Vascular Pericytes Sustain Hematopoietic Stem Cells

Abstract Abstract 2394 Mesenchymal stromal (or stem-) cells (MSC) are culture-selected, heterogeneous supporting cells that can differentially regulate hematopoietic stem cell (HSC) behavior in vitro. The elusive identity of native MSC has obscured the contribution, if any, of these cells to HSC support in vivo. Having previously demonstrated that vascular pericytes (ubiquitous cells encircling endothelial cells in capillaries and microvessels) are ancestors of human MSC, we now hypothesize that pericytes are a critical component of the HSC “niche”. Consequently, pericyte isolation from total stroma would allow to develop co-culture systems for human HSC maintenance. In the present study, human cord blood CD34+ cells were cultured onto confluent human pericytes isolated from adipose tissue as CD146+CD34-CD45-CD56- cells. Co-culture of CD34+ cells on pericytes, for up to 6 weeks in the absence of any added growth factor, produced significantly i) higher numbers of CD45+ and CD34+ cells (p<0.05), ii) higher percentages of primitive CD34+CD33-CD10-CD19- progenitors (p<0.05), iii) higher percentages of single- and multi-lineage CFU (p<0.05) and iv) lower percentages of mature myeloid and lymphoid cells (p<0.05), compared to control co-cultures on unfractionated adipose stromal cells (ASC) (n=10 individual experiments, n=4 biological replicates). Most importantly, only pericytes could maintain HSC with self-renewal and long-term repopulating potential, as demonstrated by transplantation into primary and secondary NOD/SCID/IL2Rg−/− mouse recipients (n=3 individual experiments). In the latter setting, none of the mice receiving CD34+ cells co-cultured with ASC engrafted (n=10), whereas all recipients of CD34+ cells cultured in the presence of pericytes developed lympho-myeloid hematopoietic human cells (n=10). Altogether, these results support the hypothesis that pericytes maintain hematopoietic cell stemness. Conversely, unfractionated stromal cell cultures may promote HSC differentiation at the expense of self-renewal. Both tentative scenarios were explored. Co-cultures with pericytes in a transwell system revealed that cell-to-cell contact is required for HSC survival. Since Notch signaling regulates stem cell maintenance by inhibiting cell differentiation through cell-cell interactions, we hypothesized that pericytes purified from total stroma express specific Notch ligands. As shown by qPCR, the expression of Jagged-1 is 2 fold higher in pericytes compared to unfractionated ASC. Addition of a Notch inhibitor (DAPT) to pericyte/HSC co-cultures resulted in the significant reduction of CFU numbers (p<0.05) and increase in B-cell development. Furthermore, increased myeloid differentiation was observed when ASC conditioned medium was added to pericytes/HSC co-cultures. In conclusion, we demonstrate that vascular pericytes sustain HSC by promoting survival and preventing differentiation via cell-to-cell interactions involving Notch activation, whereas unfractionated stroma promotes HSC differentiation through a paracrine mechanism. We thus infer that HSC-supporting stromal cells are not confined within blood-forming organs (similar observations, not reported here, have been made on skeletal muscle pericytes). This novel concept is not easy to reconcile with normal hematopoiesis, but may be highly relevant in the context of the dissemination of malignant hematopoietic cells. Of important note, adipose tissue used in this study represents a convenient, safe and often abundant source of autologous therapeutic cells. Therefore, human fat-derived pericytes emerge as a candidate cell product for HSC ex vivo manipulation in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Expansion of Cord Blood Stem Cells and Enhancing Their Mobilization and Homing Potential Using Mesenchymal Stromal Cells

Blood ◽

10.1182/blood-2018-99-117779 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3344-3344

Author(s):

