CD34 KSL Cells Research Articles

Multiple cells of origin in common with various types of mouse N-Myc acute leukemia

Little is known regarding whether the cell of origin differs among different leukemia types. To address this fundamental issue, we determined the cell of origin in five distinct types of acute leukemia induced by N-Myc overexpression in mice. CD150+CD48-CD41-CD34-c-Kit+Sca-1+Lin- (KSL) (HSC1) cells, CD150-CD48-CD41-CD34-KSL (HSC2) cells, CD150+CD41+CD34-KSL (HPC1) cells, CD150+CD41+CD34+KSL (HPC2) cells, and CD150-CD41-CD34+KSL (HPC3) cells were purified from the bone marrow of adult C57BL/6 mice, transduced with the N-Myc retrovirus vector, and transplanted into lethally irradiated mice. B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), acute myeloid leukemia (AML), acute undifferentiated leukemia (AUL), and mixed phenotype acute leukemia (MPAL) developed from five populations. RNA sequencing data supported the phenotypical diagnoses of leukemia, except that AUL appeared transcriptionally close to T-ALL. Whole-genome sequencing revealed that retroviral integration sites were irrelevant to the leukemia types and that T-ALL and AML of MPAL shared the same integration site and many gene mutations, suggesting their common origin. Additionally, leukemic stem cells were identified in the KSL cell population, suggesting that the phenotypes of leukemic stem cells are irrelevant to leukemia types. This study provides experimental evidence for the similar and multiple cells of origin in acute leukemia.

Syndecan-2 enriches for hematopoietic stem cells and regulates stem cell repopulating capacity

AbstractThe discovery of novel hematopoietic stem cell (HSC) surface markers can enhance understanding of HSC identity and function. We have discovered a population of primitive bone marrow (BM) HSCs distinguished by their expression of the heparan sulfate proteoglycan Syndecan-2, which serves as both a marker and a regulator of HSC function. Syndecan-2 expression was increased 10-fold in CD150+CD48–CD34–c-Kit+Sca-1+Lineage– cells (long-term HSCs [LT-HSCs]) compared with differentiated hematopoietic cells. Isolation of BM cells based solely on syndecan-2 surface expression produced a 24-fold enrichment for LT-HSCs and sixfold enrichment for α-catulin+c-kit+ HSCs, and yielded HSCs with superior in vivo repopulating capacity compared with CD150+ cells. Competitive repopulation assays revealed the HSC frequency to be 17-fold higher in syndecan-2+CD34–KSL cells compared with syndecan-2–CD34–KSL cells and indistinguishable from CD150+CD34–KSL cells. Syndecan-2 expression also identified nearly all repopulating HSCs within the CD150+CD34–KSL population. Mechanistically, syndecan-2 regulates HSC repopulating capacity through control of expression of Cdkn1c (p57) and HSC quiescence. Loss of syndecan-2 expression caused increased HSC cell cycle entry, downregulation of Cdkn1c, and loss of HSC long-term repopulating capacity. Syndecan-2 is a novel marker of HSCs that regulates HSC repopulating capacity via control of HSC quiescence.

SINGLE CELL RNA-SEQ REVEALED CLONAL REPOPULATION DYNAMICS BY MOUSE HEMATOPOIETIC STEM CELLS

Single hematopoietic stem cells (HSCs) can reconstitute the entire hematopoietic system after transplantation. However, how HSCs reconstitute hematopoietic cells is poorly understood. We used single-cell RNA sequencing (scRNA-seq) to identify cells and study their kinetics. Single CD201+CD150+CD48-Lineage- cells from GFP-transgenic B6 mice were transplanted into lethally irradiated mice with competitor cells. From BM and spleen of recipients, single GFP+ cells were isolated 2-3 weeks after transplantation, and single GFP+ CD34-KSL cells were isolated 1, 4, 8, and 12 months after transplantation. These cells were subjected to scRNA-seq. Based on transcriptome-defined (t) cell types under homeostasis, donor-derived single cells were classified into tHSCs, tMPPs, tCPs (committed progenitors, CMP, MEP, GMP and CLP), tMEs, tGMs, and tLymphocytes (B, T, and NK cells). We detected tMEs and tGMs as the major populations 2 weeks after transplantation. We first detected tHSCs 3 weeks after transplantation. Based on the absolute number of tHSCs, their maximum expansion rate was detected between 3 and 4 weeks after transplantation. We also detected tMEs, tGMs, and tLymphocytes from 1 to 12 months, and these cells increased more 8 and 12 months after transplantation. Donor cells were assigned as a and b cells based on the ratio of myeloid cells (GM) and lymphoid cells (T+B) in peripheral blood 4 month after transplantation. a cells gave rise to tHSC1 and 2 while b cells gave rise to tHSC1, 2, and 3. These data supported the original function definition of these cells. In summary, this study not only illustrates the early differentiation phase, followed by the expansion phase of HSC after transplantation. Deep analysis of the key regulation network in clonal hematopoiesis will provide more information for molecular mechanism of HSC self-renewal and lineage differentiation.

