Autologous iPS Research Articles

Progress of iPS cell-based transplantation therapy for retinal diseases.

The discovery of induced Pluripotent Stem) (iPS) cells has instigated innovation in various fields, including ophthalmology. Cell therapy has shown tremendous progress in translational research on retinal diseases, including the first-in-human transplantation of autologous iPS cell-derived retinal pigment epithelium (RPE) cells for patients with age-related macular degeneration (AMD). Cell therapy for retinitis pigmentosa (RP) has also been developed. Retinal organoid and photoreceptor cell transplantation has been shown to incorporate into the degenerated host retina, forming synapses with host neurons and resulting in functional recovery. Based on preclinical data, first-in-human transplantation of iPS cell-derived retinal sheets has been conducted. In this review, we summarize the current progress in iPS cell-based retinal cell transplantation research for retinal diseases, addressing some remaining challenges and future prospects.

38 - Safety considerations in the generation of clinical grade autologous ips cell lines

38 - Safety considerations in the generation of clinical grade autologous ips cell lines

Risk of HLA Homozygous Cord Blood Transplantation: Implications for Induced Pluripotent Stem Cell Banking and Transplantation

Clinical application of induced pluripotent stem cells (iPS) in autologous settings has just begun. To overcome the high time and cost barriers in the individual production of autologous iPS, the use of allogeneic iPS with a homozygous human leukocyte antigen (HLA) haplotype (HLA‐homo HP) has been proposed. Cord blood transplantation (CBT) is a suitable model for evaluating the allogeneic immunogenicity of iPS transplantation from HLA‐homo donors. We analyzed 1,374 Japanese single cord blood transplant pairs who were retrospectively typed as HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1, and ‐DPB1. Among these, six pairs with donor HLA homo—patient‐HLA hetero (homo‐hetero) were found, all of which showed favorable neutrophil engraftment. Multivariate analysis revealed a significantly elevated engraftment risk (HR = 1.59) compared with hetero‐hetero pairs with HLA 1‐2 locus mismatch (789 pts) and comparative risk (HR = 1.23) compared with hetero‐hetero pairs with 0 mismatch (104 pts). These results for CBT with HLA‐homo HP cord blood carry an important implication, namely the possibility that HLA‐homo iPS transplantation results in favorable engraftment. Furthermore, we obtained detailed information on HLA alleles and haplotypes of HLA‐homo. All donor HLA‐homo HPs had a common specific ethnicity and high conservation of the HLA region, and one of two patient heterogeneous HPs invariably shared the same HP as donor HLA‐homo HP, and another non‐shared patient HP was mismatched with 1 to 4 HLA alleles of HLA‐A, ‐B, ‐C, and ‐DRB1 loci in the GVH direction. These findings indicate that patients possessing a single common HLA haplotype have a higher chance of yielding HLA‐homo iPS. Stem Cells Translational Medicine 2018;7:173–179

Generation of Hypoimmunogenic Induced Pluripotent Stem Cells for Allogeneic Cell and Tissue Transplantation

Background: The most urgent problem facing transplantation today is the lack of suitable donor organs and tissues. One possible alternative may be cell transplantation with the aim to replace, repair, or enhance the biological function of the diseased organ. Induced pluripotent stem cells are promising candidates for cell-based therapies. Transplanted autologous iPS cell do not cross histocompatibility barriers since such cells are genetically identical to the transplant recipient. However, for acute diseases such as myocardial infarction, treatment options must be readily available, and generating autologous iPSCs for treatment purposes would take too long. Therefore, identifying alternative methods for preventing rejection of iPSCs would be of great interest. In this study, we generated MHC I and MHCII -deficient mouse iPSCs (miPSCs) via disruption of beta-2-microglobulin and C2TA. The antigenicity was investigated in in vivo and in vitro assays. Methods: IPSCs were generated from C57BL/6 mouse fibroblasts by non-viral minicircle transfection of the transcription factors c-Myc, Sox-2, Klf4 and Oct4. The CRIPSR/Cas9 technology was used to generate β2m and C2TA knockout miPSCs (β2m-C2TA−/− miPSCs). Unmodified and β2m-C2TA −/− miPSCs as well as their derivates were injected in syngeneic C57BL/6 mice and in allogeneic BALB/c mice. Cellular response was quantified and bioluminescence imaging was performed to assess cell survival. Results: FACS analysis showed a lack of MHC I and MHC II expression on the β2m-C2TA−/− iPSCs but a normal expression in unmodified iPSCs. After allogeneic transplantation into BALB/c mice, β2m-C2TA−/− miPSCs generated a significantly weaker response in both IFNγ (p<0.01) and IL-4 (p<0.01) ELISPOT assays. Teratoma formation occurred in C57BL/6 mice receiving unmodified iPSCs (100%) and BALB/c mice receiving β2m-C2TA−/− iPSCs (100%), whereas BALB/c mice receiving unmodified iPSCs did not form any teratoma (0%). Indeed, also miPSC derivates, such as cardiomyocytes, endothelial and islet cells showed long-term survival after allogeneic transplantation. Conclusion: Our results clearly demonstrate the hypoimmunogenicity of β2m-C2TA−/− iPSCs after allogeneic transplantation. Thus, MHC I MHC II deficient iPSCs might serve an unlimited cell source for the generation of universally compatible “off-the-shelf” cells grafts or tissues in future clinical applications. Christiane Pahrmann.