Julian Cooney

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stromal Cells ◽

Molecular Level ◽

Ex Vivo ◽

Sox9 Expression ◽

Self Renewal ◽

Hematopoietic Stem ◽

Coculture System ◽

Cd34 Cells

Abstract Umbilical cord blood (CB) contains hematopoietic stem cells (HSC). CB can be collected easily, cryopreserved and be readily available from CB banks when needed. Transplantation with CB is associated with less graft-versus-host disease (GvHD) when compared to peripheral blood (PB) and bone marrow (BM) HSCT. Attempts have been made to overcome the issue of low and insufficient number of CB stem cells. Mesenchymal stromal cells (MSCs) has been proven as immune modulators in GvHD and other immunological diseases. MSCs may enhance HSC engraftment to improve hematopoietic recovery. There is compelling evidence that MSCs produce a wide range of cytokines that are capable of maintaining HSC in a quiescent state and other cytokines which induces proliferation and self-renewal of HSC. Recently, it has been shown that expansion of HSC in unfractionated CB is markedly enhanced by co-culture with MSCs, with the time to neutrophil engraftment and platelet recovery markedly shortened. CB stem cells expanded with allogeneic MSCs then transplanted into patients led to multiple times expansion for both total nucleated cells and CD34+ cells in vivo when compared to unmanipulated CB stem cells. Therefore, expansion of CB stem cells with MSCs has great potential. The exact mechanism of how CB cells expand at the molecular level remains unknown. This study's aim was to expand CB in an ex vivo coculture system using MSCs and selected growth factors (GFs) and to investigate expansion at the molecular level. In this study, CB cells were expanded in a coculture system for two weeks using BM-derived MSCs with and without GFs (SCF, TPO, Flt3-L, G-CSF and IL6). A 69.9 fold expansion in CB total nucleated cells and 53.2 fold expansion in CD34+ cells was achieved after 12 days using MSCs as feeders in 50 ng/ml Flt3-L, SCF, TPO, G-CSF, and IL6. The viability of CB cells was significantly higher when cocultured with MSCs regardless of GFs addition. CB expanded on MSCs expressed higher percentages of CD45+CD34+CXCR4+ and CD45+CD34+EpHB4+ populations. Interestingly, the HSC maintenance marker EpHB4 expressed by MSCs at low levels decreased significantly in the coculture system. To investigate MSCs effect on CB expression levels of other genes that are important for expansion, stemness and pluripotency hematopoietic lineage commitment, RUNX1, SOX17, HOXC8, Myc, TP53, SOX9, FOXO1, FOXO4, GATA1 genes were examined. The study demonstrated that HOXC8, SDF-1, SOX17 and SOX9 expression was suppressed in CB cells, while the expression of other genes such as CXCR4, EpHB4, FOXO1, Myc, and HPRT1 increased. The undetectable level of HOXC8 expression, which regulates self-renewal and differentiation of stem cells, may have caused less CB stem cell differentiation, favoring an increase in CD34+ cells. Equally, SOX17 and SOX9 expression, which also decreased to undetectable levels in CB cells post coculture with MSCs, plays a significant role in CB expansion. SOX17 is known as a primer of hemogenic potential, regulating hematopoietic development from hESCs/iPSCs, whereas SOX7 plays an important role in HSC differentiation. Therefore, we speculate that MSCs-CB coculture dampens the differentiation of adjacent CB cells, associated with changes of signaling in BM-like niches. Simultaneously, MSCs may be sending signalling messages to CB cells in the ex vivo niche to maintain quiescence or self-renewal capacity through other signalling pathways. FOXO family has the ability to regulate stem cells and program them to remain quiescent through cell-cycle repression via the oxidative stress-activated P66shc-Akt-FOXO pathway. In this study, FOXO1 was upregulated in CB cells cultured with MSCs supporting our hypothesis and other earlier findings (decrease in the expression of EpHB4 on MSCs and a decline in the expression of SDF1 in CB cells), suggesting that MSCs have the ability to induce stem cell quiescence and maintain stemness. In conclusion BM-derived MSCs support CB viability, expansion, and increased stemness potential, by modifying key hematopoietic, progenitor, differentiation and stemness signaling pathways. The study identified HOXC8, SDF-1, SOX17, SOX9, CXCR4, EpHB4, FOXO1, Myc, and HPRT1 as potential factors involved in CB expansion signaling pathways. Using MSCs as a feeder layer resulted in higher CB viability and proliferation rates which may increase the potential use of CB in hematopoietic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

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AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates

Blood ◽

10.1182/blood-2005-09-3592 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3772-3778 ◽

Cited By ~ 142

Author(s):

André Larochelle ◽

Allen Krouse ◽

Mark Metzger ◽

Donald Orlic ◽

Robert E. Donahue ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Hematopoietic Stem Cells ◽

