Mouse Human Hematopoietic Stem Cells Research Articles

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

The BET inhibitor CPI203 promotes ex vivo expansion of cord blood long-term repopulating HSCs and megakaryocytes

AbstractAlthough cytokine-mediated expansion of human hematopoietic stem cells (HSCs) can result in high yields of hematopoietic progenitor cells, this generally occurs at the expense of reduced bone marrow HSC repopulating ability, thereby limiting potential therapeutic applications. Because bromodomain-containing proteins (BCPs) have been demonstrated to regulate mouse HSC self-renewal and stemness, we screened small molecules targeting various BCPs as potential agents for ex vivo expansion of human HSCs. Of 10 compounds tested, only the bromodomain and extra-terminal motif inhibitor CPI203 enhanced the expansion of human cord blood HSCs without losing cell viability in vitro. The expanded cells also demonstrated improved engraftment and repopulation in serial transplantation assays. Transcriptomic and functional studies showed that the expansion of long-term repopulating HSCs was accompanied by synchronized expansion and maturation of megakaryocytes consistent with CPI203-mediated reprogramming of cord blood hematopoietic stem and progenitor cells. This approach may therefore prove beneficial for ex vivo gene editing, for enhanced platelet production, and for the improved usage of cord blood for transplantation research and therapy.

Hepatic Leukemia Factor Is An Important Regulator Of Hematopoietic Stem Cell Activity and Identity

The transcription factor Hepatic Leukemia Factor (HLF) was originally identified in a chromosomal translocation with the gene E2A causing a subset of childhood B-lineage acute lymphoid leukemia. Moreover, HLF has been described as a regulator of circadian rhythm and recent findings have implicated HLF as a candidate “stemness” gene in both normal and malignant stem cells. Accordingly, overexpression of HLF in human hematopoietic stem cells (HSC) results in an enhanced reconstitution capability in NOD-SCID mice. However, little is known about HLF's physiological role in hematopoiesis and HSC regulation. Using quantitative PCR, we found that HLF is highly expressed in mouse (C57Bl/6) HSC and is downregulated upon differentiation (HSC 3.2 (±0.95) fold (p<0.001), LSK 1.9 (±0.47) fold (p<0.05), CMP, GMP MEP all less then 0.1 fold, all values are compared to HPRT). This encouraged us to further investigate HSC function in the absence of HLF.The conventional HLF knockout (KO) mice (C57bl/6 background) were viable, born at normal Mendelian ratios and showed normal hematopoietic parameters (bone marrow cellularity: WT 2.7x107 (±5.4 x106), KO 3.3x107 (±6.4 x106), p>0.2 n=9). In addition, the HLF KO mice demonstrated normal lineage distribution of both mature cells in the peripheral blood and bone marrow as well as the frequency of immunophenotypic HSC (Lin-Sca1+ckit+CD34-Flt3-: WT 0.0005 (±0.5x10-4)%, KO 0.0005 (±0.1x10-3)%; n>10). However, in a serial competitive transplantation assay using whole bone marrow (200 000 cells 1:1 ratio), HLF KO cells demonstrated a significant reduction in reconstitution capacity in primary recipients (WT 56 (±15)%, KO 40.2 (±16)%, p=0.028, n>10), which was further increased in the secondary recipients (WT 87.2 (±26)%, KO 8.7 (±5.8)%, p<0.001, n>10). Almost no engraftment was detected from the HLF KO cells in tertiary recipients. To further evaluate stem cell activity in the absence of HLF, we next enumerated the number of competitive repopulating units (CRU) by limiting dilution assay, which revealed a 2.6 fold reduction, of CRU in the HLF KO mice compared to WT controls (WT 1.6 (±0.4)/105 bone marrow cells, KO 0.6 (±0.2)/105 bone marrow cells). Similarly, transplantation of sorted HSC (Lin-Sca1+ckit+CD34-Flt3-) also showed a 2.4 fold (WT 47.3 (±24)%, KO 19.4 (±25)%, p=0.16, n=9) reduced engraftment of total cells but with enhanced T cell frequency in peripheral blood (WT 19.5 (±6.2)%, KO 40.8 (±7.4)%, p=0.01, n=9).Since we also found that HLF was highly expressed in fetal liver derived HSC, we transplanted fetal liver HLF KO cells from E14.5 in a competitive repopulation setting. In line with the phenotype seen in the adult HLF KO mice, the fetal liver HLF KO cells demonstrated impaired reconstitution ability (WT 52.8 (±16)%, KO 0.9 (±1.4)%, n>10). Intriguingly, the phenotype was stronger than in the adult HLF KO HSC, indicating that HLF is particularly important during the expansion phase of HSC in embryonic development.The underlying mechanism of the reduced HSC activity is still unclear, but preliminary findings show that HLF KO HSC have enhanced ROS levels (WT 337 (±33), KO 510 (±55), p<0.05, n=3) and increased cycling HSC (G0: WT 66.5 (±6.4)%, KO 58.5 (±4.7)%; G1/S/G2/M: WT 33.6 (±6.6)%, KO 41.7 (±4.9)%, n=3). We are currently performing global gene expression analysis to further understand the mechanism of HLF in HSC regulation.Interestingly, we also found that HLF appears to regulate the identity of HSC by modulating the expression of the SLAM code on the cell surface of the HLF KO HSC. In contrast to the normal frequency of LSK Flt3-CD34- cells, the HLF KO mice displayed a 3.5 fold reduction in the frequency of LSK CD150+CD48- cells (WT 1.94x10-4 (±4.4x10-5)%, KO 0.56x10-4 (±1.5x10-5)%, p<0.001 n>10). Strikingly, transplantation of as many as 150 LSK CD150+CD48-HLF KO cells showed a complete lack of repopulating capacity in vivo. This did not correlate to the number of functional HSC seen when transplanting whole bone marrow and indicates that HLF affects the identity of HSC by modulating the expression of the SLAM markers.Taken together, we show here for the first time that HLF has a fundamental role in HSC biology during both fetal and adult hematopoiesis by regulating HSC activity and identity. Disclosures:No relevant conflicts of interest to declare.

