Primitive Murine Hematopoietic Cells Research Articles

An NFIX-mediated regulatory network governs the balance of hematopoietic stem and progenitor cells during hematopoiesis.

The transcription factor (TF) nuclear factor I-X (NFIX) is a positive regulator of hematopoietic stem and progenitor cell (HSPC) transplantation. Nfix-deficient HSPCs exhibit a severe loss of repopulating activity, increased apoptosis, and a loss of colony-forming potential. However, the underlying mechanism remains elusive. Here, we performed cellular indexing of transcriptomes and epitopes by high-throughput sequencing (CITE-seq) on Nfix-deficient HSPCs and observed a loss of long-term hematopoietic stem cells and an accumulation of megakaryocyte and myelo-erythroid progenitors. The genome-wide binding profile of NFIX in primitive murine hematopoietic cells revealed its colocalization with other hematopoietic TFs, such as PU.1. We confirmed the physical interaction between NFIX and PU.1 and demonstrated that the 2 TFs co-occupy super-enhancers and regulate genes implicated in cellular respiration and hematopoietic differentiation. In addition, we provide evidence suggesting that the absence of NFIX negatively affects PU.1 binding at some genomic loci. Our data support a model in which NFIX collaborates with PU.1 at super-enhancers to promote the differentiation and homeostatic balance of hematopoietic progenitors.

Placental Growth Factor Expression Is Required for Bone Marrow Endothelial Cell Support of Primitive Murine Hematopoietic Cells

Two distinct microenvironmental niches that regulate hematopoietic stem/progenitor cell physiology in the adult bone marrow have been proposed; the endosteal and the vascular niche. While extensive studies have been performed relating to molecular interactions in the endosteal niche, the mechanisms that regulate hematopoietic stem/progenitor cell interaction with bone marrow endothelial cells are less well defined. Here we demonstrate that endothelial cells derived from the bone marrow supported hematopoietic stem/progenitor cells to a higher degree than other endothelial or stromal cell populations. This support was dependant upon placental growth factor expression, as genetic knockdown of mRNA levels reduced the ability of endothelial cells to support hematopoietic stem/progenitor cells in vitro. Furthermore, using an in vivo model of recovery from radiation induced myelosuppression, we demonstrate that bone marrow endothelial cells were able to augment the recovery of the hematopoietic stem/progenitor cells. However, this effect was diminished when the same cells with reduced placental growth factor expression were administered, possibly owing to a reduced homing of the cells to the bone marrow vasculature. Our data suggest that placental growth factor elaborated from bone marrow endothelial cells mediates the regulatory effects of the vascular niche on hematopoietic stem/progenitor cell physiology.

Differential Expression of Transforming Growth Factor Beta Receptor 2 (TGFβR2) In Diabetic CD34+ Cells: Implications for Vascular Repair

AbstractAbstract 4795We recently reported that the vascular reparative ability of diabetic CD34+ cells can be restored by transient inhibition of transforming growth factor β1 (TGF-b1) using antisense phosphorodiamidate morpholino oligomers (TGF-β1-PMO) (Bhatwadekar 2010). TGF-β1 mediates its action by binding to TGF-β-R2 receptor homodimers, which then form heterotetrameric complexes with two TGF-βR1 subunits. We investigated whether healthy or diabetic CD34+ cells differentially express TGFβR2 receptor and whether inhibition or knock-out of endogenous TGF-β1 expression would affect the surface expression of TGF-βR2.TGF-βR2 expression of murine bone marrow (BM) lin- Sca-1+/− cells from TGF-β1 knockout mice or wild type mice was studied using flow cytometry. Unexpectedly, TGF-β-R2 expression was essentially absent in BM lin- Sca-1 +and - cells from TGF-β1 knockout mice compared to wild type cells. Because the absence of endogenous and exogenous TGF-β1 resulted in complete down regulation of TGF-βR2 in primitive murine hematopoietic cells, we explored the relationship of endogenous TGF-β1in human mature and immature CD34+ cells in blood, bone marrow and cord blood.Human CD34+CD45+ CD38+ or - cells from healthy and diabetic individuals were FACS sorted based on surface expression the analysed for TGF-βR2 expression. At the same time, TGF-βR2 and TGF-β1 mRNA expression was determined for sorted populations using quantitative real time RT-PCR.A comparison of TGF-βR2 surface expression between mature progenitors (lin- CD34+ CD45+ CD38+) from diabetic and healthy cord blood revealed that TGF-βR2 expression did not differ between these groups. In contrast, TGF-βR2 surface expression in immature cells (CD34+ CD45+ CD38-) was very low in diabetic patients compared to healthy controls (p = 0.001).Next, we inhibited endogenous TGF-β1 in healthy CD34+ cells which resulted in a significant (p<0.01) reduction in TGF-β-R2 mRNA expression. However, in diabetic CD34 cells, TGF β1-PMO treatment abolished TGFβR2 expression.Our study suggests that healthy and diabetic CD34+ cells differentially regulate TGF-βR2 expression which may be the result of the elevated intracellular and extracellular levels of TGFb1 in diabetic individuals. A reduction in TGF-βR2 expression in diabetic CD34+ CD38- cells may physiologically enhance proliferation of the primitive CD34+ CD38- cell compartment, an outcome that may be necessary to keep up with increased demand for CD34+ cells in the diabetic individual. Disclosures:Stepps: BetaStem Therapuetics: Employment. Bartelmez: BetaStem Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