Genetic Manipulation ◽

Retroviral Vectors ◽

Lymphoid Cells ◽

Alternative Source ◽

Hematopoietic Stem ◽

Cd34 Cells

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34+ hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34+ cells were phenotypically analyzed, marked with NeoR-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34+ cells with NeoR gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34+ cells are in the G1 phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34+ cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34+ cells were better than those obtained using CD34+ cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34+ cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

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Roles of Ring1A/B on Stem Cell Potential of MOZ and Other Acute Myeloid Leukemias,

Blood ◽

10.1182/blood.v118.21.3998.3998 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3998-3998

Author(s):

Haruko Shima ◽

Mika Shino ◽

Kazutsune Yamagata ◽

Yukiko Aikawa ◽

Haruhiko Koseki ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Differentiation ◽

Conflicts Of Interest ◽

Expression Profiles ◽

Leukemia Stem Cells ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cell Functions ◽

Acute Myeloid

Abstract Abstract 3998 Leukemia and other cancers possess self-renewing stem cells that help maintain cancer. Chromosomal translocations are often involved in the development of human acute myeloid leukemia (AML). The monocytic leukemia zinc finger (MOZ) gene is one of the targets of such translocations. While MOZ is essential for the self-renewal of hematopoietic stem cells, the leukemia associated MOZ-fusion proteins enable the transformation of non–self-renewing myeloid progenitors into leukemia stem cells. Ring1A and Ring1B are catalytic subunits of the polycomb-group repressive complex 1 (PRC1) complex containing Bmi1, and PRC1 complex plays an important role in the regulation of stem cell self-renewal. Using Ring1A-null and Ring1B-conditional deficient mice, we showed that Ring1A/B are required for continuous colony forming ability that is enabled by MOZ-TIF2 and other AML-associated fusions such as MLL-AF10, AML1-ETO, and PML-RARα. Furthermore, MOZ-TIF2- and MLL-AF10-induced AML development in mice were prevented by Ring 1A/B deficiency. To clarify the mechanism of stemness regulation in AML stem cells by PRC1 complex, we compared gene expression profiles of Ring1A/B deleted and non-deleted MOZ-TIF2-induced AML cells. As expected, Ink4a/Arf, a known major target of PRC1 complex involved in stem cell functions, was derepressed by deletion of Ring1A/B. Although deletion of Ink4a/Arf in Ring1A/B deficient AML cells partially restored colony formation ability, it was not substantial to initiate leukemia in recipient mice. Among several target genes which were derepressed by deletion of Ring1A/B, we focused on “Stemness inhibitory factor (SIF)”, known to be required for cell differentiation and morphogenesis in some specific organs. Enforced expression of SIF in MOZ-TIF2-induced AML cells stimulated differentiation of AML progenitors into macrophages. On the other hand, knock-down of SIF blocked cell differentiation block and restored the immortalizing ability of MOZ-TIF2-induced AML progenitors, despite of the absence of Ring1A/B. Collectively, our data demonstrate that Ring 1A/B play crucial roles in the maintenance of AML stem cells through repression of SIF, which strongly promote differentiation of leukemia stem cells. Disclosures: No relevant conflicts of interest to declare.

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JAK2 Inhibitors Do Not Affect Stem Cells Present in the Spleens of Patients with Myelofibrosis

Blood ◽

10.1182/blood.v120.21.1752.1752 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1752-1752

Author(s):

Xiaoli Wang ◽

Joseph Tripodi ◽

Jesse Novak ◽

Min Lu ◽

Yan Li ◽

...

Keyword(s):