Improved Engraftment of Human Hematopoietic Stem Cells in NOD/SCID Mice by the Antioxidant, N-Acetyl-L-Cysteine

Abstract 1884Non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice are widely used as a human-mouse xeno-transplant model for assessing engraftment of human hematopoietic stem cells (HSCs). Optimizing this model and understanding the variables that affect engraftment are critical to correctly interpret the engraftment results of human HSCs in NOD/SCID recipients. However, the engraftment efficiency of human HSCs remains very low in NOD/SCID mice. Larger efforts have been devoted to improving engraftment by increasing the immune-deficiency of the mice, but less effort has been made toward other negative parameters in the host. Our preliminary study showed that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in the bone marrow (BM) in comparison with other commonly used mouse strains (C57BL/6 and BALB/C). Given the previous studies by us and others showing that excessive ROS could impair the function of HSCs and that antioxidants were able to overcome the exhaustion of mouse HSCs in transplant recipients, we hypothesized that the poor engraftment of human hematopoietic cells in NOD/SCID recipients may be partially attributed to higher levels of ROS in NOD/SCID BM and a reduction of ROS by antioxidants may improve the engraftment of human HSCs in NOD/SCID mice.To test this hypothesis, NOD/SCID (8- to 12-week-old) mice were injected subcutaneously daily with an antioxidant, N-acetyl-L-cysteine (NAC) or PBS (control) for two weeks before being irradiated with 200 cGy from a cesium-137 source at 70cGy/min. Different doses of CD34+ cells or highly-enriched HSCs from human cord blood (CB) were injected through the tail vein or into the right tibia of the mice. The mice were maintained on NAC treated drinking water following injection, then sacrificed 12–14 weeks after transplantation to measure the engraftment levels of different hematopoietic cell lineages.We found that treatment with NAC was able to lower the levels of ROS in NOD/SCID BM. At the highest dose of injected CD34+ cells (>5×105), the NAC treated recipients displayed a significant increase of engraftment (2.1-fold) when compared with the control group (Control vs. NAC treated recipients: 11.04±3.11% vs. 23.21±4.0%, p=0.0224; n=20/each). This improvement was even more significant when injected cell numbers were reduced (2×105 and 1×105 had 3.9- and 4.9- fold higher engraftment, respectively, in NAC treated recipients), thus suggesting that saturating levels of HSCs may ease the anti-oxidant effect on engraftment. Furthermore, we also demonstrated higher levels of overall engraftment and multi-lineage differentiation of human HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+Rholow) with a limiting dilution analysis. In comparison with the control mice, NAC treated recipients displayed 2.5-, 3.5-, and 5.7-fold increases in engraftment in the injected tibia (IT), BM and spleen, respectively. The frequency of SCID-repopulating cell (SRC) in IT was approximately 3.0-fold higher in NAC treated mice than in control mice (1 in 108 vs. 1 in 36). Similar improvements (4.1- to 7.9- fold) in SRC frequencies were also detected in BM and spleen. Notably, NAC increased the probability of positive engraftment when a single human HSC was directly transplanted into the BM of NOD/SCID mice. In summary, our current study uncovers a previously unappreciated negative effect of ROS in the human-NOD/SCID xenotransplant model and reduction of ROS via antioxidants such as NAC may significantly enhance the engraftment of human hematopoietic stem cells in NOD/SCID mice. Disclosures:No relevant conflicts of interest to declare.