New Candidate Regulators of Hematopoietic Stem Cells Identified from Transcriptional Comparisons of Highly Purified Populations of Longterm Repopulating Cells with Markedly Different in Vivo Self-Renewal Activities

Significant advances have been made in the development of methods for purifying murine hematopoietic cells with longterm (>4 months) in vivo reconstituting ability although these longterm repopulating cells (LTRCs) remain heterogeneous with regard to the self-renewal (SR) activity they display when transplanted into irradiated hosts. Furthermore our group has also identified cell culture conditions that differentially alter LTRC activity without immediate effects on their proliferation or survival. Here, we show that highly purified LTRCs with high and low SR properties can be prospectively isolated from normal adult mouse bone marrow (ABM) as 2 separate populations according to their expression of CD150 within the EPCR++CD48−CD45mid fraction of cells: 56% total LTRCs and 43% of the high SR type in the CD150+ subset vs. 39% total LTRCs and 32% of the low SR type in the CD150− subset (as determined from 62 and 28 single cell transplants, respectively). As a first test of whether these populations would likely be useful to search for new molecular differences associated with their different SR properties, we compared the level of expression in these 2 populations of a small set of genes previously reported to regulate LTRC SR activity: c-Kit, Bmi1, Gata3, Rae28, Ezh2 and Lnk by quantitative real-time PCR (Q-RT-PCR). This exercise revealed transcript levels of the first 4 of these genes to be significantly higher in the CD150+ subset that is selectively enriched in high SR LTRCs, thus validating the concept that they have a distinct molecular signature. Previous evidence shows that high SR LTRCs are present in both FL LTRCs and ABM LTRCs but they differ in some properties (i.e.: cell cycle status, regeneration kinetics). We therefore began a search for ontogeny-independent components of the SR machinery by comparing tags present in 2 LongSAGE libraries produced from CD45midlin−Rho−SP ABM cells and from lin−Sca1+CD43+Mac1+ embryonic day 14.5 fetal liver (FL) cells (each 20–30% total LTRCs and 12–20% of the high SR type, as determined by 132 (FL) and 352 (ABM) single cell transplants, respectively). From these comparisons and additional data in other publicly available datasets for primitive murine hematopoietic cells, we identified 28 genes not previously shown to have a functional role in LTRC SR control. We then compared the level of expression of these 28 genes between the CD150+ subsets of EPCR++CD48−CD45mid ABM cells and FL cells (24% total LTRCs and 12% high SR LTRCs in the FL subset) and their respective downstream lin− progeny. This comparison revealed 10 of these genes to be down-regulated in the lin− populations of both ABM and FL. Further comparison of the expression of these 10 genes between the high vs. low SR LTRCs (found in the CD150+ and CD150− subsets of EPCR++CD48−CD45mid) ABM cells showed the expression of 5 (Vwf, Rhob, Pld3, Prnp and Smarcc2) to be downregulated in the CD150− (low SR LTRC) subset. Interestingly, the first 4 of these genes, as well as 2 of the preliminary set of SR regulators (Bmi1 and Gata3), were also selectively down-regulated in EPCR++CD150+CD48−CD45mid ABM cells that had been incubated for 16 hours in 1 or 10 ng/ml Steel factor + 20 ng/ml IL-11 (conditions that decrease LTRC activity in vivo 4–5-fold before any of these divide or die). Taken together, these results point to the existence of more, although a rather small number of additional genes, including Vwf, Rhob, Pld3, and Prnp, whose products may be involved in controlling the SR potential of normal mouse LTRCs.

