs. control vector 2.94 ±1.97% P=.0035) and bone marrow 6 months post transplantation (Smad4 vector 1.5 ±0.88% GFP vs. control vector 5.60 ±1.54% P=.0029). Expression of lineage surface markers (CD13, CD15 and CD19) in PB 3 month post transplantation was unaltered. In summary, our results demonstrate that increased Smad4 expression sensitizes human CB HSCs to TGF-β. This leads to growth arrest and apoptosis in vitro and reduced HSC reconstitution capacity in vivo with no effect on lineage distribution. Together, these findings demonstrate an important role for TGF-β signaling in the regulation of human HSCs in vivo. Disclosures: No relevant conflicts of interest to declare.

Human hematopoietic stem/progenitor cells overexpressing Smad4 exhibit impaired reconstitution potential in vivo

Blood ◽  
2012 ◽  
Vol 120 (22) ◽  
pp. 4343-4351 ◽  
Author(s):  
Emma Rörby ◽  
Matilda Nifelt Hägerström ◽  
Ulrika Blank ◽  
Göran Karlsson ◽  
Stefan Karlsson
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Cell System ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Smad Pathway ◽  
Smad Signaling ◽  
Smad Signaling Pathway

Abstract Hematopoietic stem cells (HSCs) constitute a rare population of tissue-specific cells that can self-renew and differentiate into all lineages of the blood cell system. These properties are critical for tissue regeneration and clinical applications of HSCs. Cord blood is an easily accessible source of HSCs. However, the number of HSCs from one unit is too low to effectively transplant most adult patients, and expansion of HSCs in vitro has met with limited success because of incomplete knowledge regarding mechanisms regulating self-renewal. Members of the TGF-β superfamily have been shown to regulate HSCs through the Smad signaling pathway; however, its role in human HSCs has remained relatively uncharted in vivo. Therefore, we asked whether enforced expression of the common-Smad, Smad4, could reveal a role for TGF-β in human hematopoietic stem/progenitor cells (HSPCs) from cord blood. Using a lentiviral overexpression approach, we demonstrate that Smad4 overexpression sensitizes HSPCs to TGF-β, resulting in growth arrest and apoptosis in vitro. This phenotype translates in vivo into reduced HSPC reconstitution capacity yet intact lineage distribution. This suggests that the Smad pathway regulates self-renewal independently of differentiation. These findings demonstrate that the Smad signaling circuitry negatively regulates the regeneration capacity of human HSPCs in vivo.

scholarly journals CD66e Enrichment Enhances Repopulation of Human Long-Term Hematopoietic Stem Cells

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2156-2156
Author(s):  
Kuiying Ma ◽  
Riguo Fang ◽  
Lingling Yu ◽  
Yongjian Zhang ◽  
Chao Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Surface Markers ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Differentiation Pattern ◽  
Human Hscs