A Novel Osteoclastic Network Determines In Vitro Niche for Mouse and Human Hematopoietic Stem Cells

Abstract 1325New innovative strategies are needed to expand human hematopoietic stem cells (HSCs) for clinical applications. In this perspective, we recently developed an in vitro/in vivo screening strategy, which revealed 18 nuclear factors that enhance HSC activity (Deneault et al., Cell 2009). During these experiments, mouse HSCs were kept for 12 days in mini-cultures that included viral producer cells for each tested factor. Interestingly, 4 of the 18 hits identified in this initial screen, i.e., Fos, Tcfec, Hmgb1 and Sfpi1 operated through non-HSC autonomous (NHA) mechanisms: expanded HSCs were not infected with retrovirus. This suggested that the transfected viral producer cells (feeder cells) produced membrane-bound or soluble molecules that promote expansion of HSCs introduced in these cultures. We now provide evidence that seven additional factors, i.e., Smarcc1, Vps72, Sox4, Klf10, Ski, Prdm16 and Erdr1 significantly enhance HSC activity through NHA mechanisms, hereafter called “NHA factors”. Moreover, we found that Vps72, Fos and Klf10 also promote expansion of human HSCs by NHA mechanisms. Interestingly at least for feeders expressing Klf10, we observed that physical contact between HSCs and these engineered support cells was not necessary, suggesting the presence of secreted molecule(s) in the medium. Expression profiling was next performed using mRNA extracted from feeder cells transduced with each of the NHA factors. Firstly, we shed light on the transcriptional make up and potential convergence of signaling pathways in the engineered feeder cells: our results reveal two independent but interconnected transcriptional regulatory subnetworks. Strikingly, some constituents of the subnetworks, i.e., Sfpi1, Fos, Klf10 and Tcfec (Mitf-related) have previously been shown to play critical roles in the regulation of osteoclasts, which are a myeloid-derived population of cells residing in the HSC bone marrow niche. In addition, all of the NHA factors act in concert to increase Prdm16 expression levels in a range from 2.5- to 54.6-fold. For this reason, Prdm16 clearly holds the position of central hub of the osteoclastic network. However, Tcfec represents the ultimate downstream effector of this pathway as Prdm16 elevates its expression levels by 18.9-fold. In parallel, Prdm16 can also increase the expression of Sfpi1, which in turn can upregulate Tcfec expression up to 874-fold. Moreover, upregulated mRNA targets corresponding to factors that are secreted or associated with the plasma membrane were considered as potential candidate agonists of HSC self-renewal. A high degree of overlap was observed between the sets of proteins produced by feeder cells engineered to overexpress Sfpi1, Fos, Klf10, Tcfec or Ski. These secreted and membrane bound proteins include Agt, Aspn, Ogn, Ptgds, Nckap1l, Rgs16 and Lcn2. Studies are ongoing to characterize the contribution of these newly identified NHA proteins in HSC expansion. Validation of these NHA proteins in human HSC expansion will have a clear potential for translational medicine. Disclosures:No relevant conflicts of interest to declare.