Induced to cure: Engineering iPS cell derived RPE scaffolds to treat degenerative eye diseases

SummaryThe recent success with embryonic stem (ES) cell derived retinal pigment epithelium (RPE) has provided hope for a cure for degenerative eye diseases. Induced pluripotent stem (iPS) cells are an autologous source of stem cells potentially with fewer immune‐challenges as compared to ES cells. Using a developmentally guided differentiation protocol we have developed fully polarized RPE tissue from iPS cells. The RPE monolayer along with its secreted ECM and a degradable scaffold form a tissue that mimics the native tissue in structural and functional properties. This RPE tissue performs several key RPE functions like phagocytosis of photoreceptor outer segments, ability to transport water from apical to basal side, and the ability to secrete cytokines in a polarized fashion. Currently, we are testing the safety and the efficacy of this tissue in animal models. We have begun Phase I Investigational New Drug (IND) enabling studies with the goal to transplant autologous iPS cell derived RPE in patients in advanced Geographic Atrophy stage of age‐related macular degeneration (AMD), one of the leading blinding diseases in the US. Our work will provide a potential personalized cell therapy for AMD patients.

Potential and limitation of HLA-based banking of human pluripotent stem cells for cell therapy.

Great hopes have been placed on human pluripotent stem (hPS) cells for therapy. Tissues or organs derived from hPS cells could be the best solution to cure many different human diseases, especially those who do not respond to standard medication or drugs, such as neurodegenerative diseases, heart failure, or diabetes. The origin of hPS is critical and the idea of creating a bank of well-characterized hPS cells has emerged, like the one that already exists for cord blood. However, the main obstacle in transplantation is the rejection of tissues or organ by the receiver, due to the three main immunological barriers: the human leukocyte antigen (HLA), the ABO blood group, and minor antigens. The problem could be circumvented by using autologous stem cells, like induced pluripotent stem (iPS) cells, derived directly from the patient. But iPS cells have limitations, especially regarding the disease of the recipient and possible difficulties to handle or prepare autologous iPS cells. Finally, reaching standards of good clinical or manufacturing practices could be challenging. That is why well-characterized and universal hPS cells could be a better solution. In this review, we will discuss the interest and the feasibility to establish hPS cells bank, as well as some economics and ethical issues.

Flk1+ and VE-Cadherin+ Endothelial Cells Derived from iPSCs Recapitulates Vascular Development during Differentiation and Display Similar Angiogenic Potential as ESC-Derived Cells