Stem Cells ◽

Chromosomal Abnormality ◽

Conflicts Of Interest ◽

Clinical Investigation ◽

Lymphoid Cells ◽

Hematopoietic Stem ◽

Cytogenetic Abnormalities ◽

Jak2 Inhibitor ◽

Cd34 Cells ◽

Jak2 Inhibitors

Abstract Abstract 1752 Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by abnormal trafficking of hematopoietic stem cells (HSC) and hematopoietic progenitor cells (HPC), resulting in their constitutive mobilization and the establishment of extra-medullary hematopoiesis. At present, there is no known therapeutic approach capable of altering the natural history of MF, except for allogeneic stem cell transplantation. Treatment with JAK2 inhibitors has been shown to lead to a rapid and dramatic reduction of splenomegaly although having only a modest effect on the JAK2V617F allele burden and not resulting in the elimination of cytogenetic abnormalities or correction of histopathological abnormalities (Verstovsek S, et al. N Engl J Med. 2010; 363:1117-27). To date, the mechanism underlying the reduction of splenomegaly observed following the treatment with a JAK2 inhibitor remains the subject of speculation. Recently, we observed the presence of MF-stem cells (MF-SC) in the spleens of MF patients and demonstrated that these splenic MF-SCs have a distinct differentiation program that distinguishes them from MF peripheral blood (PB) counterparts (Wang X, et al. Journal of Clinical Investigation, 2012. In Press). We therefore explored the effect of a JAK2 Inhibitor, AZD1480, on splenic MF-SCs in order to provide an explanation for the dramatic effects on MF spleen size. Treatment of splenic or PB MF CD34+ cells with cytokines+AZD1480 (CAZD, 150nM) for 3 days resulted in a significant reduction in the number of total cells, CD34+ cells and assayable HPC (CFU-GM, BFU-E and CFU-Mix) as compared with splenic or PB MF CD34+ cells treated with cytokines alone (CA), respectively (P all <0.05, n=6). Moreover, the numbers of CD34+CD90+ cells and CD34+CXCR4+ cells generated in cultures of splenic MF CD34+ cells with CAZD were each half of that achieved in cultures containing CA. The numbers of CD34+CD90+ cells and CD34+CXCR4+ cells present in cultures of PB MF CD34+ cells with CAZD were each 1/3 of that observed in cultures with CA. However, exposure of splenic or PB MF CD34+ cells to CAZD did not result in an alteration of the proportion of JAK2V617F positive HPCs. Furthermore, the treatment of splenic MF CD34+cells with CAZD did not affect the number of colonies with a marker chromosomal abnormality. We have reported that the transplantation of PB MF CD34+ cells into NOD/SCID/IL2R γnull mice resulted in a limited degree of donor cell chimerism and a differentiation program skewed toward myeloid lineages (Wang X, et al. Blood. 2010; 116: 5972–5982), while the transplantation of splenic MF CD34+ cells achieved a higher level of chimerism and generated both myeloid and lymphoid cells which contained molecular or cytogenetic abnormalities indicating their malignant origin. Only splenic MF CD34+ cells were able to sustain hematopoiesis for prolonged time periods (9 months) and were able to engraft secondary recipients. These data document the existence of MF-SCs that reside in the spleens of MF patients (Wang X, et al. Journal of Clinical Investigation 2012. In Press). We, therefore, examined the effect of AZD1480 on splenic MF-SCs by analyzing the behavior of splenic MF SCID repopulating cells following the transplantation of splenic MF CD34+ cells treated with CA or CAZD (n=3) into NOD/SCID/IL2R γnull mice. Six months after transplantation of splenic MF CD34+ cells treated with CA or CAZD, similar numbers of human CD45+ cells were detected in both the BM and spleens of recipient mice, respectively. Furthermore, human CD45+ cells were harvested and isolated from the BM and spleen of the mice receiving splenic CD34+ cells treated with CA or CAZD from a MF patient who had a deletion of the long arms of chromosome 20 [del (20q)] and the presence of del(20q) in human CD45+ cells were determined. All the human BM CD45+ cells isolated from the mice transplanted with splenic CD34+ cells treated with either CA or CAZD had this specific chromosomal abnormality, del (20q). These findings suggest that JAK2 inhibitor treatment does not affect splenic MF-SCs, indicating that the rapid reduction in splenomegaly following such therapy or it enlargement following the cessation of such therapy is either due to the effects of JAK2 inhibitors on more differentiated hematopoietic cells or on the splenic microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Myelodysplastic Syndrome-Derived Stromal Cells Support Leukemia-Initiating Cells and Contradirectly Eliminate Normal CD34+ Clonogenic Cells

Blood ◽

10.1182/blood.v122.21.1219.1219 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1219-1219

Author(s):

Hiroto Horiguchi ◽

Masayoshi Kobune ◽

Shohei Kikuchi ◽

Satoshi Iyama ◽

Kohichi Takada ◽

...