Adipose Tissue-Derived Mesenchymal Stem Cells Facilitate Hematopoiesis in Vitro and in Vivo: Advantages Over Bone Marrow-Derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) have emerged as a new therapeutic modality for reconstituting the hematopoietic microenvironment by improving engraftment in stem cell transplantation. However, the availability of conventional bone marrow (BM)-derived MSCs (BMSCs) is limited. Recent studies showed that a large number of MSCs can be easily isolated from fat tissue (adipose tissue-derived MSCs [ADSCs]). In this study, we extensively evaluated the hematopoiesis-supporting properties of ADSCs, which are largely unknown. In vitro coculture and progenitor assays showed that ADSCs generated significantly more granulocytes and progenitor cells from human hematopoietic stem cells (HSCs) than BMSCs. We found that ADSCs express the chemokine CXCL12, a critical regulator of hematopoiesis, at levels that are three fold higher than those with BMSCs. The addition of a CXCL12 receptor antagonist resulted in a lower yield of granulocytes from ADSC layers, whereas the addition of recombinant CXCL12 to BMSC cocultures promoted the growth of granulocytes. In vivo cell homing assays showed that ADSCs facilitated the homing of mouse HSCs to the BM better than BMSCs. ADSCs injected into the BM cavity of fatally irradiated mice reconstituted hematopoiesis more promptly than BMSCs and subsequently rescued mice that had received a low number of HSCs. Secondary transplantation experiments showed that ADSCs exerted favorable effects on long-term HSCs. These results suggest that ADSCs can be a promising therapeutic alternative to BMSCs.

CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

Expansion ex vivo des cellules souches hématopoïétiques humaines par la transduction passive de l'homéoprotéine HOXB4

Expansion of human hematopoietic stem cells (HSCs) is a challenge for cellular therapy. It currently relies on either the use of recombinant cytokines or transfer of transcription factor genes. Among these, the HOXB4 homeoprotein is of particular interest since it promotes the expansion of mouse HSCs without inducing leukemia. To prevent potential deleterious side effects associated with stable HOXB4 gene transfer into the cells, we took advantage of the ability of homeoproteins to passively pass through cell membranes. We have shown that, when co-cultured with stromal cells engineered to secrete HOXB4, human stem cells and immature progenitors clearly were expanded. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. The role that HOXB4 plays on stem cell expansion has also been tested on human lymphoid progenitors. We found that our model of protein transfer was also able to induce the expansion of the immature lympho-myeloid and pro-T/NK progenitors as well as of more mature NK progenitors. We then looked for synergistic activities between HOXB4 and other homeoproteins such as HOXC4. We found that HOXC4 was able to promote the expansion of human hematopoietic cells in vitro roughly as HOXB4 did and that the presence of both HOXB4 and HOXC4 molecules induced even higher expansion levels of these cells. Our method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia

Human hematopoietic stem cells (HSCs) are generally regarded as being devoid of the markers expressed by differentiated blood cells, the lineage-specific antigens. However, recent work suggests that genes associated with the myeloid lineage are transcribed in mouse HSCs. Here, we explore whether myeloid genes are actually translated in human HSCs. We show that CD33, CD13, and CD123, well-established myeloid markers, are expressed on human long-term repopulating cells from cord blood and bone marrow. In addition, we demonstrate that nonobese diabetic/severe combined immunodeficiency (NOD/SCID) leukemia-initiating cells (SL-ICs) are restricted to the CD33+ fraction in 11 of 12 acute myeloid leukemia (AML) samples studied, indicating that leukemic stem cells (LSCs) express this antigen. This study changes our view of HSCs and the process of differentiation. Furthermore, based on the phenotypic similarity of HSCs and LSCs, our data provide support for the hypothesis that AML derives from an HSC. Our findings also provide a challenge to contemporary attempts to improve the outcome of AML using myeloid antigen-targeted therapies, given the potential for HSC killing.

Ex vivo expansion of human hematopoietic stem cells by direct delivery of the HOXB4 homeoprotein.

Expansion of human hematopoietic stem cells (HSCs) is a major challenge in cellular therapy, and currently relies on the use of recombinant cytokines or on gene transfer of transcription factors. Of these, the HOXB4 homeoprotein protein is of particular interests as it promotes the expansion of mouse HSCs without inducing the development of leukemia. To eliminate any deleterious effects that might be associated with stable HOXB4 gene transfer into human cells, we took advantage of the ability of HOX proteins to passively translocate through cell membranes. Here we show that when cultured on stromal cells genetically engineered to secrete HOXB4, human long-term culture-initiating cells (LTC-ICs) and nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mouse repopulating cells (SRCs) were expanded by more than 20- and 2.5-fold, respectively, over their input numbers. This expansion was associated with enhanced stem cell repopulating capacity in vivo and maintenance of pluripotentiality. This method provides a basis for developing cell therapy strategies using expanded HSCs that are not genetically modified.