Impaired development and dysfunction of endothelial progenitor cells in type 2 diabetic mice

AimDysfunction of circulating endothelial progenitor cells (EPCs) has been shown to affect the development of microvascular diseases in diabetes patients. The aim of this study was to elucidate the development and mechanical dysfunction of EPCs in type 2 diabetes (T2D). MethodsThe colony-forming capacity of EPCs and differentiation potential of bone marrow (BM) c-Kit(+)/Sca-I(+) lineage-negative mononuclear cells (KSL) were examined in T2D mice, db/db mice and KKAy mice, using EPC colony-forming assay (EPC-CFA). ResultsT2D mice had fewer BM stem/progenitor cells, and proliferation of KSL was lowest in the BM of db/db mice. In T2D mice, the frequency of large colony-forming units (CFUs) derived from BM-KSL was highly reduced, indicating dysfunction of differentiation into mature EPCs. Only a small number of BM-derived progenitors [CD34(+) KSL cells], which contribute to the supply of EPCs for postnatal neovascularization, was also found. Furthermore, in terms of their plasticity to transdifferentiate into various cell types, BM-KSL exhibited a greater potential to differentiate into granulocyte macrophages (GMs) than into other cell types. ConclusionT2D affected EPC colony formation and differentiation of stem cells to mature EPCs or haematopoietic cells. These data suggest opposing regulatory mechanisms for differentiation into mature EPCs and GMs in T2D mice.

Clock gene Bmal1 is dispensable for intrinsic properties of murine hematopoietic stem cells

BackgroundCircadian rhythms are known to influence a variety of biological phenomena such as cell cycle, sleep-wake rhythm, hormone release and other important physiological functions. Given that cell cycle entry of hibernating hematopoietic stem cells (HSCs) plays a critical role in controlling hematopoiesis, we asked functional significance of the clock gene Bmal1, which plays a central role in regulating circadian rhythms as a transcription factor. Here we investigated the necessity of Bmal1 for HSC functions using Bmal1 deficient (Bmal1−/−) mice.FindingsUsing colony-forming assays in vitro, we found that the frequency of mixed colony formation between Bmal1+/+ and Bmal1−/− CD34−KSL cells does not differ significantly. Competitive bone marrow assays also revealed that Bmal1−/− bone marrow cells competed normally with wild-type cells and displayed long-term multi-hematopoietic lineage reconstitution. In addition, there were no significant differences in the frequencies and hibernation state of bone marrow HSCs between Bmal1+/+ and Bmal1−/− mice, suggesting that they are independent of circadian rhythms.ConclusionsThis paper discusses the necessity of circadian rhythms for HSC functions. Our data clearly shows that a key circadian clock gene Bmal1 is dispensable for intrinsic functions of HSCs, such as differentiation, proliferation and repopulating ability.

Haploinsufficiency of Sf3b1 leads to compromised stem cell function but not to myelodysplasia

SF3B1 is a core component of the mRNA splicing machinery and frequently mutated in myeloid neoplasms with myelodysplasia, particularly in those characterized by the presence of increased ring sideroblasts. Deregulated RNA splicing is implicated in the pathogenesis of SF3B1-mutated neoplasms, but the exact mechanism by which the SF3B1 mutation is associated with myelodysplasia and the increased ring sideroblasts formation is still unknown. We investigated the functional role of SF3B1 in normal hematopoiesis utilizing Sf3b1 heterozygous-deficient mice. Sf3b1(+/-) mice had a significantly reduced number of hematopoietic stem cells (CD34(-)cKit(+)ScaI(+)Lin(-) cells or CD34(-)KSL cells) compared with Sf3b1(+/+) mice, but hematopoiesis was grossly normal in Sf3b1(+/-) mice. When transplanted competitively with Sf3b1(+/+) bone marrow cells, Sf3b1(+/-) stem cells showed compromised reconstitution capacity in lethally irradiated mice. There was no increase in the number of ring sideroblasts or evidence of myeloid dysplasia in Sf3b1(+/-) mice. These data suggest that SF3B1 plays an important role in the regulation of hematopoietic stem cells, whereas SF3B1 haploinsufficiency itself is not associated with the myelodysplastic syndrome phenotype with ring sideroblasts.

