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Progenitor Cell Maintenance of Nonhuman Primate Marrow Cells Via a Non-Cell-Autonomous Effect of Fos and Vps72
STEM CELL THERAPIES II cells than other cell types. Here, we show that murine GBCs can be robustly expanded in vitro, and present evidence that these cells can be reprogrammed to the beta cell fate. In order to expand GBCs in culture, we seeded primary GBCs on an irradiated feeder layer on which the GBCs form distinct colonies that grow readily and can be passaged. These cells can undergo at least 14 passages, allowing a rapid generation of cells amenable to reprogramming. In order to reprogram GBCs, we chose a non-integrating vector, adenovirus, to express the relevant pancreatic transcription factors. Using an adenoviral vector expressing the EGFP transgene, we show adenovirus is able to transduce and express EGFP in GBCs. Next, we investigated whether GBCs can be reprogrammed to a different cell lineage. Using different combinations of Neurog3, Pdx1, MafA, MafB, Pax4 and Nkx6.1, we analyzed pancreatic cell specific gene expression by qPCR to identify the optimal combination of transcription factors for reprogramming. By detection of the mRNA transcripts for Ins1, Ins2, Neurod1, Nkx2.2, Sst, Ppy and others, we confirmed that GBCs are reprogrammable to the pancreatic lineage. We hypothesized that the reprogramming resulted in the generation of heterogeneous pancreatic endocrine cell types. Further experiments supported this, as we were able to FACS-sort specific populations of reprogrammed cells that were more highly enriched for beta cell mRNA transcripts. In order to isolate a more pure population of gall bladder-derived beta-like cells, we used a reporter mouse strain in which the Ins1 gene promoter drives the expression of EGFP. In normal GBCs there is no expression of EGFP; however in reprogrammed GBCs, a population of EGFP-positive cells could be FACS-sorted within 48 hours post adenovirus transduction. By qPCR analysis, these cells were highly enriched for beta cell-specific transcripts and these cells will be further characterized by RNA-Seq and immunocytochemistry methods. These results indicate that GBCs are amenable to reprogramming towards a beta-like cell fate. In addition, we are currently characterizing these gall bladder-derived beta cells at the functional level, including their transplantation into a diabetic mouse model. These experiments will allow a more thorough understanding of the potential for the gall bladder to be used for the treatment of diabetes.
372. Improved Hematopoietic Stem/Progenitor Cell Maintenance of Nonhuman Primate Marrow Cells Via a Non-Cell-Autonomous Effect of Fos and Vps72
Korashon L. Watts,1 Eric Deneault,2 Guy Sauvageau,2 Hans-Peter Kiem.1 1 Fred Hutchinson Cancer Research Center, Seattle, WA; 2 University of Montreal, Montreal, QC, Canada.
Expansion of hematopoietic stem cells has important implications for stem cell transplantation and gene therapy. For example, it can potentially facilitate engraftment in settings where cell doses are limited, allow for prescreening of expanded stem cells, or aid engraftment in a nonmyeloablative setting. To this end, we tested genes previously identified as having an enhanced role in hematopoiesis to determine if any demonstrate a non-cell-autonomous effect on Macaca nemestrina bone marrow (BM) CD34+ cells, thus rendering them potential candidates for non-integrating cell expansion techniques. NIH 3T3 cells were transduced with gamma retroviruses expressing each of the following genes: pKOf1 (control), Fos and Vps72. These cells were then FACS-sorted to greater than 95% purity to establish 3 feeder cell lines expressing each of the genes (1 control gene and 2 genes with demonstrated hematopoietic effect.) Macaque BM CD34+ cells were co-cultured for 7 days on each feeder layer. After 7 days, phenotypic analysis was performed to detect CD3+, CD13+, and CD34+ percentage. Annexin V and 7AAD staining were used to assess viability and apoptosis, and CFUs were set up in triplicate using 10,000 BM cells from each treatment. Furthermore, cytospins Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
were prepared on Day 7 from BM cells from each feeder treatment. Slides were Giemsa-stained, and the ratio of blasts to differentiated cells determined for each treatment. All experiments were in triplicate. After 7 days of co-culture, macaque BM cells grown on Vps72- and Fos-transduced NIH 3T3 cells were 8% and 10% CD3+, respectively, compared to only 1% CD3+ on control (pKOf1)-transduced NIH 3T3 cells. CD13+ percentages on Fos-, Vps72- and control-transduced feeders were 8%, 10%, and 48%. Furthermore, CD34+ percentages of 37% (control), 67% (Vps72), and 71% (Fos) were obtained after 7 days of co-culture. Viability by 7AAD stain showed that Fos and Vps72 resulted in a higher viability (95% and 96%) after 7 days, while all other treatments exhibited viabilities between 88% and 91%. Finally, annexin V staining confirmed that Fos (6%) and Vps72 (4%) resulted in lower rates of apoptosis than all other treatments (range, 16% to 22%). The majority of apoptotic cells were in early-phase, rather than late-phase, apoptosis. Co-culture on Fos-overexpressing cells resulted in a CFU plating efficiency of 2.4%; co-culture on Vps72-overexpressing cells resulted in a plating efficiency of 2.5%, and all others ranged from 1.0% to 1.4%. Morphological analysis from Giemsa-stained cytospins showed Fos and Vps72 were the only genes tested in which the ratio of blasts to differentiated cells was >1. We found that Fos and Vps72 exhibited a non-cell-autonomous effect on macaque CD34+ cells. This discovery has immense potential in the development of a large animal clinical model for stem cell transplant. We have shown that Fos and Vps72 influence macaque cells via a nonintegrating pathway to yield (a) an increase in the lymphoid lineage, (b) improved maintenance of CD34+ cells, (c) higher viability, and (d) better maintenance of a primitive, undifferentiated morphology compared to control.
