Safe and Efficient Expansion of Human HSC with Sendai Virus Vector Expressing HoxB4 in Fetal Sheep.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 693-693
Author(s):  
Shigeo Masuda ◽  
Tomoyuki Abe ◽  
Makoto Inoue ◽  
Mamoru Hasegawa ◽  
Satoshi Hayashi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Insertional Mutagenesis ◽  
Sendai Virus ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Control Group ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Colony Pcr ◽  
Cd34 Cells

Abstract Abstract 693 Background: Homeobox B4 (HoxB4) has been shown to be a potent stem cell self-renewal gene, especially in hematopoietic stem cells (HSC). Accumulating evidence from murine studies indicates that the overexpression of HoxB4 enhances in vivo and ex vivo expansion of HSC. Although no leukemia has been observed after transplantation of HoxB4-transduced cells in murine models, the study using large animals such as dogs and non-human primates with retroviral vectors expressing HoxB4 showed the frequent development of leukemia. Regarding retroviral vectors expressing HoxB4, there is another concern, that is, insertional leukemogenesis, which has been elucidated in the hematopoietic stem cell gene therapy for X-SCID. To avoid the insertional mutagenesis, other vectors may be considered, including Epstein-Barr nuclear antigen (EBNA)-1 based episomal vectors or the transposon; however, problems are left, i.e. low transduction efficiency with EBNA vectors and unclear safety with transposon vectors. To avoid both the persistent HoxB4 expression and insertional mutagenesis leading to leukemogenesis, we have developed a new type of Sendai virus (SeV) vector; it lacks the polymerase gene, namely P-defective SeV (SeV/δP) vector. SeV is an enveloped virus with a non-segmented, negative-stranded RNA genome. SeV-based vectors are non-integrating, cytoplasmic vectors. They replicate exclusively in the cytoplasm of transduced cells, and do not go through a DNA phase; therefore, there is no concern about the unwanted integration of foreign sequences into chromosomal DNA of the host. We have previously shown that the transduction efficiency of human CD34+ cells with the SeV vector was very high; around 70% (100 multiplicity of infections). On the other hand, SeV/δP vectors are incapable of self-replication, thus enabling transient gene expression without spoiling their ability to efficiently transduce CD34+ cells. Here, using the SeV/δP vector expressing HoxB4 (SeV/δP/HoxB4 vector), we examined the effectiveness and safety of human HSC expansion after in utero transplantation to fetal sheep. Methods: After enrichment of CD34+ cells from cryopreserved human umbilical cord blood, these cells were repeatedly exposed to SeV/δP/HoxB4 vector every 24 h for 4 days. The transduced cells (3.2–11.7 × 105) were transplanted into the abdominal cavity of fetal sheep at 45–50 gestational days (full term, 147 days) that have premature immune system (HoxB4 group, n = 4; control group, n = 4). The engraftment of hematopoietic cells derived from human HSC in the lambs after birth was quantitatively evaluated by colony PCR of the bone marrow. The development of leukemia was assessed by regular sampling of peripheral blood and bone marrow. Results: The human–sheep chimeric ratio in the bone marrow of HoxB4 group was calculated 4.8-times higher than that of control group after birth, as assessed by colony PCR. The SeV genome was no longer detectable in the bone marrow and peripheral blood of lambs as assessed by RNA-PCR, confirming the SeV vectors were cleared. No leukemia developed in any of the sheep in either group at present (at 12 months after transplantation). Conclusion: The SeV/δP vector would be suitable for transient expression of HoxB4 in human CD34+ cells, enabling 4.8-times expansion of human HSC as assessed by their repopulating ability in sheep. The expansion of human HSC with the SeV vector was comparable to that with HoxB4-retroviral vectors. In addition, the SeV/δP vector is free of concern about transgene-related and insertional leukemogenesis and should be safer than retroviral vectors. Disclosures: No relevant conflicts of interest to declare.