Keyword(s):

Stem Cell ◽

Myelodysplastic Syndrome ◽

Progenitor Cells ◽

Stromal Cells ◽

Conflicts Of Interest ◽

Methylation Status ◽

Gene Expressions ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Clonogenic Cells

Abstract Introduction The failure of normal hematopoiesis in myeloid neoplasm could be induced by a variety of mechanism. Regarding myelodysplastic syndrome (MDS)/acute leukemia (AML), aberrant hematopoietic stem/progenitor cells with exhibiting ineffective hematopoiesis and impaired differentiation ability gradually substitute it for normal hematopoietic stem/progenitor cells during a long term as a consequent of replacement of stem cell niche. However, it has not yet been clarified precise mechanism how MDS stem/progenitor cells could replace normal hematopoietic stem/progenitor cells. Methods In an attempt to analyze the supporting activity of bone marrow (BM) stromal cells, we first established the MDS/AML-derived stromal cells and healthy volunteer (HV)-derived-stromal cells. Next, MDS/AML-derived CD34+ cells or normal CD34+ cells were cocultured with established stromal cells using cytokines including stem cell factor, thrombopoietin, flt3-ligand in the presence of notch ligand (for normal CD34+ cells) or IL-3 (for AML/MDS derived cells). Subsequently, we analyzed clonogenic cells after 2 weeks coculture, 5 week cobblestone area-forming cells (CAFC) and repopulating cells in immunedeficient mice (NSG mice). Results The support of clonogenic cells after 2 weeks coculture and 5 weeks CAFCs was observed after coculture with normal CD34+ cells and HV-derived stromal cells. Furthermore, these cocultured cells engrafted into immunedeficient mice. Interestingly, the number of colony-forming units (CFU) mixed cells (MIXs) and CAFC derived from CD34+ cells was drastically reduced after coculture with MDS/AML-derived stromal cells. Nevertheless, MDS/AML-derived stromal cells support the proliferation of leukemia-initiating cells (L-ICs) and L-ICs were detected after third replating. These results indicate that MDS/AML-derived stromal cells preferentially support leukemia stem/progenitor cells, but not normal CD34+ cells. We compared the mRNA expression between (HV)-derived-stromal cells, MDS/AML-derived stromal cells and 5-aza-dC-treated stromal cells. The expression of several factors including hedgehog-interacting protein (HHIP) was reduced in MDS/AML-derived stromal cells. 5-aza-dC treatment restored the expression in some of genes and the stromal supporting activity for normal CD34+ cells partially recovered. Conclusion These results suggest that reduction of several gene expressions was detected in MDS/AML stromal cells by changes of methylation status. The epigenetic alteration of stromal genome may be involved in the progression of myeloid disorders. Disclosures: No relevant conflicts of interest to declare.

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Mir-125a Confers Multi-Lineage Long-Term Repopulating Stem Cell Activity to Human Hematopoietic Committed Progenitors

Blood ◽

10.1182/blood.v126.23.900.900 ◽

2015 ◽

Vol 126 (23) ◽

pp. 900-900 ◽

Cited By ~ 1

Author(s):

Eric R. Lechman ◽

Karin G. Hermans ◽

Erwin M. Schoof ◽

Aaron Trotman-Grant ◽

Stephanie M Dobson ◽

...

Keyword(s):

Stem Cell ◽

B Cell ◽

Conflicts Of Interest ◽

Cell Activity ◽

Lymphoid Cells ◽

Cell Populations ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Self Renewal ◽