373. Muscle Derived Stem Cells Transduced with BMP4 Interact Broadly with Host Cells To Promote Bone Healing in Critical Sized Skull Defect
Xueqin Gao,1 Arvydas Usas,1 Aiping Lu,1 Jonathan Proto,1 Jessica C. Tebbets,1 Hongshuai Li,1 James H. Cummins,1 Mathieu Huard,1 Johnny Huard.1 1 Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. Introduction: We have shown that muscle derived stem cells transduced with BMP4 (MDSCsBMP4) promoted ectopic bone formation and bone defect healing in mice. The aim of this study was to investigate the interaction of MDSCs with the host cells and determine their contribution to the cranial bone regeneration. Methods: We transduced male MDSCs with retroBMP4-GFP and identified chemo-attractant gene expression by RT-PCR. Transduced MDSCs were then implanted into critical sized skull defects created in 12 week old male C57BL/6J mice using Tisseel FS as a scaffold. The control mice received PBS instead of cells and a scaffold. We monitored bone healing weekly by µCT. To investigate the donor and host cell interaction dynamically, we sacrificed mice at 3, 7, 14, 21, and 28 days post implantation (PI) and harvested the skull tissues for cryosectioning. We defined donor cell contribution by colocalizing GFP with Collagen2A1 (Col2A1) and osteocalcin (OC). We identified the host response by immunofluorescense: inflammatory cells by colocalizing Gr-1 and CD68 with GFP; endothelial cells by colocalizing CD31 and CD105 with GFP; immune cells by colocalizing CD4 and CD8 with GFP at different time points. Results: (1) RT-PCR indicated that MDSCs endogenously express Sox9, Bmpr1b, Bmpr2, Cox-2, Inos, Mcp1 and Vegf, but not Cytl1. BMP4 transduction did not affect the expression of these genes except Inos. (2) µCT revealed that MDSCsBMP4 healed bone completely in 4 weeks. Co-localization of GFP with Col2A1and OC showed that MDSCs contributed to majority of the endochondral bone at the defect site. (3) At 3 d PI, we observed significantly more inflammatory cells (CD68+ macrophages and Gr-1+ S145
372. Improved Hematopoietic Stem/Progenitor Cell Maintenance of Nonhuman Primate Marrow Cells Via a Non-Cell-Autonomous Effect of Fos and Vps72
Korashon L. Watts,1 Eric Deneault,2 Guy Sauvageau,2 Hans-Peter Kiem.1 1 Fred Hutchinson Cancer Research Center, Seattle, WA; 2 University of Montreal, Montreal, QC, Canada.
