A Modified Foamy Viral Envelope Enhances Gene Transfer Efficiency and Reduces Toxicity of Lentiviral FANCA Vectors in Fanca-/- HSCs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 696-696
Author(s):  
Zejin Sun ◽  
Yan Li ◽  
Jingling Li ◽  
Shanbao Cai ◽  
Yi Zeng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Transduction Efficiency ◽  
Gene Transfer Efficiency

Abstract Abstract 696 Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow failure, genomic instability, and a predisposition to malignancies. Stem cell gene transfer technology is a potentially promising therapy, however, we have previously shown that prolonged ex vivo culture of cells using gamma retroviruses, results in a high incidence of apoptosis and predisposes the surviving reinfused cells to hematological malignancy in a murine model of FA. Here, we developed a lentiviral vector encoding the human FANCA cDNA and tested the ability of this construct pseudotyped with either VSV-G or a modified foamy virus (FV) envelope to correct murine Fanca-/- stem and progenitor cells. An overnight transduction protocol was utilized to minimize genotoxic stress due to extended ex vivo manipulations. Transduction and expression of hFANCA was confirmed by three classical functional and biochemical measures in vitro: improved survival of clonogenic progenitors in the presence of mitomycin C (MMC), correction of MMC-induced G2/M arrest, and by the restoration of Fancd2 mono-ubiquitination. Furthermore, in vivo competitive repopulation experiments demonstrated that the repopulating ability of Fanca-/- stem cells transduced with the lentivirus encoding hFANCA was equivalent to that of wild-type stem cells, and the genetically-corrected reconstituting Fanca-/- cells were resistant to MMC and TNF-αa. Importantly, while a significant toxicity was observed using the VSV-G envelope, the toxicity of the FV envelope to murine c-kit+ cells was limited. In parallel experiments, human umbilical cord blood CD34+ cord blood cells were transduced with either a VSV-G- or FV envelope-pseudotyped lentivirus encoding the EGFP reporter gene. Transplantation of 4×105 cells into NOD/SCID/gamma-chainnull yielded a peripheral blood human chimerism comparable to the untransduced control cells (∼30%) regardless of the envelope. However, a much higher gene transfer efficiency of CD34+ cells was observed prior to transplantation when the FV envelope was employed (∼60%), as compared to the VSV-G envelope (∼15%). Furthermore, a similar 4 fold increase in transduction efficiency was observed in peripheral blood and in progenitors isolated from the bone marrow of both primary and secondary long term reconstituted mice. Collectively, these data indicate that the lentiviral construct pseudotyped with FV envelope can efficiently correct murine FA HSC/progenitor cells in a short transduction protocol and that the modified foamy envelope offers significantly greater transduction efficiency at comparable titers in long term reconstituting human cells in a xenograft model. This envelope may offer significant advantages compared to VSV-G in moving forward to phase 1 clinical trials. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease.

1993 ◽  
Vol 178 (2) ◽  
pp. 529-536 ◽  
Author(s):  
T Moritz ◽  
D C Keller ◽  
D A Williams
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Severe Combined Immunodeficiency ◽  
Genetic Material ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Human Cord Blood

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.

scholarly journals Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells from Human Umbilical Cord Blood

2007 ◽  
Vol 16 (6) ◽  
pp. 579-585 ◽  
Author(s):  
Guo-Ping Huang ◽  
Zhi-Jun Pan ◽  
Bing-Bing Jia ◽  
Qiang Zheng ◽  
Chun-Gang Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45–55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

InVitro Culture of Human Cord Blood Hematopoietic Stem/Progenitor Cells Interfered with Their Distribution to Other Bone Marrow Compartments after Intra-Bone Marrow Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4868-4868
Author(s):  
Kohshi Ohishi ◽  
Kentaro Yamamura ◽  
Masahiro Masuya ◽  
Naoyuki Katayama
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Marrow Transplantation ◽  
Cultured Cells ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Left Tibia

