IL-10 Is a Novel Ligand for Hematopoietic Stem Cell Self-Renewal.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2164-2164 ◽  
Author(s):  
Il-Hoan Oh ◽  
Young-Ju Kang ◽  
Bin Cho
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Biological Effects ◽  
Control Group ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Limiting Dilution ◽  
Cells Cultured

Abstract IL-10 has been known to play a major role in modulating inflammatory and immune response. However, the anemia sign in targeted disruption of IL-10 (IL-10KO) and supportive effects of IL-10 during in-vitro culture of hematopoietic progenitor cells implicated potential role of IL-10 for hematopoietic function. To delineate the physiological significance of IL-10 for normal hematopoiesis, first, we undertook study to examine the various hematopoietic compartments in IL-10 KO mice in comparison to wild type (WT) counterparts. In the phenotypic analysis, the bone marrow cells (BMC) of IL-10 KO mice showed modest hypocellularity including TER119+ cells, but no difference were observed in the assay for in-vitro colony forming cells (CFC) (3 Exp.). In contrast, the number of more primitive hematopoietic cells as defined by long term culture (LTC-IC) was 2-fold lower in the BMCs of IL-10 KO mice compared to WT, and further decrease (7-fold) were observed in those IL-10 KO mice that had enterocolitis (2 Exp). Similarly, when equivalent numbers of BMCs from IL-10 KO or WT were transplanted into irradiated congeneic recipient (Pep3b-Ly5.1), IL-10 KO BMC showed 2-fold lower level of engraftment (88% vs 44%) than WT over the span of post-transplantation 3 to 12 weeks (2 exp, n=7 ea,). In the limiting dilution analysis to measure the HSC contents, the frequency of competitive repopulating unit (CRU) in the IL-10 KO BMC was also two fold lower than WT (1/2164 vs. 1/5931, respectively) with further decreasse in CRU frequency being observed for those from IL-10 KO with enterocolitis (< 1/17380), suggesting that IL-10 KO mice has decreased content of hematopoietic stem cell (HSC). To exclude possible indirect biological effects in IL-10 KO mice, and see if IL-10 have direct effect on quantity of HSCs, normal 5-FU prestimulated BMCs were cultured for 5 days on stromal cells that had been retrovirally transduced with IL-10 or control vector (MIG) along with TPO, SCF and FL. When the cells were transplanted into the recipients, cells cultured in the IL-10 secreting stroma exhibited significantly higher level engraftment compared to those cultured in the control stroma (23% vs 54% for PB, 16% vs 49% for BM engraftment, respectively, n=4). Secondary transplantation of these primary recipients at post-transplantation 9 month showed that the BM cells grown in IL-10 secreting stroma had 663 donor-derived CRUs, while those transplanted with control group had 215 donor-derived CRUs, a 3-fold higher CRU contents in the presence of IL-10. In further studies to see if IL-10 could be a direct ligand for primitive HSCs, first, expression of IL-10 receptor on those cells were confirmed by RT-PCR. Next, purified HSCs (c-kit+Sca-1+Lin-) without accessory cells were cultured in the stroma-free condition with or without exogeneous addition of IL-10 for 5 days, and transplanted into recipients in limiting dilution. The result showed that CRU frequency of cells cultured with IL-10 was 3-fold higher than those cultured in the absence of IL-10 (1/525 vs. 1/1465), thus suggesting that IL-10 is a direct ligand for HSC self-renewal. These results may implicate significance of IL-10 for pathogenesis and treatment of autoimmune diseases by stem cell transplantation as well as for improved ex-vivo expansion of HSCs.

scholarly journals Making HSCs in vitro: don’t forget the hemogenic endothelium

Blood ◽  
2018 ◽  
Vol 132 (13) ◽  
pp. 1372-1378 ◽  
Author(s):  
Bradley W. Blaser ◽  
Leonard I. Zon
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Autologous Transplantation ◽  
Cell Types ◽  
Therapeutic Modality ◽  
Hemogenic Endothelium ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Ontogeny