Human Embryonic Stem Cell (hESC)-Derived Hematopoietic Elements Are Capable of Engrafting Primary as well as Secondary Fetal Sheep Recipients.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2685-2685
Author(s):  
A. Daisy Narayan ◽  
Jessica L. Chase ◽  
Adel Ersek ◽  
James A. Thomson ◽  
Rachel L. Lewis ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Peripheral Blood ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Blood Samples ◽  
Human Dna ◽  
Cd34 Cells ◽  
Hematopoietic Activity

Abstract We used transplantation into 10 and 20 pre-immune fetal sheep recipients (55–65 days-old, term: 145 days) to evaluate the in vivo potential of hematopoietic elements derived from hESC. The in utero human/sheep xenograft model has proven valuable in assessing the in vivo hematopoietic activity of stem cells from a variety of fetal and post-natal human sources. Five transplant groups were established. Non-differentiated hESC were injected in one group. In the second and third group, embroid bodies differentiated for 8 days were injected whole or CD34+ cells were selected for injection. In the fourth and fifth group, hESC were differentiated on S17 mouse stroma layer and injected whole or CD34+ cells were selected for injection. The animals were allowed to complete gestation and be born. Bone marrow and peripheral blood samples were taken periodically up to over 12 months after injection, and PCR and flowcytometry was used to determine the presence of human DNA/blood cells in these samples. A total of 30 animals were analyzed. One primary recipient that was positive for human hematopoietic activity was sacrificed and whole bone marrow cells were transplanted into a secondary recipient. We analyzed the secondary recipient at 9 months post-injection by PCR and found it to be positive for human DNA in its peripheral blood and bone marrow. This animal was further challenged with human GM-CSF and human hematopoietic activity was noted by flowcytometry analyses of bone marrow and peripheral blood samples. Further, CD34+ cells enriched from its bone marrow were cultured in methylcellulose and human colonies were identified by PCR. We therefore conclude that hESC are capable of generating hematopoietic cells that engraft in 1° sheep recipients. These cells also fulfill the criteria for long-term engrafting hematopoietic stem cells as demonstrated by engraftment and differentiation in the 20 recipient.

Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 203-203
Author(s):  
Theo Gomes ◽  
Stephanie Sellers ◽  
Robert E. Donahue ◽  
Rima Adler ◽  
Andre La Rochelle ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Insertional Mutagenesis ◽  
Ex Vivo ◽  
Safety Issue ◽  
Retroviral Vectors ◽  
Ex Vivo Expansion ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Abstract There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

scholarly journals Stromal support enhances cell-free retroviral vector transduction of human bone marrow long-term culture-initiating cells

Blood ◽  
1992 ◽  
Vol 79 (6) ◽  
pp. 1393-1399 ◽  
Author(s):  
KA Moore ◽  
AB Deisseroth ◽  
CL Reading ◽  
DE Williams ◽  
JW Belmont
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Transduction Efficiency ◽  
Hematopoietic Stem ◽  
Long Term Culture ◽  
Stromal Support

Abstract Gene transfer into hematopoietic stem cells by cell-free virions is a goal for gene therapy of hematolymphoid disorders. Because the hematopoietic microenvironment provided by the stroma is required for stem cell maintenance both in vivo and in vitro, we reasoned that cell- free transduction of bone marrow cells (BMC) may be aided by stromal support. We used two high-titer replication-defective retroviral vectors to differentially mark progenitor cells. The transducing vector was shown to be a specific DNA fragment by polymerase chain reaction of colony-forming cells derived from progenitors maintained in long-term culture (LTC). BMC were infected separately by cell-free virions with or without pre-established, irradiated, allogeneic stromal layers, and in the presence or absence of exogenous growth factors (GF). The GF assessed were interleukin-3 (IL-3) and IL-6 in combination, leukemia inhibitory factor (LIF), mast cell growth factor (MGF), and LIF and MGF in combination. In addition, we developed a competitive LTC system to directly assess the effect of infection conditions on the transduction of clonogenic progenitors as reflected by the presence of a predominate provirus after maintenance in the same microenvironment. The results show gene transfer into human LTC-initiating cells by cell-free retroviral vector and a beneficial effect of stromal support allowing a transduction efficiency of 64.6% in contrast to 15.8% without a supporting stromal layer. A high transduction rate was achieved independent of stimulation with exogenous GF. We propose that autologous marrow stromal support during the transduction period may have application in clinical gene therapy protocols.