Abstract Intra-bone marrow transplantation (IBMT) is a novel strategy for transplantation of hematopoietic stem cells because it can transfer various types of cells to bone marrow regardless of their homing capacity. However, reconstitution process of these cells after IBMT remains to be fully elucidated. Here, we investigated whether in vitro culture of cord blood hematopoietic stem/progenitor cells affects their reconstitution in bone marrow after IBMT. Freshly isolated AC133+ cells (5x104 cells/mouse) or all cells derived from AC133+ cells cultured with growth factors (stem cell factor, flt-3 ligand, and thrombopoietin) for 5 days were injected into the bone marrow of the left tibia in irradiated NOD/SCID mice. In the bone marrow of the injected left tibia, the engraftment levels of human CD45+ cells at 6 weeks after transplantation was not considerably different between transplantation of noncultured and cytokine-cultured cells (54±28% vs. 69±13%). However, the migration of transplanted cells to the bone marrow of other noninjected bones was extremely lower for cytokine-treated cells compared with noncultured cells (2±2% vs. 36±10%). Similar findings were observed for engraftment of CD34+ cells. To enhance the migration of cytokine-cultured cells after IBMT, we similarly transplanted cultured AC133+ cells into the bone marrow of the left tibia, assessed the engraftment in the injected and noninjected tibiae at 7 days after transplantation, and then subcutaneously administered G-CSF (250 μg/kg/d) for 5 days. Administration of G-CSF stimulated the migration of cytokine-cultured cells to the bone marrow of previously-aspirated right tibia but failed to induce their migration to intact bone marrow of femur. These data indicate that ex vivo manipulation of hematopoietic progenitor/stem cells adversely influences their migration properties to other bone marrow compartments after IBMT. Our data raise caution for future clinical applications of the IBMT method using ex vivo-manipulated hematopoietic stem cells.

scholarly journals Correction of Fanconi Anemia Group C Hematopoietic Stem Cells Following Intrafemoral Gene Transfer

Anemia ◽  
2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Ouassila Habi ◽  
Johanne Girard ◽  
Valérie Bourdages ◽  
Marie-Chantal Delisle ◽  
Madeleine Carreau
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Hematopoietic Stem Cells ◽  
Fanconi Anemia ◽  
Ex Vivo ◽  
Direct Injection ◽  
Negative Effects ◽  
Hematopoietic Stem

The main cause of morbidity and mortality in Fanconi anemia patients is the development of bone marrow (BM) failure; thus correction of hematopoietic stem cells (HSCs) through gene transfer approaches would benefit FA patients. However, gene therapy trials for FA patients using ex vivo transduction protocols have failed to provide long-term correction. In addition, ex vivo cultures have been found to be hazardous for FA cells. To circumvent negative effects of ex vivo culture in FA stem cells, we tested the corrective ability of direct injection of recombinant lentiviral particles encoding FancC-EGFP into femurs ofFancC−/−mice. Using this approach, we show thatFancC−/−HSCs were efficiently corrected. Intrafemoral gene transfer of theFancCgene prevented the mitomycin C-induced BM failure. Moreover, we show that intrafemoral gene delivery into aplastic marrow restored the bone marrow cellularity and corrected the remaining HSCs. These results provide evidence that targeting FA-deficient HSCs directly in their environment enables efficient and long-term correction of BM defects in FA.

Hematopoietic Reconstitution of Mice Cord Blood Transplantation Combined with Bone Marrow Transient Hematopoietic Progenitor Cell

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4688-4688
Author(s):  
Jun Shi ◽  
Kazuma Ikeda ◽  
Maeda Yosinobu ◽  
Yinghua Yuan ◽  
Yehua Yu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Lymphocytes ◽  
Cord Blood ◽  
Progenitor Cells ◽  
B Lymphocytes ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Progenitor Cell ◽  
Fetal Blood