RationaleInduced pluripotent stem (iPS) cells have emerged as a source of potentially unlimited supply of autologous endothelial cells (ECs) for vascularization. However, the regenerative function of these cells relative to adult ECs and ECs derived from embryonic stem (ES) cells is unknown. The objective was to define the differentiation characteristics and vascularization potential of Fetal liver kinase (Flk)1+ and Vascular Endothelial (VE)-cadherin+ ECs derived identically from mouse (m)ES and miPS cells. Methods and ResultsNaive mES and miPS cells cultured in type IV collagen (IV Col) in defined media for 5 days induced the formation of adherent cell populations, which demonstrated similar expression of Flk1 and VE-cadherin and the emergence of EC progenies. FACS purification resulted in 100% Flk1+ VE-cadherin+ cells from both mES and miPS cells. Emergence of Flk1+VE-cadherin+ cells entailed expression of the vascular developmental transcription factor Er71, which bound identically to Flk1, VE-cadherin, and CD31 promoters in both populations. Immunostaining with anti-VE-cadherin and anti-CD31 antibodies and microscopy demonstrated the endothelial nature of these cells. Each cell population (unlike mature ECs) organized into well-developed vascular structures in vitro and incorporated into CD31+ neovessels in matrigel plugs implanted in nude mice in vivo.ConclusionThus, iPS cell-derived Flk1+VE-cadherin+ cells expressing the Er71 are as angiogenic as mES cell-derived cells and incorporate into CD31+ neovessels. Their vessel forming capacity highlights the potential of autologous iPS cells-derived EC progeny for therapeutic angiogenesis.

Long-term Safety and Efficacy of Human-Induced Pluripotent Stem Cell (iPS) Grafts in a Preclinical Model of Retinitis Pigmentosa

The U.S. Food and Drug Administration recently approved phase I/II clinical trials for embryonic stem (ES) cell-based retinal pigmented epithelium (RPE) transplantation, but this allograft transplantation requires lifelong immunosuppressive therapy. Autografts from patient-specific induced pluripotent stem (iPS) cells offer an alternative solution to this problem. However, more data are required to establish the safety and efficacy of iPS transplantation in animal models before moving iPS therapy into clinical trials. This study examines the efficacy of iPS transplantation in restoring functional vision in Rpe65(rd12)/Rpe65(rd12) mice, a clinically relevant model of retinitis pigmentosa (RP). Human iPS cells were differentiated into morphologically and functionally RPE-like tissue. Quantitative real-time polymerase chain reaction (RT-PCR) and immunoblots confirmed RPE fate. The iPS-derived RPE cells were injected into the subretinal space of Rpe65(rd12)/Rpe65(rd12) mice at 2 d postnatally. After transplantation, the long-term surviving iPS-derived RPE graft colocalized with the host native RPE cells and assimilated into the host retina without disruption. None of the mice receiving transplants developed tumors over their lifetimes. Furthermore, electroretinogram, a standard method for measuring efficacy in human trials, demonstrated improved visual function in recipients over the lifetime of this RP mouse model. Our study provides the first direct evidence of functional recovery in a clinically relevant model of retinal degeneration using iPS transplantation and supports the feasibility of autologous iPS cell transplantation for retinal and macular degenerations featuring significant RPE loss.

206 GENERATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS FROM DENTAL PULP-DERIVED MESENCHYMAL STEM CELLS

Induced pluripotent stem (iPS) cells are somatic cells that have been reprogrammed to a pluripotent state and a great source for regenerative medicine. Several types of human somatic and adult stem cells have been reprogrammed into iPS cells, including mesenchymal stem cells (MSC). Recently, human dental pulp has been considered as a valuable alternative source of MSC (hDP-MSC) with excellent proliferation capacity and multilineage differentiation potential. In this study, our objective was to establish iPS cells from hDP-MSC and evaluate the expression of transcriptional factors and in vitro differentiation potential into mesenchymal lineages. The hMSC were isolated from the dental pulp of male donor (∼18 years old) were cultured in advanced-DMEM supplemented with 10% fetal bovine serum at 37°C, 5% CO2 in a humidified atmosphere. The hDP-MSC at passage 3 were analysed for the expression of MSC-specific surface markers (CD44 and CD90) using flow cytometry and transcriptional factors (Oct4, Nanog and Sox2) by immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR). Differentiation into adipocytes and osteocytes of hDP-MSC was carried out under specific conditions for 2 and 4 weeks, respectively and assessed by cytochemical staining (Oil red O, von Kossa and Alizarin Red S, respectively). iPS cells were generated from hDP-MSC at passage 3 by using pMXs retroviral vector (Addgene, Cambridge, MA, USA) containing cDNA of c-Myc, Klf4, Nanog and Sox2. The iPS cells were evaluated for alkaline phosphatase (AP) activity, expression of human embryonic stem cells (hESC) markers (Rex1, Nanog, Oct4, SSEA-1 and TRA-160) by immunostaining. Isolated hDP-MSC expressed surface markers, such as CD44 and CD90 (86% and 93%, respectively) by flow cytometry and positively stained for transcriptional factors (Oct4, Nanog and Sox2) by immunofluorescence. Further, the cells were capable of differentiating in vitro into adipocytes and osteocytes as demonstrated by Oil red O and von Kossa and Alizarin red S staining, respectively. The iPS cells generated from hDP-MSC were positive for AP staining and clearly expressed the markers specific to hESC, including Rex1, Nanog, Oct4, SSEA-1 and TRA-160. In conclusion, hMSC derived from dental pulp could be successfully reprogrammed into iPS cells by retroviral vector systems and the generated iPS cells shared the similar characteristics of hESC. Therefore, hDP-MSC might be an ideal alternative cell source to derive autologous iPS cells for therapeutic applications. This work was supported by Grant No. 2007031034040 from Bio-organ and Grant No. 200908FHT010204005 from Biogreen21.