Role Of Sf3b1 On Hematopoiesis

Frequent pathway mutation involving multiple components of the RNA splicing machinery is a cardinal feature of myeloid neoplasms showing myeloid dysplasia, in which the major mutational targets include U2AF35, ZRSR2, SRSF2 and SF3B1. Among these, SF3B1 mutations were strongly associated with MDS subtypes characterized by increased ring sideroblasts, such as refractory anemia and refractory cytopenia with multiple lineage dysplasia with ring sideroblasts, suggesting the critical role of SF3B1 mutations in these MDS subtypes. However, currently, the molecular mechanism of SF3B1mutation leading to the ring sideroblasts formation and MDS remains unknown.The SF3B1 is a core component of the U2-small nuclear ribonucleoprotein (U2 snRNP), which recognizes the 3′ splice site at intron–exon junctions. It was demonstrated that Sf3b1 null mice were shown to be embryonic lethal, while Sf3b1 +/- mice exhibited various skeletal alterations that could be attributed to deregulation of Hox gene expression due to haploinsufficiency of Sf3b1. However, no detailed analysis of the functional role of Sf3b1 in hematopoietic system in these mice has been performed. So, to clarify the role of SF3B1 in hematopoiesis, we investigated the hematological phenotype of Sf3b1 +/- mice. There was no significant difference in peripheral blood counts, peripheral blood lineage distribution, bone marrow total cellularity or bone marrow lineage composition between Sf3b1 +/+ and Sf3b1 +/- mice. Morphologic abnormalities of bone marrow and increased ring sideroblasts were not observed. However, quantitative analysis of bone marrow cells from Sf3b1 +/- mice revealed a reduction of the number of hematopoietic stem cells (CD34 neg/low, cKit positive, Sca-1 positive, lineage-marker negative: CD34-KSL cells) measured by flow cytometry analysis, compared to Sf3b1 +/+ mice. Whereas examination of hematopoietic progenitor cells revealed a small decrease in KSL cell populations and megakaryocyte - erythroid progenitors (MEP) in Sf3b1 +/- mice, and common myeloid progenitors (CMP), granulocyte - monocyte progenitors (GMP) and common lymphoid progenitors (CLP) remained unchanged between Sf3b1 +/+ and Sf3b1 +/- mice.In accordance with the reduced number of hematopoietic stem cells in Sf3b1 +/- mice, the total number of colony-forming unit generated from equal number of whole bone marrow cells showed lower colony number in Sf3b1 +/- mice in vitro. Competitive whole bone marrow transplantation assay, which irradiated recipient mice were transplanted with donor whole bone marrow cells from Sf3b1 +/+ or Sf3b1 +/- mice with an equal number of competitor bone marrow cells, revealed impaired competitive whole bone marrow reconstitution capacity of Sf3b1 +/- mice in vivo. These data demonstrated Sf3b1 was required for hematopoietic stem cells maintenance.To further examine the function of hematopoietic stem cells in Sf3b1 +/- mice, we performed competitive transplantation of purified hematopoietic stem cells from Sf3b1 +/+ or Sf3b1 +/- mice into lethally irradiated mice together with competitor bone marrow cells. Sf3b1 +/- progenitors showed reduced hematopoietic stem cells reconstitution capacity compared to those from Sf3b1 +/+ mice. In serial transplantation experiments, progenitors from Sf3b1 +/- mice showed reduced repopulation ability in the primary bone marrow transplantation, which was even more pronounced after the second bone marrow transplantation. Taken together, these data demonstrate that Sf3b1 plays an important role in normal hematopoiesis by maintaining hematopoietic stem cell pool size and regulating hematopoietic stem cell function. To determine the molecular mechanism underlying the observed defect in hematopoietic stem cells of Sf3b1 +/- mice, we performed RNA-seq analysis. We will present the results of our biological assay and discuss the relation of Sf3b1 and hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Biological Analysis of SRSF2 Mutations in Leukemogenesis

