Stable gene transfer and expression in cord blood–derived CD34+ hematopoietic stem and progenitor cells by a hyperactive Sleeping Beauty transposon system

Blood ◽  
2009 ◽  
Vol 114 (7) ◽  
pp. 1319-1330 ◽  
Author(s):  
Xingkui Xue ◽  
Xin Huang ◽  
Sonja E. Nodland ◽  
Lajos Mátés ◽  
Linan Ma ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Cord Blood ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Here we report stable gene transfer in cord blood-derived CD34+ hematopoietic stem cells using a hyperactive nonviral Sleeping Beauty (SB) transposase (SB100X). In colony-forming assays, SB100X mediated the highest efficiency (24%) of stable Discosoma sp red fluorescent protein (DsRed) reporter gene transfer in committed hematopoietic progenitors compared with both the early-generation hyperactive SB11 transposase and the piggyBac transposon system (1.23% and 3.8%, respectively). In vitro differentiation assays further demonstrated that SB100X-transfected CD34+ cells can develop into DsRed+ CD4+CD8+ T (3.17%-21.84%; median, 7.97%), CD19+ B (3.83%-18.66%; median, 7.84%), CD56+CD3− NK (3.53%-79.98%; median, 7.88%), and CD33+ myeloid (7.59%-15.63%; median, 9.48%) cells. SB100X-transfected CD34+ cells achieved approximately 46% engraftment in NOD-scid IL2γcnull (NOG) mice. Twelve weeks after transplantation, 0.57% to 28.96% (median, 2.79%) and 0.49% to 34.50% (median, 5.59%) of total human CD45+ cells in the bone marrow and spleen expressed DsRed, including CD19+ B, CD14+ monocytoid, and CD33+ myeloid cell lineages. Integration site analysis revealed SB transposon sequences in the human chromosomes of in vitro differentiated T, B, NK, and myeloid cells, as well as in human CD45+ cells isolated from bone marrow and spleen of transplanted NOG mice. Our results support the continuing development of SB-based gene transfer into human hematopoietic stem cells as a modality for gene therapy.

Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1652-1652
Author(s):  
Patrick Ziegler ◽  
Steffen Boettcher ◽  
Hildegard Keppeler ◽  
Bettina Kirchner ◽  
Markus G. Manz
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Human Mesenchymal Stem Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Gene Transcripts

Abstract We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells. Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented. Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Bernhard Schiedlmeier ◽  
Hannes Klump ◽  
Elke Will ◽  
Gökhan Arman-Kalcek ◽  
Zhixiong Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Therapeutic Window ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Cord Blood Cd34 ◽  
The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4253-4253
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Scid Mice ◽  
Common Mechanism ◽  
Hematopoietic Stem ◽  
Variable Expression ◽  
Cd34 Cells ◽  
Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (&gt;95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (&gt;90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after &gt;6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Induced Hematopoietic Differentiation Of Common Marmoset Embryonic Stem Cells By LYL1 Can Give Rise To Hematopoietic Stem Cells Having The Enhanced Bone Marrow Reconstitution Capability