Abstract Gene-modified hematopoietic stem cells (HSCs) therapy has demonstrated remarkable success for the treatment of inherited blood disorders. As the origin of hematologic hierarchy, HSCs play an essential role in sustaining life-long hematopoiesis. HSCs identification via reliable and robust bio-markers could facilitate the development of HSC gene therapy. Previous studies showed that long-term hematopoietic stem cells (LT-HSCs) were enriched in the Lin -CD34 +CD38 -CD45RA -CD90 +CD49f + population which could support long-term hematopoietic reconstitution. However, several of these surface markers proved to be unreliable when ex vivo culturing, such as CD38 and CD49f. Thus, HSCs characterization is still hindered by lacking bona-fide bio-markers, and consequently identification of long-term HSCs still needs time-consuming in vivo transplantation. To this end, we performed in vitro screening and comprehensive functional evaluation to identify a novel surface marker of human HSCs. During initial screening, a cell surface antigen screen panel (including 242 human cell surface markers) and human CD34 and CD90 antibodies were used to perform flow cytometry analysis on CD34 + HSPCs enriched from umbilical cord blood. Compared with CD34 + cell population, we found that CD66 (a,c,d,e), CD200 and CD48 positive cells were more enriched in CD34 +CD90 + subset. Previous studies indicated that HSCs cannot be maintained during in vitro culturing. By tracking these candidate surface markers based on this principle, CD66e was selected as the potential HSCs bio-marker. Next, we examined the in vivo hematologic repopulating potential of HSCs by limiting dilution assay (LDA) on immune-deficient mouse model. We sorted CD66e + and CD66e - subsets from CD34 +CD90 +CD45RA - subpopulation, and transplanted into irradiated NOD-scid Il2rg −/− (NPG) mice respectively. At week16 post-transplantation, in contrast to the CD66e - group, CD66e + cells exhibited significantly higher reconstitution in peripheral blood (PB), bone marrow (BM) and spleen. Engraftment dynamics revealed that the CD66e - group were only capable of reconstitution 4 weeks post transplantation, even at the highest initial cell dose. Moreover, the CD66e - group displayed impaired multi-lineage differentiation pattern, especially in PB and BM samples, while the CD66e + group presented a robust multi-lineage reconstitution. Notably, LDA results showed that the CD66e + cells within CD34 +CD90 +CD45RA - population contained 1 out of 529 SCID repopulating cells (SRC), almost 60-fold greater than the CD66e - fraction. To further investigate the long-term repopulating potential of the CD66e + cells, we performed the secondary transplantation collected from the BM cells of primary recipients. CD66e + cells presented significant higher repopulating activity than CD66e- subset in the secondary recipients. These findings reveal that the major cells with homing and long-term reconstitution capacity among CD34 +CD90 +CD45RA - cells were CD66e positive. In order to determine the transcriptional profile of CD66e + cells, we performed RNA-sequencing analysis using the population of CD34 + cells, CD34 +CD90 +CD45RA - cells, CD66e + and CD66e - cells within CD34 +CD90 +CD45RA - subset. Remarkably, compared with other groups, the CD66e + cells displayed a bias toward the signature of HSC and early progenitors such as LMPP and CLP. Moreover, gene set enrichment analysis showed that hematopoietic lineage and long-term potentiation-related genes were highly enriched in the CD66e + cells. Further qRT-PCR experiment confirmed that several HSC-related genes were significantly higher expressed in CD34 +CD90 +CD45RA -CD66e + cells, compared to CD66e - population or CD34 + HSPCs, suggesting that the gene expression profile of CD66e + cells is reminiscent of HSC signature. Altogether, we demonstrate that CD66e is a robust functional HSC bio-marker that CD66e-positive population among CD34 +CD90 +CD45RA - cells exhibit typical HSC signature, enhanced in vivo engraftment potential and robust multilineage differentiation pattern, which will provide an invaluable tool to investigate the origin of human HSCs, paving the way for the therapeutic application. Figure 1 Figure 1. Disclosures Fang: EdiGene, Inc.: Current Employment.

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.

Evidence for the Involvement of CXCR4 Signaling in In Vivo Self-Renewal of Transplanted Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1888-1888 ◽  
Author(s):  
Chen-YI LAI ◽  
Makoto Otsu ◽  
Motohito Okabe ◽  
Sachie Suzuki ◽  
Satoshi Yamazaki ◽  
...  
Keyword(s):  
Post Transplantation ◽  
Gain Of Function ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cxcr4 Signaling ◽  
Hematopoietic Reconstitution