Generating a hematopoietic stem cell (HSC) in vitro from nonhematopoietic tissue has been a goal of experimental hematologists for decades. Until recently, no in vitro–derived cell has closely demonstrated the full lineage potential and self-renewal capacity of a true HSC. Studies revealing stem cell ontogeny from embryonic mesoderm to hemogenic endothelium to HSC provided the key to inducing HSC-like cells in vitro from a variety of cell types. Here we review the path to this discovery and discuss the future of autologous transplantation with in vitro–derived HSCs as a therapeutic modality.

Durable and Robust Fetal Globin Induction without Anemia in Rhesus Monkeys Following Autologous Hematopoietic Stem Cell Transplant with BCL11A Erythroid Enhancer Editing

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4632-4632 ◽  
Author(s):  
Selami Demirci ◽  
Jing Zeng ◽  
Yuxuan Wu ◽  
Naoya Uchida ◽  
Jackson Gamer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Mononuclear Cells ◽  
Hematologic Toxicity ◽  
Cell Transplant ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Significant Difference ◽  
Hbf Induction

Elevated fetal hemoglobin (HbF, α2γ2) levels are clinically beneficial for patients with β-hemoglobinopathies. Editing of the erythroid-specific BCL11A enhancer induces HbF, inhibiting sickling and restoring globin chain balance in erythroid cells derived from hematopoietic stem and progenitor cells (HSPCs) from SCD and β-thalassemia patients respectively, without detectable genotoxicity or adverse effects on hematopoietic stem cell (HSC) function (Wu Y, Nat Med, 2019). Here, we sought to evaluate engraftment and HbF induction potential of erythroid-specific BCL11A enhancer edited CD34+ HSPCs in a non-human primate transplantation model in which hemoglobin switching is conserved. We targeted the erythroid-specific +58 DNAse I hypersensitive site of BCL11A, which has identical human and rhesus sequences at the spacer and protospacer adjacent motif (PAM) of the potent #1617 sgRNA. Ribonucleoprotein complex (RNP) composed of 3x-NLS SpCas9 protein and either BCL11A enhancer targeting (#1617) or AAVS1 targeting sgRNA was electroporated into rhesus CD34+ HSPCs (n=3). Following erythroid differentiation, substantial γ-globin expression (54-77%, p<0.01) was observed in BCL11A edited cells (81-85% indels) as compared to 19-25% and 15-24% for non-electroporated and AAVS1 edited cells, respectively, with no significant difference in red blood cell (RBC) enucleation efficiency (44-47%) among groups. We tested BCL11A enhancer editing with autologous HSC transplant in two cohorts, with two macaques per cohort. For cohort 1, we performed competitive engraftment of BCL11A enhancer and AAVS1 edited HSPCs to test long-term reconstitution. For cohort 2, we evaluated BCL11A enhancer editing alone to