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1192-1192
Author(s):  
Hirotaka Kawano ◽  
Tomotoshi Marumoto ◽  
Takafumi Hiramoto ◽  
Michiyo Okada ◽  
Tomoko Inoue ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Lymphoblastic Leukemia ◽  
Embryonic Stem ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract Hematopoietic stem cell (HSC) transplantation is the most successful cellular therapy for the malignant hematopoietic diseases such as leukemia, and early recovery of host’s hematopoiesis after HSC transplantation has eagerly been expected to reduce the regimen related toxicity for many years. For the establishment of the safer and more efficient cell source for allogeneic or autologous HSC transplantation, HSCs differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) that show indefinite proliferation in an undifferentiated state and pluripotency, are considered to be one of the best candidates. Unfortunately, despite many recent efforts, the HSC-specific differentiation from ESCs and iPSCs remains poor [Kaufman, DS et al., 2001][Ledran MH et al., 2008]. In this study, we developed the new method to differentiate HSC from non-human primate ESC/iPSC. It has been reported that common marmoset (CM), a non-human primate, is a suitable experimental animal for the preclinical studies of HSC therapy [Hibino H et al., 1999]. We have been investigated the hematopoietic differentiation of CM ESCs into HSCs, and previously reported that the induction of CD34+ cells having a blood colony forming capacity from CM ESCs were promoted by lentiviral transduction of TAL1 cDNA [Kurita R et al., 2006]. However, those CD34+ cells did not have a bone marrow reconstituting ability in irradiated NOG (NOD/Shi-scid/IL-2Rγnull) mice, suggesting that transduction of TAL1 gene was not sufficient to induce functional HSCs which have self-renewal capability and multipotency. Thus, we tried to find other hematopoietic genes being able to promote hematopoietic differetiation more efficiently than TAL1. We selected 6 genes (LYL1, HOXB4, BMI1, GATA2, c-MYB and LMO2) as candidates for factors that induce the differentiation of ESCs into HSCs, based on the previous study of hematopoietic differentiation from human and mouse ESCs. And CM ESCs (Cj11) lentivirally transduced with the respective candidate gene were processed for embryoid body (EB) formation to induce their differentiation into HSCs for 9 days. We found that lentiviral transduction of LYL1 (lymphoblastic leukemia 1), a basic helix-loop-helix transcription factor, in EBs markedly increased the proportion of cells positive for CD34 (approximately 20% of LYL1-transduced cells). RT-PCR showed that LYL1-transduced EBs expressed various hematopoietic genes, such as TAL1, RUNX1 and c-KIT. To examine whether these CD34+ cells have the ability to differentiate into hematopoietic cells in vitro, we performed colony-forming unit (CFU) assay, and found that CD34+ cells in LYL1-transduced EBs could form multi-lineage blood colonies. Furthermore the number of blood colonies originated from CD34+CD45+ cells in LYL1-transduced EBs was almost the same as that from CD34+CD45+ cells derived from CM bone marrow. These results suggested that enforced expression of LYL1 in CM ESCs promoted the emergence of HSCs by EB formation in vitro. The LYL1 was originally identified as the factor of a chromosomal translocation, resulting in T cell acute lymphoblastic leukemia [Mellentin JD et al., 1989]. The Lyl1-deficient mice display the reduction of B cells and impaired long-term hematopoietic reconstitution capacity [Capron C et al., 2006]. And, transduction of Lyl1 in mouse bone marrow cells induced the increase of HSCs and lymphocytes in vitro and in vivo [Lukov GL et al., 2011]. Therefore we hypothesized that LYL1 may play essential roles in bone marrow reconstitution by HSCs differentiated from CM ESCs. To examine this, we transplanted CD34+ cells derived from LYL1-transduced CM ESCs into bone marrow of sublethally irradiated NOG mice, and found that about 7% of CD45+ cells derived from CM ESCs were detected in peripheral blood (PB) of recipient mice at 8 weeks after transplant (n=4). Although CM CD45+ cells disappeared at 12 weeks after transplant, CD34+ cells (about 3%) were still found in bone marrow at the same time point. Given that TAL1-transduced EBs derived from CM ESCs could not reconstitute bone marrow of irradiated mice at all, LYL1 rather than TAL1 might be a more appropriate transcription factor that can give rise to CD34+ HSCs having the enhanced capability of bone marrow reconstitution from CM ESCs. We are planning to do in vivo study to prove this hypothesis in CM. Disclosures: No relevant conflicts of interest to declare.

scholarly journals UM171 Regulates the Hematopoietic Differentiation of Human Acquired Aplastic Anemia-Derived Induced Pluripotent Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2500-2500
Author(s):  
Tellechea Maria Florencia ◽  
Flavia S. Donaires ◽  
Tiago C. Silva ◽  
Lilian F. Moreira ◽  
Yordanka Armenteros ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Patient Specific ◽  
Hematopoietic Differentiation ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Induced Pluripotent