Human CD34+ hematopoietic stem cells capable of multilineage engrafting NOD/SCID mice express flt3: distinct flt3 and c-kit expression and response patterns on mouse and candidate human hematopoietic stem cells

AbstractThe cytokine tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. Through its ability to promote ex vivo expansion and oncoretroviral transduction of primitive human hematopoietic progenitors, the flt3 ligand (FL) has emerged as a key stimulator of candidate human hematopoietic stem cells (HSCs). However, recent studies in the mouse suggest that though it is present on short-term repopulating cells, flt3 is not expressed on bone marrow long-term reconstituting HSCs, the ultimate target for the development of cell replacement and gene therapy. Herein we demonstrate that though only a fraction of human adult bone marrow and cord blood CD34+long-term culture-initiating cells (LTC-ICs) express flt3, most cord blood lymphomyeloid HSCs capable of in vivo reconstituting nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice are flt3+. The striking difference in flt3 and c-kit expression on mouse and candidate human HSCs translated into a corresponding difference in flt3 and c-kit function because FL was more efficient than SCF at supporting the survival of candidate human HSCs. In contrast, SCF is far superior to FL as a viability factor for mouse HSCs. Thus, the present data provide compelling evidence for a contrasting expression and response pattern of flt3 and c-kit on mouse and human HSCs.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

The low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Lentivirus-based vectors transduce mouse hematopoietic stem cells with similar efficiency to Moloney murine leukemia virus–based vectors

AbstractThe low levels of transduction of human hematopoietic stem cells (HSCs) with Moloney murine leukemia virus (MLV) vectors have been an obstacle to gene therapy for hematopoietic diseases. It has been demonstrated that lentivirus vectors are more efficient than MLV vectors at transducing nondividing cell lines as well as human CD34+ cells and severe combined immunodeficiency disease repopulating cells. We compared transduction of cell lines and Lin− bone marrow cells, using a vesicular stomatitis virus G (VSV-G)-pseudotyped lentivirus or MLV vectors carrying a green fluorescent protein marker gene. As predicted, the lentivirus vector was more efficient at transducing mouse and human growth-inhibited cell lines. The transduction of mouse HSC by lentivirus vectors was compared directly to MLV vectors in a co-transduction assay. In this assay, transduction by ecotropic MLV is a positive internal control for downstream steps in retrovirus transduction, including cell division. Both the VSV-G lentivirus and MLV vectors transduced mouse HSCs maintained in cytokine-free medium at very low frequency, as did the ecotropic control. The lentivirus vector and the MLV vector were equally efficient at transducing bone marrow HSCs cultured in interleukin 3 (IL-3), IL-6, and stem cell factor for 96 hours. In conclusion, although lentivirus vectors are able to transduce growth-inhibited cell lines, the cell cycle status of HSCs render them resistant to lentivirus-mediated transduction, and it is hypothesized that entry into cycle, not necessarily division, may be a requirement for efficient lentivirus-mediated transduction.

Superior transduction of mouse hematopoietic stem cells with 10A1 and VSV-G pseudotyped retrovirus vectors.

The inefficient transduction of human hematopoietic stem cells (HSC) with amphotropic retroviral vectors has been an obstacle to gene therapy for hematopoietic diseases. We have previously reported low levels of amphotropic retrovirus receptor (Pit-2) mRNA and higher levels of gibbon ape leukemia virus (GALV) or 10A1 retrovirus receptor (Pit-1) mRNA in mouse and human HSC. The vesicular stomatitis virus (VSV-G) uses an abundant membrane phospholipid as a receptor. We hypothesized that transduction of HSC requires relatively high levels of retrovirus receptor molecules. Because mouse HSC can be efficiently transduced by ecotropic virus through the abundant ecotropic receptor, the mouse is an ideal model to compare receptor levels and transduction. We have developed a cotransduction assay where ecotropic retrovirus transduction is a positive internal control for downstream steps in retrovirus transduction. A comparison of mouse HSC transduction with amphotropic, 10A1, and VSV-G envelopes showed that the level of amphotropic and 10A1 receptor mRNA in HSC correlated with the frequency of transduction. Transduction with VSV-G vectors was similar to that with 10A1 vectors. We conclude that the level of retrovirus receptor on HSC is critical for HSC transduction and that GALV or VSV-G vectors would be better for human HSC transduction.