Abstract 1282Emerging evidence is establishing a connection between MDS and spliceosome mutations. Spliceosome including SF3b1, U2AF1 and SRSF2 are frequently and exclusively mutated in myelodysplastic syndromes (MDS) and related myeloid neoplasms. Spliceosome mutations occur at varying frequencies in different disease subtypes. SF3B1 was shown to be highly associated with MDS characterized by increased ring sideroblasts and SRSF2 mutations are more prevalent in chronic myelomonocytic leukemia. In spite of the fact that the recent discovery constitutes a novel class of genomic lesions and defines an entirely new pathogenic pathway of leukaemogenesis, the pathogenesis of spliceosome mutation is not largely understood. To understanding the biological consequences of spliceosomal mutations, we previously reported mutant U2AF1 cause altered RNA splicing, and overexpressed mutant U2AF1 decrease in cell proliferarion. However, currently, no functional analysis of SRSF2 mutation has been published.SRSF2 belongs to the serine/arginine-rich (SR) protein family. SR proteins are a family of RNA binding proteins characterized by one or two RNA recognition motifs (RRMs) and a signature RS domain enriched with arginine and serine repeats (RS domain).Growing body of evidence suggests that SR protein may be directly involved in the process of carcinogenesis. Gene knockout experiment indicated SRSF2 is involved with specific pathways in regulating cell proliferation and genomic stability during mammalian organogenesis. In neck and head tumor, SRSF2 is frequently overexpressed. And upregulated SRSF2 increases missplicing and downregulates E-cadherin expression, which is an important tumor suppressor gene. Therefore SRSF2 potential function in tumorigenesis is suggested in epithelial cancers. SRSF2 mutations with MDS exclusively occur at P95 within an intervening sequence between RRM and RS domains, indicating a gain-of-function nature of these mutations.So, to clarify the biological role of SRSF2 mutations in leukemogenesis, we evaluated the oncogenic role of SRSF mutations by expressing a mutant SRSF2 allele in Jurkat cells. The cells transduced with a tumor-derived SRSF2 allele showed reduced cell proliferation and increased apoptosis compared to the mock and wild type SRSF2-transduced cells. Next we performed in vitro colony assay using a highly purified hematopoietic stem cell population (CD34-c-Kit+ScaI+ Lin-(CD34-KSL) cells) collected from C57BL/6 (B6)-Ly5.1 mouse that was retrovirally transduced with mock, mutant or wild-type SRSF2 construct. The mutant SRSF2-transduced cells showed reduced cell proliferation compared with mock- or wild-type SRSF2 transduced cells. Subsequently, we conducted bone marrow transplantaion assay. We collected CD34-KSL cells from B6-Ly5.1 mouse, and retrovirally transduce mock, mutant or wild-type SRSF2 construct, each harbouring the EGFP marker gene. And these cells were sorted by EGFP marker, and transplanted with competitor cells (B6-Ly5.1/5.2 F1 mice origin) into lethally irradiated B6-Ly5.2 mice. The wild-type SRSF2-transduced cells showed a lower reconstitution capacity than the mock-transduced cells. On the other hand, the recipients of the cells transduced with the mutant SRSF2 showed lower EGFP-positive cell chimaerism than those of the mock- or the wild-type SRSF2-transduced. Therefore, the mutant SRSF2 was indicated to have a negative effect on cellular proliferation capacity in vitro and in vivo, and a gain-of-function nature of these mutations is suggested. These results are similar to the effect of U2AF1 mutant, which we reported mutant U2AF1 transduced TF-1 and HeLa cells present with a decrease in cell proliferation and hematopoietic stem cells expressing mutant U2AF1 also displayed lower reconstitution capacity by competitive reconstitution assay in mice. So far, the mechanism responsible for the growth advantage of mutant cells in patient is unclear. We furthermore observe hematopoietic phenotype of the bone marrow transplanted model mouse. SRSF2 mutations can coexist with mutations in TET2, ASXL1 and RUNX1. Therefore we performed additionally bone marrow transplantation assay, utilizing hematopoietic cells derived from TET2 knockdown mice, as a model of multistep carcinogenesis. We will present the results of our biological assay on the SRSF2 mutations and discuss the pathogenesis of MDS. Disclosures:No relevant conflicts of interest to declare.