Abstract Abstract 4688 Umbilical cord blood has been increasingly used as a source of hematopoietic stem cells. However, fetal blood recipients had slower hematopoietic engraftment and impaired immune reconstitution. To accelerate myeloid and lymphoid recovery, we used an animal model of newborn blood modeled for cord blood (CB), along with transiently reconstituting progenitor cells from congenic bone marrow with recipients, as sources of stem cells. According to the previous reports that murine transiently reconstituting progenitor cells express the c-kit molecule, but not Sca-1 and lymphohematopoietic lineage markers, Lin-sca-1-c-kit+(c-kit+) were isolated by MACS method. c-kit+ cells population consisted of exclusively of medium- or large-sized blast-like cells, which displayed relatively low proliferative potential in vitro than Lin-sca-1+ (sca-1+) population. After transplantation of CB from DBA/2 mice (H-2d/d, CD45.2), with or without graded numbers of either c-kit+ or sca-1+ cells isolated from BDF1 mice (H-2d/b, CD45.1) bone marrow into lethally irradiated CD45.2 congenic BDF1 mice, hematopoietic engraftment were dynamic investigated. The intermingled transplantation of CB and c-kit+ cells or sca-1+ cells at the dosages of 1×104 or 2.5×104 or 5×104 to recipient mice leads to the quantity of white blood cells and platelets increased to 1×109/L and 1×1012/L at day12, whereas the injection of CB alone resulted in day17. By 2 weeks post-transplantation, congenic BM-derived cells were dominantly found in granulocytes and B lymphocytes, while host cells were dominantly found in T lymphocytes in CB transplantation combined either with c-kit+cells or sca-1+ cells. In cotransplantation with CB and c-kit+cells – engrafted surviving mice, the degree of donor CB cells in the peripheral blood increased progressively over time, while congenic donor BM-derived cells decreased gradually. After 60 weeks cotransplantation with CB and c-kit+ cells, a complete chimerism frequency of CB–derived cells continued to maintain in granulocytes and B lymphocytes, while T lymphocytes were dominantly derived from CB. On the other hand, congenic bone marrow or host-derived cells were the dominant population and CB-derived cells in the peripheral blood were less than 10% after 60 weeks cotransplantation with CB and sca-1+cells. In conclusion, the cotransplantation of CB and congenic c-kit+ cells was able to accelerate early hematopoietic recovery due to congenic marrow cells. But complete or main chimerism of cord blood was formed without or with fewer residual cells of host origin and congenic BM origin in long-term multilineage reconstitution. Thus, this cotransplant model in vivo may be to bring useful information for improving hematopoietic and immune reconstitution in fetal blood recipients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Primitive Long-Term Culture Initiating Cells (LTC-ICs) in Granulocyte Colony-Stimulating Factor Mobilized Peripheral Blood Progenitor Cells Have Similar Potential for Ex Vivo Expansion as Primitive LTC-ICs in Steady State Bone Marrow

Blood ◽  
1997 ◽  
Vol 89 (11) ◽  
pp. 3991-3997 ◽  
Author(s):  
Felipe Prosper ◽  
Kirk Vanoverbeke ◽  
David Stroncek ◽  
Catherine M. Verfaillie
Keyword(s):  
Bone Marrow ◽  
Steady State ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
Hla Dr ◽  
Stimulating Factor