Improved Amphotropic Retrovirus‐Mediated Gene Transfer into Hematopoietic Stem Cells

The efficiency of amphotropic retrovirus-mediated gene transfer into human Hematopoietic Stem Cells (HSC) is less than 1%. This has impeded gene therapy for hematopoietic diseases. In this study we demonstrate that populations of mouse and human HSC contain low to undetectable levels of the amphotropic virus receptor mRNA (ampho R mRNA), and are resistant to transduction with amphotropic retroviral vectors. In a subpopulation of mouse HSC expressing 7-fold higher levels of ampho R mRNA, transduction with amphotropic retrovirus vectors was 30-fold higher. We conclude that retrovirus transduction of HSC correlates with ampho R mRNA levels. Our results predict that alternative sources of HSC or retroviruses will be required for human gene therapy of hematopoietic diseases. One alternative source of stem cells is from individuals treated with cytokines. We have previously shown that mice treated with G-CSF and SCF have an immediate increase in peripheral blood HSC immediately after treatment, followed by a 10-fold increase in bone marrow HSC 14 days after treatment. In this report we show that when rhesus monkey bone marrow cells collected 14 days after G-CSF and SCF treatment were transduced with amphotropic retroviruses, gene transfer levels were approximately 10%, which was easily detected by Southern blot analysis. We conclude that the increased gene transfer may be the result of increased expression of the amphotropic retrovirus receptor, increased numbers of cycling HSC or both.

Amphotropic or Gibbon Ape Leukemia Virus Retrovirus Binding and Transduction Correlates with the Level of Receptor mRNA in Human Hematopoietic Cell Lines

ABSTRACT: The low level of amphotropic retrovirus mediated gene transfer into human hematopoietic stem cells (HSC) has been an impediment to gene therapy for hematopoietic diseases (1). We have previously shown that mouse and human HSC have low levels of the mRNA encoding PiT-2, the amphotropic retrovirus receptor. We hypothesized that the low level of PiT-2 mRNA was responsible for the low frequency of transduction of HSC by amphotropic retroviral vectors (2). In this study we compared the level of PiT-2 and PiT-1, the Gibbon Ape Leukemia Virus receptor (GaLV), in 5 human tissue culture cell lines. PiT-2 and PiT-1 mRNA levels were highest in K562 cells and lowest in HL60 cells. In hematopoietic cell lines, the level of PiT-2 or PiT-1 mRNA correlated directly with retrovirus binding and transduction with the appropriate (amphotropic or GaLV) retrovirus vector. The level of expression of PiT-2 and PiT-1 mRNA could be increased by treatment of HL60 cells with either PMA or Interleukin-1α. The increase in the level of PiT-2 and PiT-1 mRNA correlated with increased transduction with both amphotropic and GaLV retroviral vectors. We conclude that the improved transduction was a direct effect of the increased levels of receptor mRNA and unrelated to changes in the cell cycle status.

The level of mRNA encoding the amphotropic retrovirus receptor in mouse and human hematopoietic stem cells is low and correlates with the efficiency of retrovirus transduction.

The low level of amphotropic retrovirus-mediated gene transfer into human hematopoietic stem cells (HSC) has been a major impediment to gene therapy for hematopoietic diseases. In the present study, we have examined amphotropic retrovirus receptor (amphoR) and ecotropic retrovirus receptor mRNA expression in highly purified populations of mouse and human HSC. Murine HSC with low to undetectable levels of amphoR mRNA and relatively high levels of ecotropic retrovirus receptor mRNA were studied. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, ecotropic provirus sequences were detected in 10 of 13 long-term repopulated animals, while amphotropic proviral sequences were detected in only one recipient. A second distinct population of murine HSC were isolated that express 3-fold higher levels of amphoR mRNA. When these HSC were analyzed simultaneously for ecotropic and amphotropic retrovirus transduction, 11 of 11 repopulated mice contained ecotropic provirus and 6 of 11 contained amphotropic provirus sequences, a significant increase in the amphotropic retrovirus transduction (P = 0.018). These results indicate that, among the heterogeneous populations of HSC present in adult mouse bone marrow, the subpopulation with the highest level of amphoR mRNA is more efficiently transduced by amphotropic retrovirus. In a related study, we found low levels of human amphoR mRNA in purified populations of human HSC (CD34+ CD38-) and higher levels in committed progenitor cells (CD34+ CD38+). We conclude that the amphoR mRNA level in HSC correlates with amphotropic retrovirus transduction efficiency.