Aplastic anemia (AA) is characterized by a hypoplastic bone marrow associated with low peripheral blood counts. In acquired cases, the immune system promotes hematopoietic stem and progenitor cell (HSPC) depletion by the action of several pro-inflammatory Th1 cytokines. The current treatment options for severe cases consist of sibling-matched allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with anti-thymocyte globulin, cyclosporine, and eltrombopag. However, most patients are not eligible for HSCT and, although about 85% of patients respond to IST with eltrombopag, a proportion of patients eventually relapse, requiring further therapies. Failure to respond adequately to immunosuppression may be attributed to the scarcity of HSPCs at the time of diagnosis. Induced pluripotent stem cells (iPSCs) are potentially an alternative source of patient-specific hematopoietic cells. Patient-specific HSPCs derived from in vitro iPSC differentiation may serve as a tool to study the disease as well as a source of hematopoietic tissue for cell therapies. The pyrimidoindole molecule UM171 induces ex vivo expansion of HSCs of human cord and peripheral blood and bone marrow, but the pathways modulated by this molecule are not well understood. Here we evaluated the hematopoietic differentiation potential of iPSCs obtained from patients with acquired AA. We further determined the effects of UM171 on this differentiation process. First, we derived iPSCs from 3 patients with acquired AA after treatment (1 female; average age, 31 years; 2 partial responders, 1 complete responder) and 3 healthy subjects (3 females; average age, 61 years) and induced differentiation in vitro through the embryoid body system in cell feeder and serum-free medium supplemented with cytokines. The hematopoietic differentiation of healthy-iPSCs yielded 19% ± 8.1% (mean ± SEM) of CD34+cells after 16 days in culture, in contrast with 11% ± 4.9% of CD34+cells obtained from the differentiation of AA-iPSCs, which corresponds to a 1.7-fold reduction in CD34+cell yield. The total number of erythroid and myeloid CFUs was lower in the AA-iPSC group as compared to healthy-iPSCs (12±4.2 vs.24±7.2; respectively; p<0.03). These findings suggest that erythroid-derived AA-iPSC have an intrinsic defect in hematopoietic differentiation. Next, we tested whether UM171 modulated hematopoietic differentiation of AA-iPSCs. We found that UM171 significantly stimulated the differentiation of both healthy and AA-iPSCs. In the healthy-iPSC group, the percentage of CD34+cells was 1.9-fold higher when treated with UM171 compared to controls treated with DMSO (37% ± 7.8% vs.19% ± 8.1%; respectively; p<0.03) and in AA-iPSCs the increase was 3.9-fold (45% ± 11% vs. 11% ± 4.9%; p<0.07). The clonogenic capacity of progenitors to produce erythroid and myeloid colonies also was augmented in both groups in comparison to DMSO (28±11 vs. 23±7.2) for healthy-iPSCs and for AA-iPSCs (23±8.5 vs. 12±4.2, p<0.06). We then investigated the molecular pathways influenced by UM171. The transcriptional profile of differentiated CD34+cells showed that UM171 up-regulated genes involved in early hematopoiesis from mesoderm (BRACHYURY and MIXL1) and primitive streak specification (APELA and APLNR), to hemangioblasts and primitive hematopoietic progenitor commitment (TDGF1, SOX17, and KLF5). We also observed the up-regulation of pro-inflammatory NF-kB activators (MAP4K1, ZAP70, and CARD11) and the anti-inflammatory gene PROCR, a marker of cultured HSCs and an NF-kB inhibitor. This balanced network has been previously suggested to be modulated by UM171 (Chagraoui et. al. Cell Stem Cell 2019). Taken together, our results showed that acquired AA-iPSCs may have intrinsic defects that impair hematopoietic differentiation in vitro. This defect may be atavic to the cell or, alternatively, the consequence of epigenetic changes in erythroid precursors provoked by the immune attack. In addition, our findings demonstrate that UM171 significantly stimulate the hematopoietic differentiation of AA-iPSCs and identified a novel molecular mechanism for UM171 as an enhancer of early hematopoietic development programs. These observations may be valuable for improving the achievement of de novo hematopoietic cells. Disclosures No relevant conflicts of interest to declare.

scholarly journals Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Blood ◽  
1996 ◽  
Vol 88 (7) ◽  
pp. 2510-2516 ◽  
Author(s):  
M Holberg-Petersen ◽  
H Rollag ◽  
S Beck ◽  
I Overli ◽  
G Tjonnfjord ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Clinical Isolate ◽  
Human Cytomegalovirus ◽  
Mononuclear Cells ◽  
Colony Growth ◽  
Structural Protein ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

Fluorescence-Based Selection of Gene-Corrected Hematopoietic Stem and Progenitor Cells From Acid Sphingomyelinase-Deficient Mice: Implications for Niemann-Pick Disease Gene Therapy and the Development of Improved Stem Cell Gene Transfer Procedures

Blood ◽  
1999 ◽  
Vol 93 (1) ◽  
pp. 80-86 ◽  
Author(s):  
Shai Erlich ◽  
Silvia R.P. Miranda ◽  
Jan W.M. Visser ◽  
Arie Dagan ◽  
Shimon Gatt ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Fetal Liver ◽  
Acid Sphingomyelinase ◽  
Hematopoietic Stem