Abstract Abstract 1888 Hematopoietic stem cells (HSCs) represent the unique cell population capable of self-renewal and multi-lineage differentiation, thereby lifelong sustainment of the hematopoiesis. HSC transplantation has proven beneficial for various diseases, it is therefore important to elucidate the molecular determinants for successful HSC engraftment. Signaling through the chemokine receptor CXCR4 has been implicated in HSC engraftment by the observation that transplantation of HSCs lacking this molecule results in poor hematopoietic reconstitution. Because this impairment, however, can be attributed to the defects in any of the post-transplantation processes that include bone marrow (BM)-homing, -repopulation, or –retention, it is still unclear whether CXCR4 plays an essential role in HSC self-renewal upon transplantation. To elucidate the role of CXCR4 signaling in HSC self-renewal in conjunction with transplantation, we used a purified CD34neg/low c-Kit+ Sca-1+ Lineage-markerneg population as the defined stem cell source. As a loss-of-function study, CXCR4 was conditionally deleted in HSCs before transplantation. As a gain-of-function study, we generated the HSC populations overexpressing either wild-type (wt)- or C-terminal truncated (δC)-CXCR4 (OE-HSCs), the latter of which is known to exhibit enhancement in the SDF-1 signaling, by gene transfer and subsequent cell sorting. We compared these cells with control HSCs in in vitro assays with regard to the biological characteristics including chemotaxis, proliferation, colony formation, and cobblestone-area (CA) forming ability. To dissect in vivo post-transplantation processes, we investigated hematopoietic repopulation kinetics in the recipient BM at the homing/lodging phase (within 1 wk) and the early repopulation phase (2–3 wks) for the above test HSCs. The self-renewal potential of each HSC population was estimated by competitive repopulation assay. In vitro studies: OE-HSCs with wt- or δC-CXCR4 exhibited enhanced chemotaxis and proliferation in response to SDF1, confirming the gain-of-function effects of these modifications. CA forming ability was greater in OE-HSCs with δC-CXCR4 than control counterparts and absent in CXCR4-KO HSCs, suggesting the critical role of CXCR4-signaling in HSC proliferation in the presence of stromal support. In vivo studies: 1) the homing/lodging phase. Unexpectedly, we did not find significant alteration in the numbers of early progenies detectable in recipient BM 3 days after transplantation of HSCs receiving either loss- or gain-of-function modification to CXCR4, indicating that this signaling is indispensable in HSC homing. 2) the early repopulation phase. Impairment of hematopoietic repopulation in BM became evident for CXCR4-KO HSCs through 2–3 wks. On the other hand, OE-HSCs with CXCR4, more remarkably of ΔC-mutation, showed enhanced BM repopulation kinetics at ∼3 wks post transplantation, suggesting the importance of CXCR4 signaling in HSC amplification at this post-transplantation phase. 3) long-term hematopoiesis. CXCR4-KO-HSCs showed poor hematopoietic reconstitution potentials, consistent with previous observations. Interestingly, impaired peripheral repopulation was also observed with OE-HSCs with wt- or ΔC-CXCR4. Further characterization revealed that the recipients of CXCR4-overexpressing HSCs did retain their progenies, which showed multilineage differentiation, but exhibited impaired release of mature leukocytes from the BM to the peripheral blood. Most importantly, however, test-cell chimerism in the long-term HSC fraction was significantly higher in the mice receiving OE-HSCs with CXCR4, especially of ΔC-type, than those transplanted with control HSCs, indicating that the augmentation of CXCR4 signaling enhanced competitive repopulation ability of HSCs. These modified HSCs demonstrated repopulation abilities also in secondary recipients. We demonstrated that CXCR4 signaling is indispensible for HSC homing and that continuous overexpression of CXCR4 cannot benefit the peripheral reconstitution in contrary to the expectation. More importantly, our studies showed that augmentation of CXCR4 signaling leads to HSC expansion in vivo upon transplantation. We thus conclude that CXCR4 signaling has a role in HSC self-renewal and that its regulation may find the approach that will improve HSC transplantation outcomes. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem Cells Fail to Regenerate In Vivo Following Inflammatory Stress

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1472-1472
Author(s):  
Ruzhica Bogeska ◽  
Paul Kaschutnig ◽  
Stella Paffenholz ◽  
Julia Maassen ◽  
Jan-Philipp Mallm ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Fold Reduction