Hematopoietic Stem

Abstract Recent studies have shown that several miRNA are differentially expressed in hematopoietic stem cells (HSC) and involved in regulating self-renewal, pointing to a new axis of epigenetic control of HSC function. Murine studies have documented a role for miR-125a in regulating HSC as miR-125a enforced expression augments self-renewal. We examined whether these attributes are evolutionarily conserved within human hematopoiesis. Lentiviral vectors over-expressing miR-125a (miR-125OE) were developed and HSC function was investigated using xenotransplantation of CD34+ CD38- human umbilical cord blood (CB) hematopoietic stem and progenitor cells (HSPCs). miR-125OE resulted in significantly increased human bone marrow (BM) chimerism at 12 and 24 weeks post-transplantation and splenomegaly. Within enlarged spleens, there were significantly increased proportions of CD34+CD19+CD10+CD20-B lymphoid cells suggesting a partial B cell differentiation block at the pro-B cell stage. In the BM, CD41+ megakaryocytes, GlyA+ erythroid and CD3+ T cell populations were significantly expanded. Within the primitive compartment, multi-lymphoid progenitors (MLP) were massively expanded by 12 weeks, followed by a combined reduction of immuno-phenotypic HSC and multi-potent progenitors (MPP) by 24 weeks. Given this loss of immuno-phenotypic HSC, we wondered whether stem cell function was compromised in vivo. Secondary transplantation with limiting dilution (LDA) revealed that stem cell frequencies were increased by 4.5 fold in miR-125OE recipients. Using lentivirus sponge-mediated inhibition of miR-125 (miR-125KD) in CD34+CD38-human CB, we were able to directly link these effects to miR-125: B cells increased at the expense of T cells; immuno-phenotypic HSC increased with a concomitant loss of MLP; and functional HSC were decreased by 2.5 fold using secondary LDA assays. Together, these data strongly suggest that miR-125a expression levels regulate human HSC self-renewal and lineage commitment. Since HSC frequency increased so substantially upon miR-125OE, we asked whether more committed cell populations might also be endowed with enhanced self-renewal. Highly purified populations of HSC, MPP and MLP and CD34+CD38+ committed progenitors were transduced and transplanted cells into xenografts. Unexpectedly, miR-125OE transduced CD34+CD38+ progenitors produced a substantial graft after 12 weeks. Control transduced CD34+CD38+ cells did not engraft and only control transduced HSC generated a disseminating graft in recipient mice. miR-125OE transduced HSC and MPP generated robust engraftment, while MLP did not. In all cases, xenografts generated by CD34+CD38+ and MPP transduced with miR-125OE showed multi-lineage repopulation. Moreover, the miR-125OE grafts from CD34+CD38+ and MPP recipients were durable as secondary transplantation generated multi-lineage grafts for at least 20 weeks in 5/7 and 6/10 recipients, respectively; no control transduced groups generated secondary grafts. Thus, the enhancement of self-renewal by enforced expression of miR-125a occurs not only in HSC, but also in MPP and to an as yet unidentified subpopulation within the CD34+38+ committed progenitor compartment. Using protein mass spectrometry, we identified and validated a miR-125a target network in CD34+ CB that normally functions to restrain self-renewal in more committed progenitors. Together, our data suggest that increased miR-125a expression can endow an HSC-like program upon a selected set of non-self-renewing hematopoietic progenitors. Our findings offer the innovative potential to use MPP with enhanced self-renewal to augment limited sources of HSC to improve clinical outcomes. Disclosures No relevant conflicts of interest to declare.

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High Mobility Group A1 Chromatin Remodeling Protein Regulates Self-Renewal, Niche Formation, and Regenerative Function in Adult Stem Cells through Wnt/β-Catenin Signaling

Blood ◽

10.1182/blood.v128.22.2647.2647 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2647-2647 ◽

Cited By ~ 1

Author(s):

Linda Resar ◽

Lingling Xian ◽

Tait Huso ◽

Amy Belton ◽

Leslie Cope ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Chromatin Remodeling ◽