evaluate HbF induction and hematopoietic reconstitution. For each cohort, purified CD34+ HSPCs were electroporated with RNP one day after G-CSF and plerixafor mobilization and cultured for two days prior to cryopreservation. HSPCs were thawed and infused following 2×5 Gy total body irradiation. For cohort 1 (n=2, ZL25 and ZL22, 1.34-1.39×106 CD34+ HSPCs/kg), we observed reduced indel frequencies (8-41%) at early post-infusion time points compared to cell products (18-49%), suggesting indels in unfractionated HSPCs may overestimate those in engrafting cells and/or hematopoietic ablation was incomplete. From weeks 6 to 83, stable indel frequencies were detected in both BCL11A (~3-18%) and AAVS1 (~10-45%), suggesting no selective advantage for BCL11A enhancer edited, AAVS1 edited, or non-edited HSCs. For cohort 2 (BCL11A enhancer editing alone (n=2, ZM17 and ZM26, 1.78-6.06×106 CD34+ cells/kg), cell products showed improved editing with ~95% indels and ~65-78% γ-globin protein after in vitro erythroid culture. Animals engrafted with typical kinetics and displayed stable indel ratios up to 28 weeks post-transplantation. A significant correlation was detected between γ-globin level and indel frequency comparing all 4 transplanted animals and unedited controls (R2=0.76, p<0.01). In both edited and unedited animals γ-globin levels peaked in the first two months after transplantation and subsequently declined and plateaued. In ZM17 (~70% BCL11A enhancer indels at ~24 weeks), ~12% γ-globin was observed in peripheral blood (PB) at last measurement (compared to 0.5% γ-globin in RBC prior to transplant). In the same animal, editing ranged from 78-81% across all PB and bone marrow (BM) lineages (excluding CD3+ T-cells with 63% indels), including B-lymphoid, myeloid, erythroid, and HSPCs (in particular including 78% indels in CD71+ CD45- erythroblasts). Hemoglobin, hematocrit, and reticulocyte counts and peripheral smear appearance were all normal, suggesting no erythroid toxicity. Colony-forming ability of BM-derived mononuclear cells was similar in edited and control animals. In summary, we evaluated the clinical potential of autologous BCL11A erythroid enhancer editing in rhesus macaques. BCL11A enhancer edited HSCs can persist for at least 83 weeks post-transplant and provide therapeutic levels of HbF in peripheral RBCs without anemia or other apparent hematologic toxicity. Furthermore, these results emphasize input CD34+ HSPC dose and conditioning intensity as critical variables that influence gene editing following autologous HSCT. Overall, these findings support BCL11A erythroid enhancer genome editing as a promising strategy for therapeutic HbF induction. Disclosures Weiss: GlaxoSmithKline: Consultancy; Cellarity INC: Consultancy; Esperian: Consultancy; Beam Therapeutics: Consultancy; Rubius INC: Consultancy.