Abstract An often-cited defining property of hematopoietic stem cells (HSCs) is their extensive or unlimited in vivo self-renewal capacity. We have recently described a novel mouse disease model forFanconi anemia, in which serial challenge with pro-inflammatory agonists that mimic infection, such aspolyinosinic:polycytidylic acid (pI:C), results in HSC attrition followed by a highly penetrant severe aplastic anemia, closely recapitulating the disease in patients (Walter et al., 2015, Nature). In order to explore the broader implications of these findings in the context of HSC self-renewal, we conducted apI:Cdose escalation regimen using standard C57BL6 mice. A single injection withpI:Cprovoked transient peripheral blood (PB)cytopenias, with the recovery of mature blood cell numbers correlating with HSCs being forced into active cell cycle. Injection with 1-3 rounds ofpI:C(1-3 x 8 injections) led to no discernable sustained impact on blood production as, at 5 weeks post-treatment, PB frequencies were in the normal range, as were the absolute numbers of HSCs and all progenitor compartments in the bone marrow (BM), as determined by flowcytometry. However, in vitro analysis of the proliferation and differentiation potential of 411 individual sorted long-term (LT)-HSCs 5 weeks after 3 rounds of pI:C challenge, revealed a decrease in the frequency of LT-HSCs able to generate progeny in vitro (1.6-fold reduction, p<0.05), and a 2-fold reduction in the total number of progeny produced per HSC, which was even more pronounced inmultilineage potential clones (2.6-fold decrease, p<0.0001) compared touni- or bi-lineage clones. In line with this data, competitive repopulation assays demonstrated a progressive depletion of functional HSC numbers with increasing rounds ofpI:C treatment, with a 1.8, 3.4 and 15.3-fold decrease in donorchimerism across all lineages at 6 months post-transplantation (p<0.01) following 1, 2 or 3 rounds ofpI:C treatment, respectively. Notably, robust engraftment (up to 65% donorchimerism, 6 months post-transplantation, p<0.01) was achieved when mice exposed to 3 rounds ofpI:C treatment were used as a recipient for non-treated BM cells in the absence of any irradiation conditioning, while engraftment was always <1% when non-treated controls were used as recipients. This excludes the possibility that the observed progressive depletion of functional HSCs was the result of artifacts associated with a compromised niche or the non-physiologic stress imposed on donor cells during transplantation. In order to test the kinetics of HSC recovery following HSC challenge, BM was harvested from mice at either 5, 10 or 20 weeks after treatment with 3 rounds of pI:C, and both competitive and limiting dilution transplantation assays (Table 1) were used to quantify HSC frequencies. Surprisingly, both assays demonstrated that HSCs failed to regenerate at all following pI:Cchallenge, directly contradicting the canonical view that HSCs possess extensive self-renewal capacity in vivo. The physiologic relevance of this observation was illustrated when we measured the hematologic parameters of aged mice that had been exposed to chronicpI:C treatment in early to mid-life. Although these mice had normal PB counts at 4 weeks post-treatment, at 2 years of age, peripheral bloodcytopenias and bone marrow aplasia became evident (Table 2), recapitulating clinically relevant features of non-malignant aged human hematopoiesis that are never seen in standard laboratory mice. Together, these data suggest that functional HSCs can be progressively and irreversibly depleted in response to environmental agonists, such as infection and inflammation, which force HSCs to reconstitute mature blood cells consumed by such stimuli. This model has clear implications relating to the role of adult stem cells in tissue maintenance and regeneration during ageing, and how stress agonists that are absent in most laboratory animal models, but would be ubiquitous in the wild, are likely key mediators of age-associated disease pathologies. Disclosures Frenette: PHD Biosciences: Research Funding; Pfizer: Consultancy; GSK: Research Funding.