Abstract We have recently shown that more than 90% of long-term culture initiating cells (LTC-IC) mobilized in the peripheral blood (PB) of normal individuals express HLA-DR and CD38 antigens and can sustain hematopoiesis for only 5 weeks. However, 10% of LTC-IC in mobilized PB are CD34+HLA-DR− and CD34+CD38− and can sustain hematopoiesis for at least 8 weeks. We now examine the ex vivo expansion potential of CD34+HLA-DR+ cells (rich in mature LTC-IC) and CD34+HLA-DR− cells (rich in primitive LTC-IC) in granulocyte colony-stimulating factor (G-CSF ) mobilized PB progenitor cells (PBPC). Cells were cultured in contact with M2-10B4 cells (contact) or in transwells above M2-10B4 (noncontact) without and with interleukin-3 (IL-3) and macrophage inflammatory protein (MIP-1α) for 2 and 5 weeks. Progeny were evaluated for the presence of colony-forming cells (CFC) and LTC-IC. When CD34+HLA-DR+ PB cells were cultured in contact cultures without cytokines, a threefold expansion of CFC was seen at 2 weeks, but an 80% decrease in CFC was seen at week 5. Further, the recovery of LTC-IC at week 2 was only 17% and 1% at week 5. This confirms our previous observation that although CD34+HLA-DR+ mobilized PB cells can initiate long-term cultures, they are relatively mature and cannot sustain long-term hematopoiesis. In contrast, when CD34+HLA-DR− mobilized PB cells were cultured in contact cultures without cytokines, CFC expansion persisted until week 5 and 49% and 11% of LTC-IC were recovered at week 2 and 5, respectively. As we have shown for steady state bone marrow (BM) progenitors, recovery of LTC-IC was threefold higher when CD34+HLA-DR− PBPC were cultured in noncontact rather than contact cultures, and improved further when IL-3 and MIP-1α were added to noncontact cultures (96 ± 2% maintained at week 5). We conclude that although G-CSF mobilizes a large population of “mature” CD34+HLA-DR+ LTC-IC with a limited proliferative capacity, primitive CD34+HLA-DR− LTC-IC present in mobilized PB have similar characteristics as LTC-IC from steady state BM: (1) they can be maintained in noncontact cultures containing IL-3 and MIP-1α for at least 5 weeks; (2) they are subject to the same proliferation inhibitory influences of contact with stroma. Since the absolute number of primitive LTC-IC (week 8 LTC-IC) per mL of G-CSF mobilized PB is similar to that per mL of steady state BM, these studies further confirm that G-CSF mobilized PBPC may have similar long-term repopulating abilities as steady state BM.

scholarly journals Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3693-3701 ◽  
Author(s):  
Ponnazhagan Veena ◽  
Christie M. Traycoff ◽  
David A. Williams ◽  
Jon McMahel ◽  
Susan Rice ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Retroviral Vector ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Transduction Efficiency ◽  
Cd34 Cells

Abstract Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

scholarly journals Delayed Targeting of Cytokine-Nonresponsive Human Bone Marrow CD34+ Cells With Retrovirus-Mediated Gene Transfer Enhances Transduction Efficiency and Long-Term Expression of Transduced Genes

Blood ◽  
1998 ◽  
Vol 91 (10) ◽  
pp. 3693-3701
Author(s):  
Ponnazhagan Veena ◽  
Christie M. Traycoff ◽  
David A. Williams ◽  
Jon McMahel ◽  
Susan Rice ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Retroviral Vector ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Transduction Efficiency ◽  
Cd34 Cells

Primitive hematopoietic progenitor cells (HPCs) are potential targets for treatment of numerous hematopoietic diseases using retroviral-mediated gene transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, a delicate balance between cellular activation and proliferation and maintenance of hematopoietic potential must be established. We have demonstrated that a subpopulation of human bone marrow (BM) CD34+ cells, highly enriched for primitive HPCs, persists in culture in a mitotically quiescent state due to their cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient RMGT of these cells. To evaluate and possibly circumvent this, we designed a two-step transduction protocol usingneoR-containing vectors coupled with flow cytometric cell sorting to isolate and examine transduction efficiency in different fractions of cultured CD34+ cells. BM CD34+ cells stained on day 0 (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were transduced with the retroviral vector G1Na and sorted on d6 into cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence relative to d0 sample) and d6 CNR cells that had not divided since d0. The other half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells were cultured in secondary long-term cultures (LTCs) and assayed weekly for transduced progenitor cells. Significantly higher numbers of G418-resistant colonies were produced in cultures initiated with d5 and d6 CNR cells compared with respective CR fractions (P < .05). At week 2, transduction efficiency was comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers ofneoR progenitor cells relative to the respective CR fractions (P < .05), while no difference in transduction efficiency between d5 and d6 CNR cells could be demonstrated. Polymerase chain reaction (PCR) analysis of the origin of transducedneoR gene in clonogenic cells demonstrated that mature progenitors (CR fractions) contained predominantly LNL6 sequences, while more primitive progenitor cells (CNR fractions) were transduced with G1Na. These results demonstrate that prolonged stimulation of primitive HPCs is essential for achieving efficient RMGT into cells capable of sustaining long-term in vitro hematopoiesis. These findings may have significant implications for the development of clinical gene therapy protocols.