Busulfan Conditioning In Sheep In Utero Transplantation Confers Engraftment Potential on Human Hematopoietic Stem Cells as Efficiently as HoxB4 Transduction.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3716-3716
Author(s):  
Shigeo Masuda ◽  
Tomoyuki Abe ◽  
Satoshi Hayashi ◽  
Yujiro Tanaka ◽  
Hiroshi Ban ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Fetal Liver ◽  
Optimal Dose ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Interval Group ◽  
In Utero Transplantation ◽  
Cd34 Cells ◽  
Human Hscs

Abstract Abstract 3716 Background: In utero transplantation (IUT) of hematopoietic stem cells (HSCs) has been pursued as a treatment for congenital hematologic and genetic disorders. Although the engraftment of HSCs following IUT has been achieved, the levels of engraftment have been generally low. In order to achieve clinically relevant levels of HSC engraftment, there are many potential ways including the HSC expansion via HoxB4 transduction, and the niche expansion either by co-transplantation with mesenchymal stem cells (MSCs) or by myeloablation using busulfan (BU). BU is an alkylating agent and has application as an alternative to total body irradiation to create open niches for HSC transplantation. However, BU conditioning in IUT has not been reported. Here, we examined the safety and efficacy of BU conditioning in sheep IUT. Moreover, we compared the efficacy of BU conditioning with that of HoxB4 transduction or co-transplantation with MSCs in the context of sheep IUT. Methods: To determine the optimal dose of BU in sheep IUT, fetal sheep at 40–47 gestation days (full term, 147 days) were treated with BU at 0, 3, or 7.5 mg/kg (calculated by maternal body weight) via maternal vein. (BU can pass through placentas.) In the fetuses receiving 3 mg/kg BU, the numbers of colony-forming units (CFUs) in the fetal liver at 2 weeks after BU administration significantly decreased compared with those of the control group (p < 0.01), and there was no trouble in maintenance of pregnancy, or no developmental abnormalities in the fetuses. In the fetuses receiving 7.5 mg/kg BU, four out of the five fetuses died, with severe suppression in the maternal hematopoiesis. We therefore determined that the optimal dose of BU for sheep IUT was 3 mg/kg. Next, to determine the appropriate timing of BU treatment in IUT, BU treatment at 3 mg/kg via maternal vein was conducted at 2 or 6 days prior to IUT, followed by transplantation of human CD34+ cells from umbilical cord blood, into the fetal liver (6-day-interval group [BU-6], n = 4; 2-day-interval group [BU-2], n = 4; control group, n = 6). Furthermore, we performed IUT of CD34+ cells together with MSCs in addition to 6-day-interval BU treatment (BU-6 + MSC co-transplantation group, n = 4). IUT of CD34+ cells transduced with HoxB4 by Sendai virus vector was also conducted (HoxB4 group, n = 4), as reported in the previous annual meeting by our group. After birth, the engraftment of human hematopoietic cells in the lambs was quantitatively evaluated by colony PCR of the bone marrow. Results: Human CFUs were detected in the lamb bone marrow at 1 week after birth as shown in Table. In summary, the BU-6 group showed increased levels of engraftment compared to the control group (p < 0.01), although the BU-2 group did not show increased levels of the engraftment. Thus, the engraftment was improved by transplanting cells at 6 days, not at 2 days, after BU treatment. The failure to improve engraftment in the BU-2 group might be attributable to the effects of remaining BU on transplanted cells, resulting from not fully developed enzymes of drug metabolism in fetuses as is especially known in sheep. Notably, the BU conditioning resulted in improved engraftment to the similar levels as HoxB4 transduction (cf. HoxB4 group). There was no additional effect of MSCs on engraftment (cf. BU-6 + MSC co-transplantation group), presumably because hematopoietic niches had already restored at 6 days after BU administration. Discussion: BU treatment at 3 mg/kg via maternal vein 6 days prior to transplant can enhance the engraftment of human HSCs after sheep IUT, the efficacy of which would be comparable to that of HoxB4 transduction of human HSCs. In the context of IUT, we suggest that non cell-autonomous enhancement of human HSC engraftment by treatment with BU would be as effective as cell-autonomous enhancement such as HoxB4 transduction. The synergistic effects by both methods remain to be elucidated. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Fully Closed, Large-Scale, and Clinical Grade Cell Sorting of Hematopoietic Stem Cell (HSC)-Enriched CD90+ Cells for Transplantation and Gene Therapy