Abstract The general utility of a novel, fluorescence-based procedure for assessing gene transfer and expression has been demonstrated using hematopoietic stem and progenitor cells. Lineage-depleted hematopoietic cells were isolated from the bone marrow or fetal livers of acid sphingomyelinase–deficient mice, and retrovirally transduced with amphotropic or ecotropic vectors encoding a normal acid sphingomyelinase (ASM) cDNA. Anti–c-Kit antibodies were then used to label stem- and progenitor-enriched cell populations, and the Bodipy fluorescence was analyzed in each group after incubation with a Bodipy-conjugated sphingomyelin. Only cells expressing the functional ASM (ie, transduced) could degrade the sphingomyelin, thereby reducing their Bodipy fluorescence as compared with nontransduced cells. The usefulness of this procedure for the in vitro assessment of gene transfer into hematopoietic stem cells was evaluated, as well as its ability to provide an enrichment of transduced stem cells in vivo. To show the value of this method for in vitro analysis, the effects of retroviral transduction using ecotropic versus amphotropic vectors, various growth factor combinations, and adult bone marrow versus fetal liver stem cells were assessed. The results of these studies confirmed the fact that ecotropic vectors were much more efficient at transducing murine stem cells than amphotropic vectors, and that among the three most commonly used growth factors (stem cell factor [SCF] and interleukins 3 and 6 [IL-3 and IL-6]), SCF had the most significant effect on the transduction of stem cells, whereas IL-6 had the most significant effect on progenitor cells. In addition, it was determined that fetal liver stem cells were only approximately twofold more “transducible” than stem cells from adult bone marrow. Transplantation of Bodipy-selected bone marrow cells into lethally irradiated mice showed that the number of spleen colony-forming units that were positive for the retroviral vector (as determined by polymerase chain reaction) was 76%, as compared with 32% in animals that were transplanted with cells that were nonselected. The methods described within this manuscript are particularly useful for evaluating hematopoietic stem cell gene transfer in vivo because the marker gene used in the procedure (ASM) encodes a naturally occurring mammalian enzyme that has no known adverse effects, and the fluorescent compound used for selection (Bodipy sphingomyelin) is removed from the cells before transplantation.

Increased Engraftment after Double Cord Blood Transplantation in NOD/SCID Mice.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1199-1199
Author(s):  
Alma J. Nauta ◽  
Alwine B. Kruisselbrink ◽  
Roelof Willemze ◽  
Willem E. Fibbe
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
Cord Blood ◽  
Cord Blood Transplantation ◽  
Human Platelets ◽  
Hematopoietic Stem ◽  
Cell Dose ◽  
Blood Transplantation ◽  
Cd34 Cells

Abstract Umbilical cord blood (UCB) is considered as an attractive alternative source of hematopoietic stem cells for allogeneic stem cell transplantations in patients who lack HLA-matched donors. However, the low cell dose adversely affects the speed of hematopoietic recovery and therefore limits the application of UCB transplantation in adults. Although ex-vivo expansion of cord blood cells has been explored as a strategy to increase the cell dose, compromised engraftment potential of expanded cells has been demonstrated. Another approach to overcome cell dose limitations is transplantation of multiple, unrelated UCB units. To investigate the effect of multiple cord transplantation on engraftment, NOD/SCID mice were transplanted with human hematopoietic progenitor cells (CD34+) derived from two UCB units with HLA disparity. During the first six weeks after transplantation the number of human platelets in peripheral blood was quantified by flow cytometry. Six weeks after transplantation, the mice were sacrificed and the percentage and donor origin of human CD45+ cells in blood, and in bone marrow was determined by flow cytometry. Transplantation of CD34+ cells derived from two UCB donors resulted in significantly higher number of human platelets in peripheral blood than transplantation of CD34+ cells from either donor alone, ranging from 3.92x106/ml to 10.29x106/ml (mean 6.4x106 ± 2.55x106/ml) and 0.11x106/ml to 3.12.106/ml (mean 1.42x106 ± 1.17x106/ml), respectively. Furthermore, the overall human cell engraftment level in bone marrow after double cord blood transplantation ranged from 7.01% to 64.34% (mean 29.6 ± 21.5%) a nearly 7-fold increase compared to single cord blood transplantation ranging from 0.27% to 13.5% (mean 4.6 ± 3.8%) Although consistently higher engraftment levels were reached after double cord blood transplantation, two different patterns were observed: in 2 out of 4 experiments cells from one donor predominated the engraftment (ratio 3:1), while in two other experiments the two units contributed equally to BM engraftment. The mechanism underlying these effects are &lt;S&gt;is&lt;/S&gt; not yet clear. It is not very likely that the single donor predominance results from an unequal amount of hematopoietic stem cells in the cord blood units because each cord blood showed comparable levels of engraftment as a single unit. Alternatively, the unequal engraftment may result from an immunological competition or a graft versus graft stimulatory effect between the cords during the engraftment process and further studies are required to determine if the contribution of both units is dependent on the degree of HLA matching between the two cords. Taken together, these results demonstrate that double cord blood transplantation may represent a means of achieving increased engraftment, making multiple cord blood transplantation a promising strategy to improve the outcome of UCB transplantation. Studies are underway to unravel the mechanisms underlying the enhanced engraftment.

