scholarly journals Selective activation of STAT5 unveils its role in stem cell self-renewal in normal and leukemic hematopoiesis

2005 ◽  
Vol 202 (1) ◽  
pp. 169-179 ◽  
Author(s):  
Yuko Kato ◽  
Atsushi Iwama ◽  
Yuko Tadokoro ◽  
Kazuya Shimoda ◽  
Mayu Minoguchi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Leukemic Stem Cells ◽  
Myeloproliferative Disease ◽  
Self Renewal ◽  
Hematopoietic Stem

Although the concept of a leukemic stem cell system has recently been well accepted, its nature and the underlying molecular mechanisms remain obscure. Constitutive activation of signal transducers and activators of transcription 3 (STAT3) and STAT5 is frequently detected in various hematopoietic tumors. To evaluate their role in normal and leukemic stem cells, we took advantage of constitutively active STAT mutants to activate STAT signaling selectively in hematopoietic stem cells (HSCs). Activation of STAT5 in CD34–c-Kit+Sca-1+ lineage marker– (CD34–KSL) HSCs led to a drastic expansion of multipotential progenitors and promoted HSC self-renewal ex vivo. In sharp contrast, STAT3 was demonstrated to be dispensable for the HSC maintenance in vivo, and its activation facilitated lineage commitment of HSCs in vitro. In a mouse model of myeloproliferative disease (MPD), sustained STAT5 activation in CD34–KSL HSCs but not in CD34+KSL multipotential progenitors induced fatal MPD, indicating that the capacity of STAT5 to promote self-renewal of hematopoietic stem cells is crucial to MPD development. Our findings collectively establish a specific role for STAT5 in self-renewal of normal as well as leukemic stem cells.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1686-1686
Author(s):  
Hideyuki Oguro ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Target Genes ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture ◽  
Deficient Mice

Abstract The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.

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.

Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2433-2433
Author(s):  
Tarik Moroy ◽  
Cyrus Khandanpour ◽  
Joseph Krongold
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Mouse Bone Marrow ◽  
Paracrine Factors ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow. We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells. Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Targeting miRNA-551b, a "Stemness"-like microRNA, to Eradicate AML (Stem) Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1494-1494
Author(s):  
Tània Martiáñez ◽  
Noortje Van Gils ◽  
David Christian De Leeuw ◽  
Eline Vermue ◽  
Arjo Rutten ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Research Funding ◽  
Leukemic Stem Cells ◽  
Hematopoietic Stem ◽  
Inhibition Of Proliferation ◽  
Novel Target

Abstract Despite high complete remission (CR) rates achieved after chemotherapy, only 30-40% of patients with Acute Myeloid Leukemia (AML) survive five years after diagnosis. The main cause of this treatment failure is insufficient eradication of a subpopulation of chemotherapy-resistant leukemia cells with stem cell properties, named "leukemic stem cells" (LSCs). LSCs use a variety of mechanisms to resist chemotherapy and targeting them is one of the major challenges in AML treatment. Since miRNAs can target multiple genes/pathways simultaneously, their modulation (downregulation or upregulation) may have great potential for the successful elimination of therapy-resistant leukemic (stem) cells (Martiañez Canales et al. Cancers 2017). Here, we show that miRNA-551b, previously identified by us as a stem cell-like miRNA, can be a potential novel target to specifically eradicate AML stem-like cells. Aiming at identification of miRNA-based therapy to specifically eradicate LSCs, while sparing normal Hematopoietic Stem Cells (HSCs), we determined expression of miRNAs in normal HSCs, Leukemic Stem Cells (LSCs) and leukemic progenitors (LP) all derived from the same AML patient's bone marrow. Using this approach, we identified miRNA-551b as being highly expressed in normal HSCs residing both in healthy and AML bone marrows. In AML, high expression of miR551b demonstrated to be associated with an adverse prognosis. Moreover, miRNA-551b was highly expressed in immature AML cases and its expression in a cohort of patients coincided with the expression of stem cell genes (De Leeuw et al. Leukemia 2016). To further elucidate the link between miRNA-551b and AML "stemness" and to test whether downregulation of miRNA-551b affects the survival of AML (stem/progenitor) cells, proliferation and the balance between differentiation and "stemness", we reduced miRNA-551b expression, either by lentiviral transduction of antagomirs or by adding locked nucleotide acid (LNA)-oligonucleotides to AML cell lines and primary AML cells. Downregulation of miRNA-551b in the stem cell-like AML cell line KG1a led to inhibition of cell growth in vitro, which was due to inhibition of proliferation rather than induction of apoptosis. KG1a tumor growth in an in vivo mouse model was also reduced when miRNA-551b was downregulated. In primary AML, miRNA-551b knockdown resulted in a significant decrease in the survival of leukemic progenitors and LSCs, while hematopoietic stem cells (HSCs) and normal progenitors from healthy bone marrows were not affected. These results suggest that a therapeutic approach inhibiting miRNA-551b expression might specifically eradicate leukemic progenitors and LSCs from primary AML, while sparing HSCs. We are currently studying miRNA-551b targets which can be responsible for this specific LSCs elimination. In conclusion, our results suggest that inhibition of miRNA-551b could be a promising approach to eliminate stem cell-like AML cells, thereby decreasing relapse rates and improving AML treatment outcome. Disclosures Ossenkoppele: Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Jazz: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Karyopharm: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding; Genmab: Research Funding.