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3246-3246
Author(s):  
Stefan Radtke ◽  
Margaret Cui ◽  
Anai M Perez ◽  
Yan-Yi Chan ◽  
Stefanie Schmuck ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Transduction Efficiency ◽  
Target Cells ◽  
Gene Modification ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Cell Fractions

Introduction: Hematopoietic stem cell (HSC) gene therapy/editing is a viable treatment option for various hematological diseases and disorders including hemoglobinopathies and HIV/AIDS. Most if not all currently available approaches target CD34-enriched cell fractions, a heterogeneous mix of mostly committed progenitor cells and only very few true HSCs with long-term multilineage engraftment potential. As a consequence, gene therapy/editing approaches are currently limited in their HSC targeting efficiency, very expensive consuming huge quantities of modifying reagents, and can lead to unwanted side-effects in non-target cells. We recently described a novel HSC-enriched CD34 subset (CD90+CD45RA-) that is exclusively responsible for rapid recovery onset, robust long-term multilineage engraftment, as well as entire reconstitution of the bone marrow stem cell compartment in the nonhuman primate (NHP) stem cell transplantation and gene therapy model (Radtke et al. 2017, STM). Most importantly, we demonstrate that this CD34 subset reduces the number of target cells, modifying reagents and costs by more than 10-fold without compromising the long-term efficiency of gene-modification in the NHP (Humbert and Radtke et al. 2019, STM). Here, we aimed to develop a clinical protocol to reliably purify and efficiently gene-modify human HSC-enriched CD90+ cell fractions. Methods: Large-scale enrichment of CD34+ cells from GCSF-mobilized leukapheresis products was initially performed on the Miltenyi CliniMACS Prodigy according to previously established protocols (Adair et al. 2017, Nat. Comm.). Yield, purity, quality, and feasibility of CD90 sorting was then comprehensively tested on two different commercially available cell sorting systems comparing the jet-in-air sorter FX500 from Sony and the cartridge-based closed-system sorter MACSQuant Tyto from Miltenyi Biotech with our clinically approved gold-standard CD34-mediated gene therapy approach. Sorted CD90+ and bulk CD34+ cells were transduced with a clinical-grade lentivirus encoding for GFP and the multilineage differentiation as well as engraftment potential tested using in vitro assays and the NSG mouse xenograft model, respectively. Results: Flow-cytometric sort-purification of CD90+ cells was similarly efficient in purity and yield using either the FX500 or Tyto (Figure A,B). Both approaches reliably reduced the overall target cell count by 10 to 15-fold without impacting the cells viability and in vitro colony-forming cell potential. Unexpectedly, the transduction efficiency of sort-purified CD90+ cells was significantly improved compared to bulk-transduced CD34+ cells and especially the CD34+CD90+ subset (Figure C). All cell fractions demonstrated robust mouse xenograft potential (Figure D). Most importantly, significantly higher levels of GFP+ expression in the peripheral blood, bone marrow, spleen and thymus were observed after transplantation of gene-modified CD90+ compared to bulk CD34+ cells in NSG mice (Figure E). Conclusion: Here, we show that sort-purification of our HSC-enriched CD34+CD90+ cell subset is technically feasible and highly reproducible in two different systems. Purification of human CD90+ cell fractions significantly increased the gene-modification efficiency of primitive human HSCs with multilineage mouse engraftment potential. These findings should have important implications for currently available as well as future HSC gene therapy and gene editing protocols. Isolation of an HSC-enriched phenotype will allow more targeted gene modification and thus likely reduce unwanted off target effects. Our approach further reduced the overall costs for gene modifying reagents, can be combined with a closed transduction system, increase the portability and ultimately make HSC gene therapy GMP-facility independent and affordable. Finally, this stem cell selection strategy may also allow efficient and effective depletion of donor T cells in the setting of allogeneic stem cell or organ transplantation. Figure: A) Purity and B) yield of CD90+ cells after sort-purification. C) Transduction efficiency of bulk-transduced CD34+CD90+ cells and sort-purified CD90+ cells. Frequency of D) human chimerism and E) GFP+ human CD45+ cells in the peripheral blood (PB), bone marrow, spleen and thymus after transplantation of gene-modified bulk CD34+ or sort-purified CD90+ cells. Figure Disclosures Kiem: CSL Behring: Consultancy; Rocket Pharma: Consultancy, Equity Ownership; Homology Medicines: Consultancy, Equity Ownership; Magenta Therapeutics: Consultancy.