Frequent Pathway Mutations of Splicing Machinery in Myelodysplasia

Abstract 458MDS and related disorders comprise a group of myeloid neoplasms characterized by deregulated blood cell production and a predisposition to AML. Although currently, a number of gene alterations have been implicated in the pathogenesis of MDS, they do not fully explain the pathogenesis of MDS, because many of them are also found in other myeloid malignancies and roughly 20% of MDS cases have no known genetic changes. So, in order to clarify a complete registry of gene mutations in MDS and identify those discriminate MDS from other myeloid neoplasms, we performed whole-exome sequencing of 29 cases showing myelodysplasia. A total of 268 somatic mutations or 9.2 mutations per sample were identified. Among these 41 occurred in recurrent gene targets, which not only included a spectrum of known gene targets in MDS, such as TET2, EZH2, NRAS/KRAS, RUNX1, TP53 and DNMT3A, but also affected previously unknown genes that are commonly mapped to the RNA splicing pathway, including U2AF35, SRSF2 and ZRSR2. Together with additional three (SF3A1, SF3B1 and PRPF40B) found in single cases, 16 (55.2%) of the 29 discovery cases carried a mutation affecting the component of the splicing machinery.To confirm the observation, we examined 9 spliceosome genes for mutations in a large set of myeloid neoplasms (N=582) using a high-throughput mutation screen of pooled DNA followed by confirmation/identification of candidate mutations. In total, 219 mutations were identified in 209 out of the 582 specimens of myeloid neoplasms. Mutations of the splicing machinery were highly specific to diseases showing myelodysplastic features, including 19 of 23 (83%) cases with RARS, 43 of 50 (86%) RCMD-RS, 68 of 155 (44%) other MDS, 48 of 88 (55%) CMML, and 16 of 62 (26%) secondary AML with MDS features with a string preference of SF3B1 mutations to RARS and RCMD-RS and of SRSF2 to CMML, while they were rare in cases with de novo AML (N=151) and MPD (N=53). The mutations among 4 genes, U2AF35 (N = 37), SRSF2 (N = 56), SF3B1 (N = 79) and ZRSR2 (N = 23), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 8), PRPF40B (N = 7), U2AF65 (N = 4) and SF1 (N = 5). Interestingly, mutations in the former three genes showed clear hot spots, indicating a gain-of-function nature of these mutations. On the other hand, two thirds of the ZRSR2 mutations are nonsense or frameshift changes causing premature truncation of the protein. Significantly, these mutations occurred in an almost completely mutually exclusive manner among mutated cases, and commonly affected those components of the splicing complex that are engaged in the 3' splice site recognition during RNA splicing, strongly indicating production of unspliced or aberrantly spliced RNA species are incriminated for the pathogenesis of MDS.In fact, when transduced into HeLa cells, the recurrent S34F U2AF35 mutant induced the increase in the production of unspliced RNA species and elicited the activation of the nonsense mediated decay pathway. Functionally, the U2AF35 mutants seemed to cause deregulated stem cell functions, because CD34(−) KSL cells transduced with various U2AF35 mutants invariably showed reduced chimerism in competitive reconstitution assay. In accordance with this, the S34F U2AF35 mutant lead to suppression of cell growth in a variety of cell types, including HeLa cells, in which expression of the mutant induced a G2/M cell cycle arrest and increased apoptosis.In conclusion, whole-exome sequencing unexpectedly revealed the high frequency of the splicing pathway mutations in MDS and related myeloid neoplasms, providing the first evidence indicating that compromised RNA splicing by gene mutations are responsible for human pathogenesis. Disclosures:Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Cripto Regulates Hematopoietic Stem Cells as a Hypoxic-Niche-Related Factor through Cell Surface Receptor GRP78

Hematopoietic stem cells (HSCs) are maintained in hypoxic niches in endosteal regions of bones. Here we demonstrate that Cripto and its receptor GRP78 are important regulators of HSCs in the niche. Flow cytometry analyses revealed two distinct subpopulations of CD34(-)KSL cells based on the expression of GRP78, and these populations showed different reconstitution potential in transplantation assays. GRP78(+)HSCs mainly reside in the endosteal area, are more hypoxic, and exhibit a lower mitochondrial potential, and their HSC capacity was maintained invitro by Cripto through induction of higher glycolytic activity. Additionally, HIF-1α KO mice have decreased numbers of GRP78(+)HSCs and reduced expression of Cripto in the endosteal niche. Furthermore, blocking GRP78 induced a movement of HSCs from the endosteal to the central marrow area. These data suggest that Cripto/GRP78 signaling is an important pathway that regulates HSC quiescence and maintains HSCs in hypoxia as an intermediary of HIF-1α.

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

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

CD61/ Integrin β3 Ligation Contributes to the Thrombopoietin-Mediated Niche Function of Mouse Hematopoietic Stem Cells.