scholarly journals Slug deficiency enhances self-renewal of hematopoietic stem cells during hematopoietic regeneration

Blood ◽  
2010 ◽  
Vol 115 (9) ◽  
pp. 1709-1717 ◽  
Author(s):  
Yan Sun ◽  
Lijian Shao ◽  
Hao Bai ◽  
Zack Z. Wang ◽  
Wen-Shu Wu
Keyword(s):  
Hematopoietic Cells ◽  
Stress Conditions ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steady State Condition ◽  
Therapeutic Benefits ◽  
Evolutionarily Conserved ◽  
Novel Strategy

Abstract Both extrinsic and intrinsic mechanisms tightly govern hematopoietic stem cell (HSC) decisions of self-renewal and differentiation. However, transcription factors that can selectively regulate HSC self-renewal division after stress remain to be identified. Slug is an evolutionarily conserved zinc-finger transcription factor that is highly expressed in primitive hematopoietic cells and is critical for the radioprotection of these key cells. We studied the effect of Slug in the regulation of HSCs in Slug-deficient mice under normal and stress conditions using serial functional assays. Here, we show that Slug deficiency does not disturb hematopoiesis or alter HSC homeostasis and differentiation in bone marrow but increases the numbers of primitive hematopoietic cells in the extramedullary spleen site. Deletion of Slug enhances HSC repopulating potential but not its homing and differentiation ability. Furthermore, Slug deficiency increases HSC proliferation and repopulating potential in vivo after myelosuppression and accelerates HSC expansion during in vitro culture. Therefore, we propose that Slug is essential for controlling the transition of HSCs from relative quiescence under steady-state condition to rapid proliferation under stress conditions. Our data suggest that inhibition of Slug in HSCs may present a novel strategy for accelerating hematopoietic recovery, thus providing therapeutic benefits for patients after clinical myelosuppressive treatment.

Interleukin-3 supports expansion of long-term multilineage repopulating activity after multiple stem cell divisions in vitro

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1748-1755 ◽  
Author(s):  
David Bryder ◽  
Sten E. W. Jacobsen
Keyword(s):  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Calf Serum ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Divisions ◽  
Stem Cell Divisions

Abstract Although long-term repopulating hematopoietic stem cells (HSC) can self-renew and expand extensively in vivo, most efforts at expanding HSC in vitro have proved unsuccessful and have frequently resulted in compromised rather than improved HSC grafts. This has triggered the search for the optimal combination of cytokines for HSC expansion. Through such studies, c-kit ligand (KL), flt3 ligand (FL), thrombopoietin, and IL-11 have emerged as likely positive regulators of HSC self-renewal. In contrast, numerous studies have implicated a unique and potent negative regulatory role of IL-3, suggesting perhaps distinct regulation of HSC fate by different cytokines. However, the interpretations of these findings are complicated by the fact that different cytokines might target distinct subpopulations within the HSC compartment and by the lack of evidence for HSC undergoing self-renewal. Here, in the presence of KL+FL+megakaryocyte growth and development factor (MGDF), which recruits virtually all Lin−Sca-1+kit+ bone marrow cells into proliferation and promotes their self-renewal under serum-free conditions, IL-3 and IL-11 revealed an indistinguishable ability to further enhance proliferation. Surprisingly, and similar to IL-11, IL-3 supported KL+FL+MGDF-induced expansion of multilineage, long-term reconstituting activity in primary and secondary recipients. Furthermore, high-resolution cell division tracking demonstrated that all HSC underwent a minimum of 5 cell divisions, suggesting that long-term repopulating HSC are not compromised by IL-3 stimulation after multiple cell divisions. In striking contrast, the ex vivo expansion of murine HSC in fetal calf serum-containing medium resulted in extensive loss of reconstituting activity, an effect further facilitated by the presence of IL-3.