Forced Overexpression of Ahi-1 Confers a Proliferative Advantage on Primitive Murine Hematopoietic Cells and Enhances the Effects of BCR-ABL.

Ahi-1 (Abelson helper integration site-1) is a novel gene that is commonly activated by proviral insertional mutagenesis in v-abl or myc-induced murine leukemias and lymphomas. Ahi-1 encodes a unique protein with known signaling features including SH3 and WD40-repeat domains but its normal function is unknown. Involvement of Ahi-1 in leukemogenesis is suggested by the high frequency of Ahi-1 mutations seen in certain virus-induced murine leukemias and by the gross perturbations seen in the expression of human AHI-1 and its isoforms in several human leukemic cell lines, as well as in the primary lin−CD34+CD38− leukemic stem cell-enriched population in patients with chronic phase CML. To further investigate the role of Ahi-1 as a potential co-operating oncogene relevant to BCR-ABL-mediated leukemogenesis, we compared the biological behavior of primitive murine hematopoietic cells from the bone marrow of 5-FU-treated adult C57BL/6 mice after their transduction with MSCV-Ahi-1-IRES-YFP, MSCV-BCR-ABL-IRES-GFP retroviruses, either alone or in combination. Quantitative real-time RT-PCR analysis of RNA from FACS-purified lin−YFP+ (Ahi-1+), lin−GFP+ (BCR-ABL+) and lin−YFP+GFP+ (Ahi-1+ + BCR-ABL+)-transduced bone marrow cells showed that Ahi-1 transcripts were present at 40-fold higher levels in the Ahi-1-transduced cells by comparison to the control cells transduced with the empty MIY vector. Immediately post-transduction, the Ahi-1-transduced cells produced a similar number of colonies as the MIY-transduced control cells in semi-solid cultures containing Steel factor (SF) + IL-3 + IL-6 + EPO, although a small proportion of the Ahi-1+ CFCs (~10%) were already growth factor-independent. In addition, the proliferative activity of the FACS-purified lin−YFP+ (Ahi-1+) cells (as indicated by the rate of expansion of viable cells in a week in liquid cultures containing SF, IL-3, and IL-6) was ~3-fold higher than in cultures initiated with control (lin−YFP+) cells. Moreover, after 4 weeks in longterm culture-initiating cell (LTC-IC) assays, the Ahi-1+ cells produced 2x more CFCs than the control cells. All of these endpoints (proliferative activity, growth factor-dependence and CFC output in LTC-IC assays) are also perturbed by BCR-ABL transduction. Interestingly, in cells that were co-transduced with Ahi-1 and BCR-ABL, all of these effects were further enhanced as compared to cells transduced with either BCR-ABL alone (2–4-fold) or Ahi-1 alone (3–6-fold). Thus, overexpression of Ahi-1 alone deregulates the proliferation control of primitive murine hematopoietic cells and this is additive with the effects of BCR-ABL, suggesting that Ahi-1 and BCR-ABL can cooperate to promote the progression of BCR-ABL-associated diseases like CML.

303. Impaired Nuclear Transport and Virus Uncoating Limit Recombinant Adeno-Associated Virus 2 Vector-Mediated Transduction of Primary Murine Hematopoietic Cells