Abstract 383Hematopoietic stem cells (HSCs) reside in a specialized microenvironment, bone marrow (BM) ‘niche' for maintaining self-renewal and multi-lineage differentiation capacities through the life. We recently reported that murine HSCs (CD34- c-Kit+ Sca-1+Lin-: CD34-KSL) but not CD34+KSL hematopoietic progenitors highly express CD61 (integrin β3), correlating well with long-term repopulating activity (LTRA) of HSCs (Umemoto et al., J. Immunol, 2006). However, the roles of CD61 on HSC regulation and underlying molecular mechanisms in the BM niche are to date unknown. We herein demonstrate a novel role of CD61 using CD61-deficient (CD61-/-) and mutant CD61 mice that exhibit 747 tyrosine residue substituted with alanine in the cytoplasmic tail of β3 integrin subunit (CD61Y747A KI) whereby the integrin inside-out and outside-in signaling are impaired. We found that the CD61-deficiency influences the sensitivity to 5-fluorouracil (5-FU) administration (0.25 mg/g i.v. one shot), increasing the mortality of CD61-/- mice (CD61-/- mice died within 14 days, n=20, vs. WT mice survive over 90 days, n=20, P<0.0001). HSCs per se appeared to be responsible for these results because CD61-/- CD34-KSL cells but not CD61-/- CD34+KSL cells showed accelerated cell-cycle progression compared to those of WT, suggesting that CD61 might contribute to maintenance of quiescent state of HSCs within the BM. It is known that thrombopoietin (TPO) acts on not only potent hematopoietic differentiation from HSCs but also for maintaining quiescent state of HSCs. Accordingly, we examined the relationship between TPO effect and integrin signaling through CD61 in HSCs. The in vitro single cell culture system showed the decreased cell proliferation and survival in CD61-/- CD34-KSL cells that were cultured in the presence of TPO (50 ng/mL), compared to the WT CD34-KSL cells (P<0.01). In contrast, CD61-deficency did not influence the phenotype of cultured CD34-KSL cells in the presence of stem cell factor (SCF, 50 ng/ml). Interestingly, these results were mirrored by CD61Y747A KI CD34-KSL cells and their littermate control CD34-KSL cells. Furthermore, when 40 CD34-KSL cells after 5 days culture in the presence of TPO and they were then applied into long-term competitive repopulation assay, CD61Y747A KI CD34-KSL cells showed less LTRA than that of WT (P<0.01). These results indicated that tyrosine at 747 of the β3 integrin subunit was required for the TPO-dependent expansion/maintenance of HSC in vitro. Whereas, to initiate integrin outside-in signaling in the presence of TPO, CD34-KSL cells were cultured on a well of 96-well plates pre-coated with various CD61 ligands (either vitronectin, 5 μg/ml or osteopontin, 5 μg/mL), resulting in the enhancement of their LTRA, compared to without these ligands. Since these ligands could interact with integrin receptors besides CD61, we utilized the antibody to CD61, 2C9.G2 that is capable of phosphorylating CD61 to mimic the actions of outside-in signaling. Using CD34-KSL cells were cultured with 2C9.G2, we further confirmed that CD61 signaling allowed CD34-KSL cells to maintain undivided state in vitro and increase LTRA in vivo, which were dependent upon the presence of TPO but neither SCF nor other growth factors including insulin growth factor-2 and fibroblast growth factor-1. More interestingly, both stimulation with CD61 ligation and TPO influenced ex vivo expansion of HSCs when started at 40 CD34-KSL cells in a 96-well culture well. Collectively, our results indicate that CD61/β3 integrin signaling plays important roles in TPO-mediated expansion/maintenance of HSC within the BM niche, which are possibly mediated through the tyrosine phosphorylation at 747 tyrosine residue of the β3 integrin cytoplasmic domain. Disclosures:No relevant conflicts of interest to declare.

TIMP-3 Inhibition of Angiopoietin-1 Signaling Recruits Hematopoietic Stem Cells from the Bone Marrow Niche.

Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production in the adult bone marrow. Quiescent HSCs are rapidly recruited into the cell cycle when they face hematopoietic demands such as myelosuppression, returning to quiescence once they produce enough progenitors. It was previously shown that quiescent HSCs express Tie2 and that Tie2/angiopoietin-1 (Ang-1) signaling plays a critical role for maintaining HSC quiescence. However, molecular cues for recruiting HSCs from a quiescent state into cycle remain poorly understood. Extracellular signals are often regulated by the extracellular matrix environment, which is modulated by metalloproteinase (MMP) activities. TIMP-3 is an endogenous inhibitor of MMPs, and we have previously proposed that TIMP-3 may play a critical role in HSC physiology. In addition, TIMP-3 has been reported to suppress angiogenesis by inhibiting vascular endothelial growth factor (VEGF) signaling. By analogy with VEGF inhibition, we reasoned that TIMP-3 might suppress Ang-1 signaling in HSC and act as a molecular cue for HSC recruitment. In order to investigate a role of TIMP-3 in the HSC recruitment, we first examined whether TIMP-3 is regulated in the BM upon myelosuppression. Analyses by reverse transcription polymerase chain reaction (RT-PCR) and immunostaining revealed that the injection of 5-fluorouracil (5-FU) or irradiation induced TIMP-3 at the endosteal surface of the BM after 3-days of treatment. We next tested the hypothesis that TIMP-3 might be regulating Ang-1 signals by using cell line models. This revealed that the pre-treatment of cells with TIMP-3 suppressed autophosphorylation of Tie-2 in response to Ang-1. BIAcore and in vitro binding assay revealed that TIMP-3 directly interacted with Ang-1 and Tie-2, indicating that TIMP-3 suppressed Ang-1 signaling through interfering ligand-receptor interaction. Next we examined the effect of TIMP-3 on HSC physiology. TIMP-3 promoted the proliferation of CD34-KSL cells in vitro by approximately 2–3 fold. This was mainly due to the enhanced production of multipotential progenitors from CD34-KSL cells, which was accomplished by an enhanced symmetrical cell division of multipotential progenitors as revealed by paired-daughter cell analysis. Bone marrow transplantation study of TIMP-3-treated CD34-KSL cells showed that they sustained long-term repopulating potential comparable to the control-treated cells. Furthermore, in vivo administration of TIMP-3 into mice accelerated recovery and protected mice from myelosuppression, and in turn, the bone marrow recovery after myelosuppression was delayed in TIMP-3-deficient animals. In summary, TIMP-3 is induced by myelosuppression in the BM niche, stimulates HSC proliferation by inhibiting Ang-1 signaling, and thereby promotes production of multipotential progenitors from HSCs. These results demonstrate that TIMP-3 acts as a molecular cue for recruiting quiescent HSCs from the BM niche.