Rapid and Irreversible Alteration of the Ability of Hematopoietic Stem Cells To Execute Both Symmetric and Asymmetric Self-Renewal Divisions by Exposure to Reduced Steel Factor Concentrations with No Effect on Their Survival or Mitogenesis.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 684-684
Author(s):  
David G. Kent ◽  
Brad Dykstra ◽  
Connie J. Eaves
Keyword(s):  
Bone Marrow ◽  
Adult Mouse ◽  
Single Cells ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
High Concentrations

Abstract Hematopoietic stem cells (HSCs) are present in the marrow of adult mice at a frequency of 1/104, as measured by limiting dilution transplantation assays for individual cells that produce lymphoid (B and T) as well as myeloid (GM) cells for at least 4 months in irradiated recipients. HSCs thus defined can be reproducibly isolated in the CD45midlin−Rho−SP fraction of adult mouse bone marrow at a purity of &gt;30%. In mice, mutations in c-kit, the receptor for Steel factor (SF) lead to substantial reductions in the adult HSC population. In vitro, SF has been identified as a potent regulator of HSC self-renewal divisions. High concentrations of SF in combination with IL-11 allow adult HSCs to divide with a net 2–4 fold expansion in HSC numbers after 10 days and low concentrations of SF result in loss of HSC activity. To investigate the cellular mechanisms underlying these different outcomes, we cultured 114 CD45midlin−Rho−SP adult mouse bone marrow cells in single cell cultures containing serum-free medium + 20 ng/ml IL-11 and either 300 or 10 ng/ml of SF. Each culture was then examined every 4–6 hr. The kinetics of division of these cells under both conditions was identical with completion of the 1st division occurring between 22–68 hr. During that time none of the input cells died (&lt;1%). After 10 days of culture, during which time all input cells divided at least 5 times (&gt;50 cells), the HSC content of pooled clones (as measured by in vivo transplantation assays) was found to be &gt;10-fold higher in the clones generated under high vs. low SF conditions (p&lt;0.05). To characterize the types of self-renewal divisions undertaken, 9 doublets generated under the high SF condition were harvested between 4 and 8 hr after they underwent their 1st division and then each of the daughters was injected into a separate irradiated mouse. Analysis of the 18 mice showed that for one of the input cells both daughters were HSCs (evidence of a symmetric self-renewal division) and for 3 more, only one of the 2 daughters was an HSC (evidence of an asymmetric self-renewal division). In contrast no daughter HSCs were identified when 6 doublets produced under the low SF condition were assayed. To determine whether the loss of HSC activity under low SF conditions was a pre- or post-mitotic event, additional in vivo HSC assays were performed on cells harvested from individual wells after 8, 16 and 96 hours of incubation. The results revealed no change in the proportion of wells with either low or high concentrations of SF that contained HSCs after 8 hr of incubation (10/36 positive mice injected with starting single cells and 5/17 (low SF) vs. 6/17 (high SF) positive mice injected with 8-hr single cells, respectively). However, a significant difference (p&lt;0.01) was seen after 96 hr (5/35 vs. 2/43 positive mice, respectively) and, after only 16 hr, before a first mitosis was seen under either condition, a decline in HSCs was apparent under the low SF condition (4/15 vs. 1/15 positive mice injected with cells from the high vs. low SF condition). Together, these studies indicate that HSC exposure to different SF concentrations can rapidly and irreversibly alter the ability of HSCs to execute symmetric as well asymmetric self-renewal divisions in vitro.