Lentiviral FANCA Vectors Pseudotyped with a Modified Prototype Foamy Viral Envelope Corrects Murine Fanca−/− Hematopoietic Stem Cells with Reduced Toxicity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1677-1677
Author(s):  
Zejin Sun ◽  
Yanzhu Yang ◽  
Yan Li ◽  
Daisy Zeng ◽  
Jingling Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Gene Transfer ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Ex Vivo Culture

Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by congenital abnormalities, bone marrow failure, genomic instability, and a predisposition to malignancies. As the majority of FA patients ultimately acquires severe bone marrow failure, transplantation of stem cells from a normal donor is the only curative treatment to replace the malfunctioning hematopoietic system. Stem cell gene transfer technology aimed at re-introducing the missing gene is a potentially promising therapy, however, prolonged ex vivo culture of cells, that was utilized in clinical trials with gammaretroviruses, results in a high incidence of apoptosis and at least in mice predisposes the surviving reinfused cells to hematological malignancy. Consequently, gene delivery systems such as lentiviruses that allow a reduction in ex vivo culture time are highly desirable. Here, we constructed a lentiviral vector expressing the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSVG or a modified prototype foamyvirus (FV) envelope to correct Fanca−/− stem and progenitor cells in vitro and in vivo. In order to minimize genotoxic stress due to extended in vitro manipulations, an overnight transduction protocol was utilized where in the absence of prestimulation, murine Fanca−/− bone marrow cKit+ cells were co-cultured for 16h with FANCA lentivirus on the recombinant fibronectin fragment CH296. Transduction efficiency and transfer of lentivirally expressed FANCA was confirmed functionally in vitro by improved survival of consistently approximately 60% of clonogenic progenitors in serial concentrations of mitomycin C (MMC), irregardless of the envelope that was utilized to package the vector. Transduction of fibroblasts was also associated with complete correction of MMC-induced G2/M arrest and biochemically with the restoration of FancD2 mono-ubiquitination. Finally, to functionally determine whether gene delivery by the recombinant lentivirus during such a short transduction period is sufficient to correct Fanca−/− stem cell repopulation to wild-type levels, competitive repopulation experiments were conducted as previously described. Follow-up of up to 8 months demonstrated that the functional correction were also achieved in the hematopoietic stem cell compartment as evidenced by observations that the repopulating ability of Fanca−/− stem cells transduced with the recombinant lentivirus encoding hFANCA was equivalent to that of wild-type stem cells. Importantly, despite the fact that the gene transfer efficiency into cells surviving the transduction protocol were similar for both pseudotypes, VSVG was associated with a 4-fold higher toxicity to the c-kit+ cells than the FV envelope. Thus, when target cell numbers are limited as stem cells are in FA patients, the foamyviral envelope may facilitate overall greater survival of corrected stem cells. Collectively, these data indicate that the lentiviral construct can efficiently correct FA HSCs and progenitor cells in a short transduction protocol overnight without prestimulation and that the modified foamy envelope may have less cytotoxicity than the commonly used VSVG envelope.