Adult Stem Cells ◽

Cell Function ◽

High Mobility ◽

Transcriptional Networks ◽

Self Renewal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Introduction: Nuclear chromatin structure is a key determinant of stem cell function and cell fate, although factors that regulate this are only beginning to emerge. While High Mobility Group A1(HMGA1) chromatin remodeling proteins are among the most abundant, nonhistone chromatin binding proteins in adult stem cells (ASCs), their role in this setting has been unknown. HMGA1/2 proteins modulate gene expression by binding to DNA, bending chromatin, and recruiting transcription factor complexes to enhancers throughout the genome. The HMGA1 gene is highly expressed during embryogenesis with low or undetectable levels in mature, differentiated tissues. In cancer, HMGA1 re-expression occurs through oncogenic transcription factors, other epigenetic alterations, or in rare cases, chromosomal translocation events. Importantly, HMGA1 levels correlate with adverse clinical outcomes in diverse malignancies. We previously reported that Hmga1 transgenic mice develop leukemic transformation by inducing transcriptional networks involved in stem cell function and cell cycle progression. Methods: To elucidate the role of Hmga1 in normal development and ASCs in vivo, we generated mouse models with transgenic overexpression or deletion of Hmga1. To define the function of Hmga1 in adult stem cells (ASCs), we used gain-of-function (overexpression) and loss-of-function (silencing or genetic deletion) approaches in human and murine intestinal stem cells (ISCs) and hematopoietic stem and progenitor cells. Results:Transgenic mice overexpressing Hmga1 in ISCs develop hyperproliferation, aberrant crypt formation, and polyposis in the intestinal epithelium by expanding the ISC and niche compartments. Hmga1 enhances self-renewal in ISCs by amplifying Wnt/β-catenin signaling, inducing genes that encode both Wnt agonist receptors and downstream Wnt effectors. Surprisingly, Hmga1 also "builds" an epithelial niche by directly up-regulating Sox9 to induce Paneth cell differentiation. Paneth cells constitute the epithelial ISC niche by secreting Wnt agonists. This is the first example of Hmga1 fostering terminal differentiation to establish a stem cell niche. In human intestine, HMGA1 and SOX9 are highly correlated, and both become up-regulated in colorectal cancer. Human CD34+ cells engineered to overexpress Hmga1 expand more efficiently, while those with Hmga1 deficiency have defective proliferation and colony forming capability. Both colony number and size were decreased, and differentiation was skewed towards myeloid lineages. In mice, Hmga1 deletion causes partial embryonic lethality; over 50% of expected offspring die before mid-gestation. Those that survive develop premature aging phenotypes with early kyphosis, decreased bone density, grip strength, gait velocity, and hearing deficits. Knock-out mice also have early thymic aplasia, decreased numbers of early T-cell precursors, as well as decreased B-cell differentiation. Long-term (LT)-hematopoietic stem cells were decreased and preliminary data suggests aberrant regenerative function in serial, competitive transplant experiments.Preliminary ChIP-seq and gene expression studies in CD34+ cells suggest that Hmga1 regulates transcriptional networks involved in Wnt, JAK-STAT, and PI3K signaling. Conclusions:Our results in ASCs reveal a novel role for Hmga1 in tissue homeostasis by inducing pathways involved in Wnt and regenerative function. In ISCs, Hmga1 maintains both the stem cell pool and niche compartment whereas deregulated Hmga1 may perturb this equilibrium during carcinogenesis. Functional studies in HSCs suggest that Hmga1 also regulates self-renewal, regenerative potential, and the capacity for balanced differentiation. These findings indicate that HMGA1 is required for normal stem cell function, both during embryogenesis, and postnatally, in ASCs. Our prior work in tumor models demonstrates that a subset of HMGA1 stem cell pathways are hi-jacked by cancer cells to drive tumor progression. Together, these studies provide compelling rationale for further research to determine how to harness HMGA1 for regenerative medicine and to target it in cancer therapy. Disclosures No relevant conflicts of interest to declare.

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Evidence for MPL W515L/K mutations in hematopoietic stem cells in primitive myelofibrosis

Blood ◽

10.1182/blood-2007-05-089003 ◽

2007 ◽

Vol 110 (10) ◽

pp. 3735-3743 ◽

Cited By ~ 66

Author(s):

Ronan Chaligné ◽

Chloé James ◽

Carole Tonetti ◽

Rodolphe Besancenot ◽

Jean Pierre Le Couédic ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Lymphoid Cells ◽

Molecular Event ◽

Erythroid Progenitors ◽

Normal Response ◽

Hematopoietic Stem ◽

Nonobese Diabetic ◽

Cd34 Cells

Abstract The MPL (W515L and W515K) mutations have been detected in granulocytes of patients suffering from certain types of primitive myelofibrosis (PMF). It is still unknown whether this molecular event is also present in lymphoid cells and therefore potentially at the hematopoietic stem cell (HSC) level. Toward this goal, we conducted MPL genotyping of mature myeloid and lymphoid cells and of lymphoid/myeloid progenitors isolated from PMF patients carrying the W515 mutations. We detected both MPL mutations in granulocytes, monocytes, and platelets as well as natural killer (NK) cells but not in T cells. B/NK/myeloid and/or NK/myeloid CD34+CD38−-derived clones were found to carry the mutations. Long-term reconstitution of MPL W515 CD34+ cells in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice was successful for as long as 12 weeks after transplantation, indicating that MPL W515 mutations were present in HSCs. Moreover, the 2 MPL mutations induced a spontaneous megakaryocytic growth in culture with an overall normal response to thrombopoietin (TPO). In contrast, erythroid progenitors remained EPO dependent. These results demonstrate that in PMF, the MPL W515L or K mutation induces a spontaneous megakaryocyte (MK) differentiation and occurs in a multipotent HSCs.