The transduction efficiency of adeno-associated virus 2 (AAV) vectors varies greatly in different cells and tissues. Whereas muscle and brain cells are transduced efficiently, controversies abound regarding hematopoietic cell transduction by AAV vectors. For human hematopoietic cells, we documented this problem to be related to the extent of AAV receptor expression (J. Virol., 71: 8262–8267, 1997). Murine hematopoietic cells express the receptor, yet are transduced poorly. Using a murine fibroblast cell line, we reported that the lack of transduction was due to inefficient intracellular trafficking of AAV from cytosol into the nucleus (J. Virol., 74: 992–996, 2000), and that treatment with hydroxyurea (HU) facilitated nuclear transport of AAV in these cells (J. Virol., 75: 4080–4090, 2001). In the present studies, we extended these observations to primitive murine hematopoietic cells. Murine c-kit+, lin− hematopoietic cells were transduced with recombinant AAV-lacZ vectors. Forty-eight hrs. post-transduction, nuclear and cytoplasmic fractions were isolated and low Mr DNA samples extracted from these fractions were analyzed on Southern blots. Approximately 85% of AAV genomes were present in the cytoplasmic fraction. However, when donor mice were injected intra-peritoneally with HU at 1 mg/g body weight, 24 hrs. prior to isolation of cells, the extent of AAV genomes detected in the cytoplasmic fraction was reduced to ~40%, with a corresponding increase in the nuclear fraction, which increased to ~60%, indicating that in vivo administration of HU facilitated nuclear transport of AAV. However, when DNA samples were electrophoresed on denaturing gels and analyzed on Southern blots, it was apparent that a significant fraction of the AAV genome present in the nuclear fraction from cells obtained from HU-treated mice was single-stranded. We tested the hypothesis whether single-stranded genomes were derived from virions that failed to undergo uncoating in the nucleus. Primitive hematopoietic cells, with and without HU-administration in vivo, were transduced with recombinant AAV-lacZ vectors, and 48-hrs. post-transduction, equivalent fractions were either mock-treated, or digested exhaustively with DNase I, and analyzed on DNA slot-blots using a lacZ probe. Whereas majority of the signal, which was resistant to DNase I, was detected in the cytoplasmic fraction in cells obtained from untreated mice, as expected, a substantial fraction of the signal in the nuclear fraction in cells obtained from HU-treated mice was also resistant to DNase I, suggesting that although HU facilitated nuclear transport of AAV, most of the virions failed to undergo uncoating, thereby leading to only a partial improvement in viral second-strand DNA synthesis and transgene expression. Thus, the identification of a yet another obstacle — virus uncoating — has implications in the optimal use of recombinant AAV vectors in hematopoietic stem cell gene therapy.

Evidence for a positive role of SHIP in the BCR-ABL–mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia

AbstractPrevious studies suggested that the SH2-containing inositol-5-phosphatase (SHIP) may play a tumor suppressor-like function in BCR-ABL–mediated leukemogenesis. To investigate this possibility, we first developed a new assay for quantitating transplantable multilineage leukemia-initiating cells (L-ICs) in hematopoietic stem cell (HSC)–enriched mouse bone marrow (BM) cells transduced with a BCR-ABL–GFP (green fluorescent protein) retrovirus. The frequency of L-ICs (1 of 430 Sca-1+lin– cells) was 7-fold lower than the frequency of HSCs in the Sca-1+lin– subset transduced with a control virus (1 of 65 cells). Forced BCRABL expression was also accompanied by a loss of regular HSC activity consistent with the acquisition of an increased probability of differentiation. Interestingly, the frequency and in vivo behavior of wild-type (+/+) and SHIP–/– L-ICs were indistinguishable, and in vitro, Sca-1+lin– BCR-ABL–transduced SHIP–/– cells showed a modestly reduced factor independence. Comparison of different populations of cells from patients with chronic myeloid leukemia (CML) in chronic phase and normal human BM showed that the reduced expression of full-length SHIP proteins seen in the more mature (CD34–lin+) leukemic cells is not mirrored in the more primitive (CD34+lin–) leukemic cells. Thus, SHIP expression appears to be differently altered in the early and late stages of differentiation of BCR-ABL–transformed cells, underscoring the importance of the cellular context in which its mechanistic effects are analyzed.

Abrogation of TGF-beta activity during retroviral transduction improves murine hematopoietic progenitor and repopulating cell gene transfer efficiency.

Transforming growth factor-beta has complex activities on hematopoietic cells. We have previously shown that murine long-term repopulating activity is compromised by ex vivo culture in TGF-beta 1 and conversely is increased by abrogating endogenous TGF-beta activity with a neutralizing antibody. In the current study, we investigated the effect of abrogation of autocrine or paracrine TGF-beta present during retroviral transduction on gene transfer efficiency to primitive hematopoietic cells. Murine marrow cells were cultured and retrovirally transduced for 4 days in the presence of interleukin-3, interleukin-6 and stem cell factor, and either a neutralizing anti-TGF-beta antibody or an isotype control. Committed progenitor cells were analyzed for gene transfer efficiency, and cells were also injected into W/Wv recipient mice for analysis of transduction of long-term repopulating cells. The progenitor (CFU-C) transduction efficiency in the presence of anti-TGF-beta was significantly greater. Semiquantitative PCR analysis and Southern blot analysis for the retroviral marker gene in the blood and bone marrow of recipient mice revealed a significant increase in the transduction efficiency of long-term repopulating cells after culture and transduction in the presence of the anti-TGF-beta. Thus neutralization of TGF-beta activity during retroviral transduction allows more efficient gene transfer into primitive murine hematopoietic cells and may prove beneficial in future clinical gene transfer or therapy trials.