The Pan-Bcl-2 Family Inhibitor 97C1 Targets Blast Crisis Chronic Myeloid Leukemia Stem Cells but Spares Normal Cord Blood Progenitor Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 516-516 ◽  
Author(s):  
Daniel Goff ◽  
Alice Shih ◽  
Angela Court Recart ◽  
Larisa Balaian ◽  
Ryan Chuang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Blast Crisis ◽  
Chemotherapy Resistance ◽  
Chronic Phase ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 516 Introduction: Several studies have demonstrated the role of leukemia stem cells (LSC) in the development and maintenance of human chronic myeloid leukemia (CML). These cells, which first develop in chronic phase CML (CP CML) with acquisition of the BCR-ABL fusion protein, are often quiescent and can be highly resistant to apoptosis induced by drugs and radiotherapy that target rapidly dividing cells. Data has also shown that CML LSC become increasingly resistant to BCR-ABL inhibition with progression to blast crisis CML (BC CML). Bcl-2 family proteins are key regulators of apoptosis and have been shown by numerous studies to regulate cancer resistance to chemotherapy. This family of proteins has also been implicated in the development of BC CML, however most studies have focused on CML cell lines and their expression of Bcl-2 family proteins in vitro. Thus, there is relatively little data on expression of Bcl-2 family proteins in primary CML LSC and on the role of these proteins in regulating chemotherapy resistance in CML LSC in vivo. As Bcl-2 family proteins are known regulators of chemotherapy resistance we hypothesized that human BC CML LSC may overexpress these proteins compared to normal hematopoietic stem cells. We analyzed Bcl-2 family mRNA and protein expression in CP CML and BC CML LSC and compared this expression to normal cord blood stem and progenitor cells. We also analyzed whether these cells were sensitive to chemotherapy treatment in vitro. Finally, we tested whether a high potency pan-Bcl-2 inhibitor, 97C1, could effectively kill CML LSC in vitro and in vivo. Methods: Bcl-2 and Mcl-1 protein expression was measured in primary CP CML, BC CML, and normal cord blood cells using intracellular FACS. We also measured Bcl-2, Mcl-1, Bcl-X, and Bfl-1 mRNA expression in FACS sorted CD34+CD38+lin− cells (LSC) from these samples. For all drug studies we used either serially transplanted CD34+ cells derived from primary BC CML patient samples or primary CD34+ normal cord blood cells. In vitro drug responses were tested by culturing CD34+ cells either alone or in co-culture with a mouse bone marrow stromal cell line (SL/M2). Effects on colony formation and replating were also tested by culturing sorted CD34+CD38+lin− cells in methylcellulose in the presence and absence of drug. For in vivo testing of 97C1 we transplanted neonatal RAG2-/-yc-/- mice with CD34+ cells from 3 different BC CML and cord blood samples. Transplanted mice were screened for peripheral blood engraftment at 6–8 weeks post-transplant and engrafted mice were then treated for 2 weeks with 97C1 by IP injection. Following the treatment period the mice were sacrificed and hemotapoietic organs were analyzed for human engraftment by FACS. Results: BC CML progenitors expressed higher levels of Bcl-2 and Mcl-1 protein compared to normal cord blood and chronic phase CML cells. mRNA expression of Mcl-1, Bcl-X, and Bfl-1 was also increased in BC CML progenitors compared to CP CML progenitors. While BC CML LSC cultured in vitro were resistant to etoposide and dasatinib-induced cell death, 97C1 treatment led to a dose-dependent increase in cell death along with a dose-dependent decrease in the frequency of CD34+CD38+lin− cells compared to vehicle treated controls. While cord blood progenitor cells were also sensitive to 97C1 treatment they had an IC50 around 10 times higher than that for the BC CML cells (100nM versus 10nM). Importantly, 97C1 treatment did not inhibit cord blood colony formation or colony replating in vitro. Mice transplanted with BC CML LSC developed CML in 6–8 weeks post-transplant with diffuse myeloid sarcomas and engraftment of human CD34+CD38+lin− cells in the peripheral blood, liver, spleen, and bone marrow. In vivo treatment with 97C1 led to a significant reduction in both total human engraftment and engraftment of CD34+CD38+lin− cells in all hematopoietic organs analyzed. Conclusion: Our results demonstrate that BC CML LSC are resistant to conventional chemotherapy but are sensitive to 97C1 in vitro and in vivo. Broad-spectrum inhibition of Bcl-2 family proteins may help to eliminate CML LSC while sparing normal hematopoietic stem and progenitor cells. Disclosures: Jamieson: CoronadoBiosciences: Research Funding; CIRM: Research Funding.

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