scholarly journals Cohesin Genes Are Negative Regulators of HSC Renewal

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 605-605
Author(s):  
Roman Galeev ◽  
Aurelie Baudet ◽  
Anders Kvist ◽  
Therese Törngren ◽  
Shamit Soneji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Myeloid Malignancies ◽  
Genome Wide ◽  
Cd34 Cells ◽  
Recurrent Mutations ◽  
Major Player

Abstract The molecular principles regulating hematopoietic stem cells (HSCs) remain incompletely defined. To gain deeper insights into the mechanisms underlying renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), we have developed global RNAi screens targeted to human cord blood derived CD34+ cells. In previous work such screens have allowed us to identify novel druggable targets to facilitate ex vivo expansion of HSPCs. Recently, we employed a near genome-wide screen (targeting 15 000 genes) to identify genes with an impact on renewal/differentiation of HSPCs, in a completely unbiased manner. Among the most prominent hits from this screen were many transcription factors and epigenetic modifiers and we found a strong enrichment of genes known to be recurrently mutated in hematopoietic neoplasms. A striking finding, was the identification of several members of the cohesin complex (STAG2, RAD21, STAG1 and SMC3) among our top hits (top 0.5%). Cohesin is a multimeric protein complex that mediates adhesion of sister chromatids as well as long-range interactions of chromosomal elements to regulate transcription. Recent large-scale sequencing studies have identified recurrent mutations in the cohesin genes in myeloid malignancies. Upon individual validation and targeting of the cohesin genes by lentiviral shRNA in human CD34+ cells, we found that their knockdown by independent shRNAs led to an immediate and profound expansion of primitive hematopoietic CD34+CD90+ cells in vitro. A similar expansion phenotype was observed in vivo following transplantation to primary and secondary immundeficient mice. Transplantation of CD34+CD38lowCD90+CD45RA- cells transduced with shRNA targeting STAG2 (the cohesin component with the strongest in vitro phenotype) into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice resulted in a significant increase in human reconstitution in the bone marrow 16 weeks post-transplantation compared to controls (31.3±4.4% vs 11.6±2.8% p=0.001). The engrafted mice showed a marked skewing towards the myeloid lineage as analyzed by CD33/CD15 expression in bone marrow (27.0±5.0% vs 13.0±2.6% p=0.013), as well as an increase in the more primitive CD34+CD38- population (2.8±0.6% vs 1.3±0.4% p=0.036). In secondary transplanted mice, 3/6 recipients in the STAG2 group maintained detectable levels of human chimerism while no engraftment was detected in the control group, indicating an increased expansion of HSPCs in vivo upon knockdown of STAG2. Global transcriptome analysis of cohesin deficient CD34+ cells 36 hours post shRNA transduction showed a distinct up-regulation of HSC specific genes coupled with down-regulation of genes specific for more downstream progenitors, demonstrating an immediate shift towards a more stem-like gene expression signature upon cohesin deficiency. This observation was consistent for all cohesin genes tested (STAG2, RAD21, STAG1 and SMC3). Our findings implicate cohesin as a novel major player in regulation of human HSPCs and, together with the recent discovery of recurrent mutations in myeloid malignancies, point toward a direct role of perturbed cohesin function as a true driver event in myeloid leukemogenesis. Our findings illustrate how global RNAi screens targeted to primary human HSPCs can identify novel modifiers of cell fate and may complement genome-wide sequencing approaches to guide the identification of functionally relevant disease-related genes in hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow

Blood ◽  
2002 ◽  
Vol 100 (10) ◽  
pp. 3553-3560 ◽  
Author(s):  
Wolf-K. Hofmann ◽  
Sven de Vos ◽  
Martina Komor ◽  
Dieter Hoelzer ◽  
William Wachsman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
High Risk ◽  
Bone Marrow Cells ◽  
Low Risk ◽  
Control Group ◽  
Expression Data ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Marrow Cells

Gene patterns of expression in purified CD34+ bone marrow cells from 7 patients with low-risk myelodysplastic syndrome (MDS) and 4 patients with high-risk MDS were compared with expression data from CD34+ bone marrow cells from 4 healthy control subjects. CD34+ cells were isolated by magnetic cell separation, and high-density oligonucleotide microarray analysis was performed. For confirmation, the expression of selected genes was analyzed by real-time polymerase chain reaction. Class membership prediction analysis selected 11 genes. Using the expression profile of these genes, we were able to discriminate patients with low-risk from patients with high-risk MDS and both patient groups from the control group by hierarchical clustering (Spearman confidence). The power of these 11 genes was verified by applying the algorithm to an unknown test set containing expression data from 8 additional patients with MDS (3 at low risk, 5 at high risk). Patients at low risk could be distinguished from those at high risk by clustering analysis. In low-risk MDS, we found that the retinoic-acid–induced gene (RAI3), the radiation-inducible, immediate-early response gene (IEX1), and the stress-induced phosphoprotein 1 (STIP1) were down-regulated. These data suggest that CD34+cells from patients with low-risk MDS lack defensive proteins, resulting in their susceptibility to cell damage. In summary, we propose that gene expression profiling may have clinical relevance for risk evaluation in MDS at the time of initial diagnosis. Furthermore, this study provides evidence that in MDS, hematopoietic stem cells accumulate defects that prevent normal hematopoiesis.

scholarly journals CD34+ human marrow cells that express low levels of Kit protein are enriched for long-term marrow-engrafting cells

Blood ◽  
1996 ◽  
Vol 87 (10) ◽  
pp. 4136-4142 ◽  
Author(s):  
I Kawashima ◽  
ED Zanjani ◽  
G Almaida-Porada ◽  
AW Flake ◽  
H Zeng ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Marrow Cell ◽  
In Utero ◽  
Fetal Sheep ◽  
Hematopoietic Stem ◽  
Show Evidence ◽  
Marrow Cells

Using in utero transplantation into fetal sheep, we examined the capability of human bone marrow CD34+ cells fractionated based on Kit protein expression to provide long-term in vivo engraftment. Twelve hundred to 5,000 CD34+ Kit-, CD34+ Kit(low), and CD34+ Kit(high) cells were injected into a total of 14 preimmune fetal sheep recipients using the amniotic bubble technique. Six fetuses were killed in utero 1.5 months after bone marrow cell transplantation. Two fetuses receiving CD34+ Kit(low) cells showed signs of engraftment according to analysis of CD45+ cells in their bone marrow cells and karyotype studies of the colonies grown in methylcellulose culture. In contrast, two fetuses receiving CD34+ Kit(high) cells and two fetuses receiving CD34+ Kit- cells failed to show evidence of significant engraftment. Two fetuses were absorbed. A total of six fetuses receiving different cell populations were allowed to proceed to term, and the newborn sheep were serially examined for the presence of chimerism. Again, only the two sheep receiving CD34+ Kit(low) cells exhibited signs of engraftment upon serial examination. Earlier in studies of murine hematopoiesis, we have shown stage-specific changes in Kit expression by the progenitors. The studies of human cells reported here are in agreement with observations in mice, and indicate that human hematopoietic stem cells are enriched in the Kit(low) population.

scholarly journals Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4363-4363
Author(s):  
Alexandre Janel ◽  
Nathalie Boiret-Dupré ◽  
Juliette Berger ◽  
Céline Bourgne ◽  
Richard Lemal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Confocal Microscopy ◽  
Hematopoietic Stem Cell ◽  
Biological Samples ◽  
Mesenchymal Cells ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

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