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Gpx3 Determines Competitiveness of Normal and Leukemic Stem Cells.

Blood ◽

10.1182/blood.v116.21.1587.1587 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1587-1587 ◽

Cited By ~ 1

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.

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Utility of Plerixafor In Addition to Chemotherapy and G-CSF Mobilization Regimens

Blood ◽

10.1182/blood.v116.21.4443.4443 ◽

2010 ◽

Vol 116 (21) ◽

pp. 4443-4443

Author(s):

Farrukh Awan ◽

David Deremer ◽

Elaine Mebel ◽

Samith Thomas Kochuparambil ◽

Anand P. Jillella

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Peripheral Blood ◽

Conflicts Of Interest ◽

Chemotherapeutic Agents ◽

Phase Iii ◽

Cell Transplant ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Significant Difference

Abstract Abstract 4443 Introduction: Various chemotherapeutic agents particularly cyclophosphamide (CY) are utilized in combination with growth factors in an attempt to increase the number of stem cells available for collection in the peripheral blood. Plerixafor (P) is a reversible antagonist of CXCR4 and interrupts its interaction with SDF-1. This results in a rapid release of hematopoietic stem cells from the marrow to the circulation. Recent pivotal phase III trial data has established the efficacy of P in combination with G-CSF (G) in patients who had failed prior attempts at stem cell collection. However, there is limited data about the utility of plerixafor in patients who are being mobilized with chemotherapy and G. Method: In this single institution study of uniformly treated patients we describe our experience with the use of P as a salvage option in patients who fail to optimally mobilize CD34+ cells (>5 × 106 CD34+ cells/kg). Patients received CY (3-4 g/m2) followed by GCSF (10 mcg/kg) from day 1 to day 10. Thirteen patients (6 NHL, 4 MM, 2 Hodgkin lymphoma, 1 Ewings sarcoma) received salvage P from 2008–2010. Their outcomes were compared with 10 matched, historic controls mobilized with (CY n=8; CY + etoposide n=1; CY + topotecan n=1) plus G-CSF (10mcg/kg/d) identified from our institutional database. Data was collected on mobilization and transplant outcomes and analyzed utilizing SPSS version 13.0. Patients receiving P were closely matched to historic controls (CY+G). Result: Both groups were similar with regards to age, gender, disease type, prior therapies and performance status (p>0.05 for all). Patients in the P arm received a median of 2.5 doses (range 1–8). The mean CD34+ count was 21.5cells/ul in the P arm and 32.5 cells/ul in the CY+G arm (p=0.2). Similarly, no significant difference was observed in the average number of apheresis sessions in the P vs. CY+G arms (4.2 vs. 4.4, p=0.8) or the total number of CD34+ stem cells collected (4.0×106/kg vs. 3.9×106/kg, p=0.9). However, 7 out of the 13 patients who received P did have an increase of >10 CD34+ cells/ul in their peripheral blood. Utilizing a cut-off of 5×106 CD34+/kg, 3 (23%) patients in the P arm and 3 (30%) patients in the CY+G arm had a successful harvest. Three NHL patients required >4 doses of P, but all eventually collected >2 × 106 CD34+ cells/kg. Neutrophil and platelet engraftment dynamics were similar in both groups of patients. Median time to neutrophil engraftment was 10 days for both groups, p=0.8, and to platelet engraftment was 22 days vs. 20.5 days, p=0.1, respectively for P vs. CY+G. Conclusion: Our limited single-center retrospective case-controlled outcomes data, suggests that when compared with CY+G, the addition of P as a salvage agent does not significantly improve mobilization outcomes. Further evaluation is needed to combine P with CY+G in terms of optimal timing and potentially dosing of chemotherapy agents utilized. We suggest that the combination P+G would provide better potential outcomes such as improved collection and less hospitalization and reduce the use of chemo-mobilization prior to an Autologous Hematopoietic Stem Cell Transplant. Disclosures: No relevant conflicts of interest to declare.

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