The Role of the Antiapoptotic Protein c-FLIP in Multiple Myeloma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1966-1966
Author(s):  
Colin D. Crean ◽  
Genglin Zhang ◽  
Lauren B VanRyckeghem ◽  
Helmut Hanenberg ◽  
Sherif Farag ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Therapeutic Target ◽  
Protein C ◽  
Lentiviral Vector ◽  
Post Transplantation ◽  
Antiapoptotic Protein ◽  
Treated Water ◽  
Doxycycline Treatment

Abstract Abstract 1966 Poster Board I-989 The Effect of Targeting the Antiapoptotic Protein c-FLIP in Multiple Myeloma Background: Multiple myeloma (MM) is an incurable blood cancer. Treatments which target key proteins that play a role in drug resistance should improve treatment outcomes. The cellular FLICE-inhibitory protein (c-FLIP) is an antiapoptotic protein which confers resistance to death receptor-mediated apoptosis. Further, c-FLIP overexpression has been identified in various cancers, and in MM specifically, gene expression profiling has demonstrated that c-FLIP is overexpressed in a patient's MM cells compared to the normal plasma cells of an identical twin. However, the precise clinical significance of c-FLIP overexpression and its potential as a therapeutic target in MM have not been established. Objectives: To determine the potential of c-FLIP as a therapeutic target by exploring the in vitro and in vivo effects of c-FLIP knockdown in MM cells, using a doxycycline-inducible lentiviral vector containing c-FLIP shRNA. Results: Doxycycline treatment in vitro induced c-FLIP shRNA transcription and reduced c-FLIP levels more than 80% in H929 cells. Correspondingly, we observed a greater than 95 % reduction in the growth of H929 cells, compared to the cell line containing an empty vector control. In addition, immunoblots of MM cell lysates showed an accumulation of the cleaved products of caspases 8 and 3, suggesting that c-FLIP knockdown sufficiently induced caspase activation and apoptosis in MM cells. To explore the cytotoxicity of c-FLIP knockdown in MM cells in vivo, we used two different mouse models. In the first model, MM cells containing the doxycycline inducible, c-FLIP shRNA containing, lentiviral vector, were transplanted subcutaneously into NOD/Scid mice. Two weeks post transplantation, when accurate tumor measurements could be made, the mice were divided into two groups, of approximately equal tumor volumes, and one group was given doxycycline treated water. A significant reduction in tumor size was observed in doxycycline treated mice compared to the controls (Figure 1). In a second mouse model, NOD/Scid mice were transplanted with MM cells by tail vein injection and divided into two groups. The first group (“early dox”; n=10) was given doxycycline treated water 4 hours post transplantation, while the second group (n=20) was given regular water until they developed tumors which could be determined by measuring human IgA kappa levels in the mouse serum. Within this group, mice were randomized base on the IgA levels to receive doxycycline treatment (“late dox”; n=6) or regular water (n=6). ELISA measurements of human IgA kappa light chain from treated mice are shown in Figure 2. The “early dox” group had barely detectable IgA kappa levels while the “late dox” group had significantly lower levels than the mice that never received any doxycycline. Summary: An inducible c-FLIP shRNA system was created in order to observe the effects of c-FLIP knockdown on the viability of MM cells both in vitro and in vivo. A nearly complete c-FLIP knockdown could be generated in vitro which had a dramatic effect on the viability of the MM cells, and this translated to a significant reduction in tumor volumes when these cells were later transplanted into mice, both subcutaneously and intravenously, and then treated with doxycycline. This work provides evidence that targeting c-FLIP in MM, either directly or via gene regulation, holds significant promise. Disclosures: Abonour: Celgene: Honoraria; Millennium: Honoraria.

Cripto Selectively Expands a Distinct Population of Hematopoietic Stem Cells Expressing the Cell Surface Receptor GRP78 and Strongly Induces An Immature Phenotype In Vivo After Ex Vivo Culture

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 405-405
Author(s):  
Kenichi Miharada ◽  
Göran Karlsson ◽  
Jonas Larsson ◽  
Emma Larsson ◽  
Kavitha Siva ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Peripheral Blood ◽  
Distinct Population ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Total Bone Marrow