Sustained high-level reconstitution of the hematopoietic system by preselected hematopoietic cells expressing a transduced cell-surface antigen.

Despite improvements in retrovirus-mediated gene transfer to primitive murine hematopoietic cells, high-level reconstitution with provirally marked cells with continued expression of the transferred gene(s) remains a challenge in many situations. We evaluated a physical preselection strategy for isolating transduced cells after their infection with different vectors. The small (240-bp) cDNA coding region for the human CD24 cell-surface antigen was inserted into myeloproliferative sarcoma virus (MPSV) and murine stem cell virus (MSCV)-based retroviral vectors such that expression of CD24 was under the control of the viral long terminal repeat (LTR). After infection of (Ly-5.1) mouse bone marrow (BM), those expressing CD24 were isolated by fluorescence-activated cell sorting (FACS) and a transplant dose estimated to contain approximately 12 +/- 4 long-term competitive repopulating cells (CRU) injected into lethally irradiated congenic Ly-5.2 recipients. Six months later, virtually all recipients showed high-level (> 80%) reconstitution of their BM and thymus with Ly-5.1 (transplant-derived) cells, the majority of which were also transduced (mean of 2.5 or 2.6 proviral copies for the two vectors). All spleen colonies generated in secondary recipients of cells obtained from the BM of the 6-month-old primary mice contained the provirus. However, only in recipients of MSCVCD24-infected marrow was a correspondingly high level of CD24 expression seen: a maximum of 88% for whole BM (all mice positive), 58% for peripheral blood leukocytes (all mice positive), and 21% for thymocytes (11 of 13 mice positive). CD24 was also readily detected on the regenerated Sca-1+Lin- cells present in the primary and secondary recipients when these were studied 6 months post-transplant, but again on more of the Sca-1+Lin- cells in recipients of MSCVCD24-infected cells as compared to recipients of MPSVCD24-infected cells. These results point to the utility of preselection strategies and suggest an approach for the development of better vectors for achieving regulated, lineage-specific or stage-specific gene expression patterns in particular subsets of hematopoietic cells.

Primitive Hematopoietic Cells in Murine Bone Marrow Express the CD34 Antigen

The CD34 antigen is expressed on most, if not all, human hematopoietic stem cells (HSCs) and hematopoietic progenitor cells, and its use for the enrichment of HSCs with repopulating potential is well established. However, despite homology between human and murine CD34, its expression on subsets of primitive murine hematopoietic cells has not been examined in full detail. To address this issue, we used a novel monoclonal antibody against murine CD34 (RAM34) to fractionate bone marrow (BM) cells that were then assayed in vitro and in vivo with respect to differing functional properties. A total of 4% to 17% of murine BM cells expressed CD34 at intermediate to high levels, representing a marked improvement over the resolution obtained with previously described polyclonal anti-CD34 antibodies. Sixty percent of CD34+ BM cells lacked lineage (Lin) markers expressed on mature lymphoid or myeloid cells. Eighty-five percent of Sca-1+Thy-1(10)Lin-/10 cells that are highly enriched in HSCs expressed intermediate, but not high, levels of CD34 antigen. The remainder of these phenotypically defined stem cells were CD34-. In vitro colony-forming cells, day-8 and -12 spleen colony-forming units (CFU-S), primitive progenitors able to differentiate into B lymphocytes in vitro or into T lymphocytes in SCID mice, and stem cells with radioprotective and competitive long-term repopulating activity were all markedly enriched in the CD34+ fraction after single-parameter cell sorting. In contrast, CD34-BM cells were depleted of such activities at the cell doses tested and were capable of only short-term B-cell production in vitro. The results indicate that a significant proportion of murine HSCs and multilineage progenitor cells express detectable levels of CD34, and that the RAM34 monoclonal antibody is a useful tool to subset primitive murine hematopoietic cells. These findings should facilitate more direct comparisons of the biology of CD34+ murine and human stem and progenitor cells.