Expansion of hematopoietic stem cells has important implications for stem cell transplantation and gene therapy. For example, it can potentially facilitate engraftment in settings where cell doses are limited, allow for prescreening of expanded stem cells, or aid engraftment in a nonmyeloablative setting. To this end, we tested genes previously identified as having an enhanced role in hematopoiesis to determine if any demonstrate a non-cell-autonomous effect on Macaca nemestrina bone marrow (BM) CD34+ cells, thus rendering them potential candidates for non-integrating cell expansion techniques. NIH 3T3 cells were transduced with gamma retroviruses expressing each of the following genes: pKOf1 (control), Fos and Vps72. These cells were then FACS-sorted to greater than 95% purity to establish 3 feeder cell lines expressing each of the genes (1 control gene and 2 genes with demonstrated hematopoietic effect.) Macaque BM CD34+ cells were co-cultured for 7 days on each feeder layer. After 7 days, phenotypic analysis was performed to detect CD3+, CD13+, and CD34+ percentage. Annexin V and 7AAD staining were used to assess viability and apoptosis, and CFUs were set up in triplicate using 10,000 BM cells from each treatment. Furthermore, cytospins Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
were prepared on Day 7 from BM cells from each feeder treatment. Slides were Giemsa-stained, and the ratio of blasts to differentiated cells determined for each treatment. All experiments were in triplicate. After 7 days of co-culture, macaque BM cells grown on Vps72- and Fos-transduced NIH 3T3 cells were 8% and 10% CD3+, respectively, compared to only 1% CD3+ on control (pKOf1)-transduced NIH 3T3 cells. CD13+ percentages on Fos-, Vps72- and control-transduced feeders were 8%, 10%, and 48%. Furthermore, CD34+ percentages of 37% (control), 67% (Vps72), and 71% (Fos) were obtained after 7 days of co-culture. Viability by 7AAD stain showed that Fos and Vps72 resulted in a higher viability (95% and 96%) after 7 days, while all other treatments exhibited viabilities between 88% and 91%. Finally, annexin V staining confirmed that Fos (6%) and Vps72 (4%) resulted in lower rates of apoptosis than all other treatments (range, 16% to 22%). The majority of apoptotic cells were in early-phase, rather than late-phase, apoptosis. Co-culture on Fos-overexpressing cells resulted in a CFU plating efficiency of 2.4%; co-culture on Vps72-overexpressing cells resulted in a plating efficiency of 2.5%, and all others ranged from 1.0% to 1.4%. Morphological analysis from Giemsa-stained cytospins showed Fos and Vps72 were the only genes tested in which the ratio of blasts to differentiated cells was >1. We found that Fos and Vps72 exhibited a non-cell-autonomous effect on macaque CD34+ cells. This discovery has immense potential in the development of a large animal clinical model for stem cell transplant. We have shown that Fos and Vps72 influence macaque cells via a nonintegrating pathway to yield (a) an increase in the lymphoid lineage, (b) improved maintenance of CD34+ cells, (c) higher viability, and (d) better maintenance of a primitive, undifferentiated morphology compared to control.
373. Muscle Derived Stem Cells Transduced with BMP4 Interact Broadly with Host Cells To Promote Bone Healing in Critical Sized Skull Defect
Xueqin Gao,1 Arvydas Usas,1 Aiping Lu,1 Jonathan Proto,1 Jessica C. Tebbets,1 Hongshuai Li,1 James H. Cummins,1 Mathieu Huard,1 Johnny Huard.1 1 Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA. Introduction: We have shown that muscle derived stem cells transduced with BMP4 (MDSCsBMP4) promoted ectopic bone formation and bone defect healing in mice. The aim of this study was to investigate the interaction of MDSCs with the host cells and determine their contribution to the cranial bone regeneration. Methods: We transduced male MDSCs with retroBMP4-GFP and identified chemo-attractant gene expression by RT-PCR. Transduced MDSCs were then implanted into critical sized skull defects created in 12 week old male C57BL/6J mice using Tisseel FS as a scaffold. The control mice received PBS instead of cells and a scaffold. We monitored bone healing weekly by µCT. To investigate the donor and host cell interaction dynamically, we sacrificed mice at 3, 7, 14, 21, and 28 days post implantation (PI) and harvested the skull tissues for cryosectioning. We defined donor cell contribution by colocalizing GFP with Collagen2A1 (Col2A1) and osteocalcin (OC). We identified the host response by immunofluorescense: inflammatory cells by colocalizing Gr-1 and CD68 with GFP; endothelial cells by colocalizing CD31 and CD105 with GFP; immune cells by colocalizing CD4 and CD8 with GFP at different time points. Results: (1) RT-PCR indicated that MDSCs endogenously express Sox9, Bmpr1b, Bmpr2, Cox-2, Inos, Mcp1 and Vegf, but not Cytl1. BMP4 transduction did not affect the expression of these genes except Inos. (2) µCT revealed that MDSCsBMP4 healed bone completely in 4 weeks. Co-localization of GFP with Col2A1and OC showed that MDSCs contributed to majority of the endochondral bone at the defect site. (3) At 3 d PI, we observed significantly more inflammatory cells (CD68+ macrophages and Gr-1+ S145