Adult mouse hematopoietic stem cells: purification and single-cell assays

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.

Non-side-population hematopoietic stem cells in mouse bone marrow

AbstractMost hematopoietic stem cells (HSCs) are assumed to reside in the so-called side population (SP) in adult mouse bone marrow (BM). We report the coexistence of non-SP HSCs that do not significantly differ from SP HSCs in numbers, capacities, and cell-cycle states. When stained with Hoechst 33342 dye, the CD34-/low c-Kit+Sca-1+lineage marker- (CD34-KSL) cell population, highly enriched in mouse HSCs, was almost equally divided into the SP and the main population (MP) that represents non-SP cells. Competitive repopulation assays with single or 30 SP- or MP-CD34-KSL cells found similar degrees of repopulating activity and frequencies of repopulating cells for these populations. Secondary transplantation detected self-renewal capacity in both populations. SP analysis of BM cells from primary recipient mice suggested that the SP and MP phenotypes are interconvertible. Cell-cycle analyses revealed that CD34-KSL cells were in a quiescent state and showed uniform cell-cycle kinetics, regardless of whether they were in the SP or MP. Bcrp-1 expression was similarly detected in SP- and MP-CD34-KSL cells, suggesting that the SP phenotype is regulated not only by Bcrp-1, but also by other factors. The SP phenotype does not specify all HSCs; its identity with stem cell function thus is unlikely.

Waking Stem Cells

Hematopoietic stem cells (HSCs) reside in bone marrow in a nondividing state from which they are occasionally roused to enter the cell cycle. Noting the similarity of HSC dormancy to mammalian hibernation and Caenorhabditis elegans dauer formation, Yamazaki et al. wondered whether the PI3K (phosphatidylinositol 3-kinase)-Akt-FOXO signaling pathway, implicated in regulation of both of these states, might be involved in HSC "hibernation." In quiescent CD34 – Kit + Sca-1 + lineage marker-negative (CD34 – KSL) cells freshly isolated from mouse bone marrow, no phosphorylated (activated) Akt was apparent, and its downstream target FOXO3a was found in the nucleus. In contrast, Akt was phosphorylated and FOXO3a was present in the cytoplasm of cycling CD34 + KSL progenitor cells. Cytokine treatment of CD34 – KSL cells led to polarization of the lipid raft marker GM1 ganglioside as well as phosphorylation of Akt and relocation of FOXO3a to the cytoplasm. Depleting cholesterol with β-cyclodextrin (MβCD) to inhibit lipid raft clustering in response to cytokine treatment in CD34 – KSL cells suppressed Akt activation and FOXO3a relocation; inhibited cytokine-dependent translocation of cyclins D1, D2, and D3 to the nucleus; and prevented disappearance from the cytoplasm of the cyclin-dependent kinase inhibitor p57 Kip2 . When single HSCs that had survived without dividing for several days in the presence of MβCD, stem cell factor, and thrombopoietin were placed in MβCD-free medium containing stem cell factor, thrombopoietin, interleukin-3, and erythropoietin, they retained the ability to proliferate and differentiate along various myeloid lineages in vitro and to repopulate the hematopoietic system of lethally irradiated mice. Thus, the authors conclude that lipid raft clustering plays a key role in HSC emergence from dormancy and that the signaling pathway involved in mediating HSC hibernation resembles that involved in dauer formation. S. Yamazaki, A. Iwama, S. Takayanagi, Y. Morita, K. Eto, H. Ema, H. Nakauchi, Cytokine signals modulated via lipid rafts mimic niche signals and induce hibernation in hematopoietic stem cells. EMBO J. 25 , 3515-3523 (2006). [PubMed]

Isolation of Murine Hematopoietic Stem Cells and Progenitor Cells

Protocols for the purification of CD34-KSL cells, CD34+KSL cells, SP cells, and Tip-SP CD34-KSL cells are described. Mouse hematopoietic stem cells are highly enriched in the CD34-KSL and SP fractions. These two populations overlap one another. A nearly homogenous population of HSCs can be obtained by isolating Tip-SP CD34-KSL cells. One of the earliest progenitor cell populations is CD34+KSL cells.