Mobilization of Hematopoitic Stem/Progenitor Cells by a Cdc42 Activity-Specific Inhibitor

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 68-68 ◽  
Author(s):  
Wei Liu ◽  
Lei Wang ◽  
Xun Shang ◽  
Fukun Guo ◽  
Marnie A. Ryan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Actin Polymerization ◽  
Specific Inhibitor ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cell transplantation has become a standard of care for the treatment of many hematological diseases. Transplantation of mobilized peripheral blood stem cells has replaced bone marrow (BM) transplantation as the preferred method for hematopoietic recovery. To date, G-CSF mobilized hematopoietic stem/progenitor cell (HSPC) harvest is the main FDA-approved preparative regiment for transplantation protocols, but this application has several limitations in utilities including diverse individual variability and potential side effects in several patient populations. Although AMD3100, a chemical CXCR4-blocker, has been found effective for HSPC mobilization, the development of additional HSPC mobilization agents that work through well defined molecular mechanisms remains in need. Previously our laboratory has shown in a conditional knockout mouse model that deficiency of the Rho GTPase Cdc42 in the BM causes impaired adhesion, homing, lodging and retention of HSPCs, leading to massive egress of HSPCs from BM to the peripheral blood without compromising their proliferative potential. From an array of small molecule inhibitors of PIP2-induced actin-polymerization discovered in a high throughput screening, we identified CASIN, a novel Cdc42 Activity-Specific Inhibitor, that is effective in suppressing Cdc42 activity in a dose-dependent manner in murine fibroblasts and low density bone marrow (LDBM) cells and human CD34+ umbilical cord blood (HCB) cells in vitro, and in murine LDBM cells in vivo. The inhibitory effect by CASIN appears to be specific to Cdc42 and is reversible. We subsequently tested the hypothesis that pharmacological targeting Cdc42 by CASIN may transiently mimic the Cdc42 knockout phenotype leading to HSPC mobilization. In the dose range of 5–10 uM, CASIN does not show detectable toxicity in wild type or Cdc42 knockout HSPCs in cell survival and colony-forming unit activity assays. CASIN treatment of 32D murine myeloid progenitor cells or freshly isolated progenitor cells results in a reversible inhibition of F-actin polymerization induced by SDF-1α and blockade of α5β1 integrin mediated adhesion to fibronectin fragment CH296. Its effects on actin organization and adhesion are associated with an inhibition of directional migration of the colony-forming cells toward SDF-1α. In contrast, CASIN does not show a detectable effect on the adhesion and migration activities of Cdc42 knockout HSPCs, suggesting that it works specifically through Cdc42 to affect cell actin structure and adhesion. Upon injection into mice (5mg/Kg, intraperitoneally), CASIN is effective in stimulating mobilization of progenitor activity into the peripheral blood (~ 6-fold increase compared to control at 40 hrs post injection). Subsequent serial transplantation experiments show that the PB harvested from CASIN treated mice could reconstitute various lineages of blood cells in primary, secondary, and tertiary recipients, indicating that long-term hematopoietic stem cells were mobilized from the BM of CASIN-treated donor mice. Consistent with the mobilization phenotype, FACS analysis shows that intravenous injection of CASIN can cause transient reduction of long-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34−) and short-term hematopoietic stem cells (IL7Ra−Lin−Sca-1+c-Kit+CD34+) from BM. Similar to the effects on murine HSPCs, CASIN is active on CD34+ HCB cells in transiently suppressing F-actin assembly, adhesion to fibronectin, and SDF-1α induced migration without detectable toxicity in vitro. Whether CASIN is effective in mobilizing HCB-engrafted NOD/SCID mice is currently under investigation. Our studies suggest that the novel concept of pharmacological targeting of Cdc42, that transiently and reversibly mimics the effect of Cdc42 knockout, may be developed into a mobilization regiment with a well defined molecular and cellular mechanism.

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