scholarly journals Growth Factor Receptor-Bound Protein 10 (Grb10) Regulates Hematopoietic Stem Cell (HSC) Self-Renewal Via Control of mTOR Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1160-1160
Author(s):  
Xiao Yan ◽  
Heather A Himburg ◽  
Phuong L Doan ◽  
Mamle Quarmyne ◽  
Evelyn Tran ◽  
...  
Keyword(s):  
Stem Cell ◽  
Growth Factor ◽  
Hematopoietic Stem Cell ◽  
Growth Factor Receptor ◽  
Post Transplantation ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Duke University

Abstract Elucidation of the mechanisms governing HSC regeneration has been impeded by difficulty in isolating HSCs early following genotoxic injury, such as total body irradiation (TBI). Using multiparametric flow cytometric cell sorting of BM ckit+sca-1+lin- cells coupled with gene expression analysis, we identified growth factor receptor-bound protein 10 (Grb10), a co-receptor which regulates Insulin Receptor/IGF-1 signaling, to be significantly overexpressed by BM KSL cells at the earliest detectable point of regeneration (day +10) following TBI (3.3-fold, p<0.0001). Grb10 is a member of the imprinted gene family which is predominately expressed in the stem cells of a variety of tissues, including embryonic stem cells, bone marrow, skin and muscle. Viral shRNA-mediated knockdown of Grb10 in BM KSL cells caused a significant decrease in KSL cells and colony forming cells (CFCs) detected in 7-day culture (p=0.03 and p=0.002, respectively). Furthermore, mice competitively transplanted with Grb10-deficient HSCs displayed 10-fold lower donor, multilineage hematopoietic cell engraftment than mice transplanted with Grb10-expressing HSCs (p=0.007 for %CD45.1+ donor cells). Secondary competitive repopulation assays confirmed a greater than 10-fold deficit in long-term repopulating capacity in Grb10-deficient KSL cells compared to Grb10-expressing KSL cells (p=0.006 for %CD45.1+ donor cells). In order to determine if Grb10 was necessary for HSC maintenance and normal hematopoiesis in vivo, we generated maternally-derived Grb10-deficient mice. Heterozygous 8 week old Grb10m/+ (1 mutant allele, 1 wild type allele) had 10-fold decreased Grb10 expression in BM lin-cells. BM CFCs and SLAM+ KSL cells were significantly decreased in Grb10m/+ mice compared to Grb10+/+ mice (p=0.006 and p=0.04, respectively). Competitive repopulation assays demonstrated significantly decreased donor hematopoietic cell repopulation in recipient mice transplanted with Grb10m/+ BM cells versus mice transplanted with Grb10+/+ BM cells (p=0.003 for %CD45.1+ donor cells). Mice transplanted with BM cells from homozygous Grb10-/- mice showed a similar decrease in donor-derived hematopoietic repopulation compared to mice transplanted with BM cells from Grb10+/+ mice (p=0.02 at 20 weeks post-transplantation). These results confirmed that Grb10 regulates HSC self-renewal capacity in vivo. To determine whether Grb10 regulates HSC regeneration after myelotoxic injury, we irradiated Grb10m/+ mice with 550cGy TBI, and monitored hematopoietic recovery over time in comparison to Grb10+/+ controls. Interestingly, Grb10m/+ mice displayed accelerated hematopoietic regeneration early following TBI. At day+10 after 550cGy, Grb10m/+ mice contained significantly increased numbers of BM SLAM+ KSL cells (p=0.04) and CFCs (p=0.009), compared to Grb10+/+ littermates. Similarly, mice transplanted with BM cells from irradiated, Grb10m/+ mice displayed 5-fold increased donor hematopoietic repopulation at 20 weeks post-transplantation compared to mice transplanted with BM cells from irradiated, Grb10+/+ mice (p=0.006). These data suggest that Grb10 deficiency accelerates hematopoietic recovery in the early period following myelosuppressive radiation injury. Mechanistically, Grb10-deficiency caused an increase in the percentage of BM KSL cells in G1 and G2/S/M phase of cell cycle compared to Grb10+/+ KSL cells (p=0.003). We also observed significantly increased levels of mTOR activation in Grb10m/+ BM KSL cells compared to Grb10+/+ BM KSL cells (p=0.001 for pS6, p=0.001 for pS6k and p=0.02 for p4EBP1). Furthermore, mTOR inhibition via siRNA-mTOR targeting rescued the defect in BM hematopoietic progenitor content (colony forming cells) in Grb10-deficient BM cells (p<0.0001). Taken together, our results suggest that Grb10 is necessary for HSC maintenance in steady state, while, paradoxically, Grb10 inhibition accelerates HSC regeneration early following injury. Furthermore, our data suggest that Grb10 mediates these effects via regulation of mTOR signaling. Selective modulation of Grb10 signaling has the potential to augment HSC self-renewal in steady state and to accelerate HSC regeneration following myelotoxic injury. Disclosures Himburg: Duke University: Patents & Royalties: Patent Application for use of Pleiotrophin as a hematopoietic stem cell growth factor. Chute:C2 Regenerate: Equity Ownership; Duke University: Patents & Royalties: Application to use PTN as growth factor as hematopoietic stem cell growth factor.

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.

scholarly journals Mitochondrial Activity Determines Hematopoietic Stem Cell Fate Decisions

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4327-4327
Author(s):  
Nicola Vannini ◽  
Mukul Girotra ◽  
Olaia M. Naveiras ◽  
Vasco Campos ◽  
Evan Williams ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Mitochondrial Activity ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions

Abstract A tight control of hematopoietic stem cell (HSC) quiescence, self-renewal and differentiation is crucial for lifelong blood production. The mechanisms behind this control are still poorly understood. Here we show that mitochondrial activity determines HSC fate decisions. A low mitochondrial membrane potential (Δψm) predicts long-term multi-lineage blood reconstitution capability, as we show for freshly isolated and in vitro-cultured HSCs. However, as in vivo both quiescent and cycling HSCs have comparable Δψm distributions, a low Δψm is not per se related to quiescence but is also found in dividing cells. Indeed, using divisional tracking, we demonstrate that daughter HSCs with a low Δψm maintain stemness, whereas daughter cells with high Δψm have undergone differentiation. Strikingly, lowering the Δψm by chemical uncoupling of the electron transport chain leads to HSC self-renewal under culture conditions that normally induce rapid differentiation. Taken together, these data show that mitochondrial activity and fate choice are causally related in HSCs, and provides a novel method for identifying HSC potential after in vitro culture. Disclosures No relevant conflicts of interest to declare.

Lymphoid, Long-Term Repopulating, and Endothelial Potential of the Embryonic Hemangioblast.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1657-1657
Author(s):  
Mitsujiro Osawa ◽  
Michael Kyba
Keyword(s):  
Endothelial Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Embryoid Bodies ◽  
Endothelial Differentiation ◽  
Hematopoietic Progenitors ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hematopoietic and endothelial cells are thought to arise from a common progenitor termed the hemangioblast. Direct evidence for the hemangioblast was first obtained from embryonic stem cells differentiated in vitro as embryoid bodies (EBs). Flk-1+ cells from early EBs generate colonies in response to VEGF and SCF (the BL-CFC assay) which can be replated to give secondary hematopoietic or endothelial cells. Bipotent BL-CFCs have also recently been derived from the posterior primitive streak of neural plate stage mouse embryos. However, a direct relationship between the early embryonic hemangioblast defined by the BL-CFC assay and the hematopoietic stem cell remains unproven. Hemangioblast-derived hematopoiesis in vitro is transient and restricted to myelo-erythroid differentiation. Lymphoid potential and long-term repopulation, two hallmarks of the definitive hematopoietic stem cell, have eluded detection to date. Previous work has shown that the homeodomain transcription factor, HoxB4, by enhancing self-renewal in vitro, can reveal latent definitive HSC activity of transient embryonic hematopoietic progenitors. Using an ES cell line with doxycycline-inducible HoxB4 expression, we have investigated the definitive hematopoietic and endothelial potential of individual hemangioblast colonies. BL-CFC numbers were unaffected by HoxB4 expression during EB differentiation, however they were increased threefold by induction during the BL-CFC assay. By replating one half of the cells from an individual blast colony in endothelial medium and the other half on an OP9 monolayer with hematopoietic cytokines, we show that the majority (60%) of HoxB4-induced BL-CFCs are bipotent. HoxB4 expression was compatible with endothelial differentiation and allowed exponential expansion of hematopoietic progenitors on OP9 cocultures. When switched to OP9-DL1 with lymphoid cytokines, T-lymphopoiesis was observed characterized by CD25 expression followed by CD4, CD8, and CD3epsilon expression. To assay long-term repopulation, individual blast colonies were picked and divided into endothelial medium and OP9 monolayers. The hematopoietic arms of colonies defined retrospectively to have been bipotent (endothelial differentiation was observed in vitro) were transplanted into sublethally irradiated Rag2; gamma-c; CD45.1 immunodeficient mice. Mice with long-term hematopoietic engraftment were identified by the presence of CD45.2 cells in peripheral blood 3 months post-transplant. Lymphoid and myeloid contribution was evaluated by costaining with Gr-1, B220, CD19, CD4, and CD8. The donor-derived component of these hematopoietic chimeras, including their entire lymphoid arm (approximately 1/3 of engrafted mice showed lymphoid differentiation) is by definition clonally derived from a single hemangioblast. These results clearly show that the embryonic hemangioblast is not intrinsically limited in its hematopoietic potential. Under conditions that favor self-renewal, lymphoid differentiation and long-term repopulation become evident, revealing the link between endothelial development and definitive hematopoiesis at the clonal level.

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.

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