Isolation and clonal characterization of hematopoietic and liver stem cells.

Prospective isolation of stem cells is essential to understanding the mechanisms that control their proliferation and differentiation. Using 9 monoclonal antibodies and fluorescence-activated cell sorting (FACS), we have succeeded in prospectively identifying hematopoietic stem cells (HSCs) in adult mouse bone marrow. Mouse HSCs were exclusively enriched in CD34 negative, c-Kit Sca-1 Lineage Marker (CD34 KSL) cells representing 0.004% of bone marrow (BM) mononuclear cells. When single CD34-KSL cells were transplanted individually into a lethally irradiated mouse, 25% of the recipient mice survived and showed long-term reconstitution of the BM, providing evidence for multipotency and a self-renewal capacity of HSCs. Using a similar approach, we also prospectively identified hepatic stem cells with multilineage differentiation potential and self-renewal capability in the c-Met CD49f c-Kit CD45 Ter119 fraction of cells isolated from day 13.5 fetal mouse liver. On cell transplantation, these cells differentiated into hepatocytes and cholangiocytes. As an alternative to the antibody based stem cell isolation, Hoechst33342 staining is useful. To understand the mechanism responsible for SP phenotype, we performed an expression cloning and identified bcrp-1/ABCG2 gene, a member of ATP binding-cassette (ABC) transporter family. Bcrp-1 is almost exclusively expressed in CD34 KSL cells among blood cells; however their expression in other tissue specific stem cells remains to be studied. With the use of FACS and monoclonal antibodies, hematopoietic and liver stem cells were prospectively isolated and characterized. HSCs could also be purified by Hoechst 33342 staining. By expression cloning, we identify bcrp-1/ABCG2 transporter as a molecule responsible for SP phenotype. Elucidation of the physiological role of bcrp-1/ABCG2 in HSCs may provide us with clues to understand the molecular mechanisms of stem cell self-renewal and differentiation.

TCDD treatment eliminates the long-term reconstitution activity of hematopoietic stem cells.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an endocrine disrupting chemical (EDC), can cause carcinogenesis, immunosuppression, and teratogenesis, through a ligand-activated transcription factor, the aryl hydrocarbon receptor (AhR). Despite remarkable recent advances in stem cell biology, the influence of TCDD on hematopoietic stem cells (HSCs), which possess the ability to reconstitute long-term multilineage hematopoiesis, has not been well investigated. In this study we examined the influence of TCDD on HSCs enriched for CD34(-), c-kit(+), Sca-1(+), lineage negative (CD34-KSL) cells. The number of the CD34-KSL cells was found to be increased about four-fold upon a single oral administration of TCDD (40 micro g/kg body weight). Surprisingly, we found that these TCDD-treated cells almost lost long-term reconstitution activity. This defect was not present in AhR(-/-) mice. These findings suggest that modulation of AhR/ARNT system activity may have an effect on HSC function or survival.

Lentiviral vector-mediated transduction of murine CD34(-) hematopoietic stem cells.

Efficient gene transfer into murine hematopoietic stem cells (HSCs) provides a powerful tool for exploring hematopoietic stem cell biology. In this study, we evaluated the efficiency of lentiviral vector-mediated gene transfer into murine CD34(-/low)c-Kit(+)Sca-1(+)Lin(-) (CD34(-) KSL) cells that are highly enriched for HSCs. FACS-sorted CD34(-) KSL cells were transduced with the vesicular stomatitis virus G glycoprotein-pseudotyped HIV-1-based lentiviral vector containing the green fluorescent protein (GFP) gene under the control of the cytomegalovirus promoter, and then 50 transduced cells were transplanted into lethally irradiated mice. Transduction efficiency was assessed by FACS analysis for GFP expression in peripheral blood (PB) cells. FACS-sorted GFP(+) KSL bone marrow (BM) cells from primary recipients were used for secondary transplantation, and GFP expression in PB cells of reconstituted mice was analyzed by FACS. GFP expression was detected in PB cells of all primary recipients (n = 10) at an average of 40% (range 26-58%) when the lentiviral vector containing the woodchuck hepatitis virus posttranscriptional regulatory element was used. GFP(+) cells were found in multilineage cells in PB, BM, spleen, and thymus for at least 8 months posttransplantation. In secondary recipients, donor-derived GFP(+) KSL BM cells could reconstitute and GFP expression was detected in both myeloid and lymphoid cells in PB. Our results indicate that lentiviral vectors can efficiently transduce highly enriched murine HSCs and sustain long-term expression of the transgene in the multilineage differentiated progeny in reconstituted mice.