scholarly journals Aging of hematopoietic stem cells

Blood ◽  
2018 ◽  
Vol 131 (5) ◽  
pp. 479-487 ◽  
Author(s):  
Gerald de Haan ◽  
Seka Simone Lazare
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Blood Cells ◽  
Aging Process ◽  
Molecular Mechanisms ◽  
Cell Aging ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Stem Cell Aging

Abstract Hematopoietic stem cells (HSCs) ensure a balanced production of all blood cells throughout life. As they age, HSCs gradually lose their self-renewal and regenerative potential, whereas the occurrence of cellular derailment strongly increases. Here we review our current understanding of the molecular mechanisms that contribute to HSC aging. We argue that most of the causes that underlie HSC aging result from cell-intrinsic pathways, and reflect on which aspects of the aging process may be reversible. Because many hematological pathologies are strongly age-associated, strategies to intervene in aspects of the stem cell aging process may have significant clinical relevance.

Selective Activation of STAT5 Unveils Its Role in the Maintenance of Hematopoietic Stem Cells and the Development of Myeloproliferative Disorder.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3549-3549
Author(s):  
Yuko Kato ◽  
Atsushi Iwama ◽  
Hiromitsu Nakauchi
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Janus Kinase ◽  
Selective Activation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Lineage Differentiation

Abstract Recent studies have implicated the janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in the maintenance of stem cells, such as mouse embryonic stem cells and Drosophila germ cells. We have previously reported that thrombopoietin (TPO) can support in vitro self-renewal division of murine hematopoietic stem cells (HSCs) (CD34−/lowc-Kit+Sca-1+lineage marker-negative; CD34−KSL cells). Signal transducers and activators of transcription 5 (STAT5) is one of the major signaling molecules that mediate TPO signals. All these findings suggest that STAT5 could be an attractive candidate for therapeutic manipulation of HSCs. Cytokines activate JAK/STAT5 pathway along with other signaling pathways, causing difficulty to dissect STAT5-specific functions in hematopoietic stem cells (HSCs). Here we took advantage of constitutively active STAT5 mutants to selectively activate STAT5 signaling pathway in HSCs. The mutants used are STAT5A 1*6 that harbors two amino acid mutations S710F and H298R in the effecter domain, and STAT5A #2 that harbors a point mutation N642H in the SH2 domain. Retroviral transduction of either STAT5 1*6 or STAT5#2 mutant into purified CD34−KSL HSCs caused a drastic expansion of multipotential progenitors in vitro and promoted multi-lineage differentiation in vitro. During 7 days of culture supplemented with SCF and TPO, the number of high proliferative potential colonies (HPPC) increased ten-fold compared with the GFP control and half of them were derived from multipotential progenitor cells. Notably, even in the culture supplemented with SCF only, expression of STAT5 mutants in HSCs supported a similar mode of expansion of progenitors cells and multi-lineage differentiation, indicating that activation of STAT5 can substitute major biological effects of TPO in HSCs. In all in vitro experiments, STAT5 1*6 showed stronger effects than STAT5#2. To evaluate the effect of STAT5A mutants in the maintenance of long-term bone marrow repopulating HSC ex vivo, cultured transduced cells corresponding to 30 initial CD34−KSL HSCs were transplanted into lethally irradiated mice 7 to 10 days after transduction. Although rapid hamatopoietic repopulation was observed with HSCs expressing STAT5A 1*6, mice developed myeloproliferative disease (MPD) and succumbed to death within two months. In contrast, HSCs expressing STAT5A #2 presented significantly higher long-term repopulating capacity than the GFP control. These data indicate that selective activation of STAT5 maintains long-term repopulating ability of HSCs ex vivo. Oncostatin M, a well known STAT5 target gene, has been postulated to be involved in the development of MPD and was actually induced STAT5A 1*6-expressing cells. However, transplantation of OSM−/− HSCs expressing STAT5A 1*6 similarly caused a lethal MPD in wild-type mice, indicating that Oncostatin is not the main target for STAT5 in MPD development. Taken together, our findings establish a role for STAT5 in the self-renewal of HSCs and provide STAT5 as novel target for therapeutic manipulation of HSCs ex vivo.

Direct Evidence for Ex Vivo Expansion of Human Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 798-798
Author(s):  
Kiyoshi Ando ◽  
Takashi Yahata ◽  
Tadayuki Sato ◽  
Hiroko Miyatake ◽  
Hideyuki Matsuzawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Scid Mouse ◽  
Direct Evidence ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (Exp Hematol, 27:904–915, 1999), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Direct evidence for ex vivo expansion of human hematopoietic stem cells

Blood ◽  
2006 ◽  
Vol 107 (8) ◽  
pp. 3371-3377 ◽  
Author(s):  
Kiyoshi Ando ◽  
Takashi Yahata ◽  
Tadayuki Sato ◽  
Hiroko Miyatake ◽  
Hideyuki Matsuzawa ◽  
...  
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Direct Evidence ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Ex Vivo Expansion ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Asymmetric Divisions

AbstractTo characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

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