Abstract Abstract 405 Cripto is a member of the EGF-CFC soluble protein family and has been identified as an important factor for the proliferation/self-renewal of ES and several types of tumor cells. The role for Cripto in the regulation of hematopoietic cells has been unknown. Here we show that Cripto is a potential new candidate factor to increase self-renewal and expand hematopoietic stem cells (HSCs) in vitro. The expression level of Cripto was analyzed by qRT-PCR in several purified murine hematopoietic cell populations. The findings demonstrated that purified CD34-KSL cells, known as highly concentrated HSC population, had higher expression levels than other hematopoietic progenitor populations including CD34+KSL cells. We asked how Cripto regulates HSCs by using recombinant mouse Cripto (rmCripto) for in vitro and in vivo experiments. First we tested the effects of rmCripto on purified hematopoietic stem cells (CD34-LSK) in vitro. After two weeks culture in serum free media supplemented with 100ng/ml of SCF, TPO and 500ng/ml of rmCripto, 30 of CD34-KSL cells formed over 1,300 of colonies, including over 60 of GEMM colonies, while control cultures without rmCripto generated few colonies and no GEMM colonies (p<0.001). Next, 20 of CD34-KSL cells were cultured with or without rmCripto for 2 weeks and transplanted to lethally irradiated mice in a competitive setting. Cripto treated donor cells showed a low level of reconstitution (4–12%) in the peripheral blood, while cells cultured without rmCripto failed to reconstitute. To define the target population and the mechanism of Cripto action, we analyzed two cell surface proteins, GRP78 and Glypican-1, as potential receptor candidates for Cripto regulation of HSC. Surprisingly, CD34-KSL cells were divided into two distinct populations where HSC expressing GRP78 exhibited robust expansion of CFU-GEMM progenitor mediated by rmCripto in CFU-assay whereas GRP78- HSC did not respond (1/3 of CD34-KSL cells were GRP78+). Furthermore, a neutralization antibody for GRP78 completely inhibited the effect of Cripto in both CFU-assay and transplantation assay. In contrast, all lineage negative cells were Glypican-1 positive. These results suggest that GRP78 must be the functional receptor for Cripto on HSC. We therefore sorted these two GRP78+CD34-KSL (GRP78+HSC) and GRP78-CD34-KSL (GRP78-HSC) populations and transplanted to lethally irradiated mice using freshly isolated cells and cells cultured with or without rmCripto for 2 weeks. Interestingly, fresh GRP78-HSCs showed higher reconstitution than GRP78+HSCs (58–82% and 8–40%, p=0.0038) and the reconstitution level in peripheral blood increased rapidly. In contrast, GRP78+HSC reconstituted the peripheral blood slowly, still at a lower level than GRP78-HSC 4 months after transplantation. However, rmCripto selectively expanded (or maintained) GRP78+HSCs but not GRP78-HSCs after culture and generated a similar level of reconstitution as freshly transplanted cells (12–35%). Finally, bone marrow cells of engrafted recipient mice were analyzed at 5 months after transplantation. Surprisingly, GRP78+HSC cultured with rmCripto showed higher reconstitution of the CD34-KSL population in the recipients' bone marrow (45–54%, p=0.0026), while the reconstitution in peripheral blood and in total bone marrow was almost the same. Additionally, most reconstituted CD34-KSL population was GRP78+. Interestingly freshly transplanted sorted GRP78+HSC and GRP78-HSC can produce the GRP78− and GRP78+ populations in the bone marrow and the ratio of GRP78+/− cells that were regenerated have the same proportion as the original donor mice. Compared to cultured cells, the level of reconstitution (peripheral blood, total bone marrow, HSC) in the recipient mice was almost similar. These results indicate that the GRP78 expression on HSC is reversible, but it seems to be “fixed” into an immature stage and differentiate with lower efficiency toward mature cells after long/strong exposure to Cripto signaling. Based on these findings, we propose that Cripto is a novel factor that maintains HSC in an immature state and may be a potent candidate for expansion of a distinct population of GRP78 expressing HSC. Disclosures: No relevant conflicts of interest to declare.

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