Induced Pluripotent Stem Cells from Individuals with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is an inherited blistering skin disorder caused by mutations in the COL7A1 gene-encoding type VII collagen (Col7), the major component of anchoring fibrils at the dermal-epidermal junction. Individuals with RDEB develop painful blisters and mucosal erosions, and currently, there are no effective forms of therapy. Nevertheless, some advances in patient therapy are being made, and cell-based therapies with mesenchymal and hematopoietic cells have shown promise in early clinical trials. To establish a foundation for personalized, gene-corrected, patient-specific cell transfer, we generated induced pluripotent stem (iPS) cells from three subjects with RDEB (RDEB iPS cells). We found that Col7 was not required for stem cell renewal and that RDEB iPS cells could be differentiated into both hematopoietic and nonhematopoietic lineages. The specific epigenetic profile associated with de-differentiation of RDEB fibroblasts and keratinocytes into RDEB iPS cells was similar to that observed in wild-type (WT) iPS cells. Importantly, human WT and RDEB iPS cells differentiated in vivo into structures resembling the skin. Gene-corrected RDEB iPS cells expressed Col7. These data identify the potential of RDEB iPS cells to generate autologous hematopoietic grafts and skin cells with the inherent capacity to treat skin and mucosal erosions that typify this genodermatosis.

Amniocytes can serve a dual function as a source of iPS cells and feeder layers.

Clinical barriers to stem-cell therapy include the need for efficient derivation of histocompatible stem cells and the zoonotic risk inherent to human stem-cell xenoculture on mouse feeder cells. We describe a system for efficiently deriving induced pluripotent stem (iPS) cells from human and mouse amniocytes, and for maintaining the pluripotency of these iPS cells on mitotically inactivated feeder layers prepared from the same amniocytes. Both cellular components of this system are thus autologous to a single donor. Moreover, the use of human feeder cells reduces the risk of zoonosis. Generation of iPS cells using retroviral vectors from short- or long-term cultured human and mouse amniocytes using four factors, or two factors in mouse, occurs in 5–7 days with 0.5% efficiency. This efficiency is greater than that reported for mouse and human fibroblasts using similar viral infection approaches, and does not appear to result from selective reprogramming of Oct4+ or c-Kit+ amniocyte subpopulations. Derivation of amniocyte-derived iPS (AdiPS) cell colonies, which express pluripotency markers and exhibit appropriate microarray expression and DNA methylation properties, was facilitated by live immunostaining. AdiPS cells also generate embryoid bodies in vitro and teratomas in vivo. Furthermore, mouse and human amniocytes can serve as feeder layers for iPS cells and for mouse and human embryonic stem (ES) cells. Thus, human amniocytes provide an efficient source of autologous iPS cells and, as feeder cells, can also maintain iPS and ES cell pluripotency without the safety concerns associated with xenoculture.

Aberrant expression of the pluripotency marker SOX-2 in endometriosis

Expression of the pluripotency factors SOX-2, OCT-4, KLF-4, and NANOG was analyzed by quantitative real-time polymerase chain reaction, immunohistochemistry, and immunofluorescence microscopy in the endometrium, myometrium, and endometriotic tissue of 36 patients. Aberrant expression of SOX-2 may indicate a stem cell origin of endometriosis, whereas the presence of all progenitor markers in endometrial tissue marks the endometrium as a potential source for induced pluripotent stem cell generation.

Regulatory Considerations for the Development of Autologous Induced Pluripotent Stem Cell Therapies

Induced pluripotent stem (iPS) cells offer tremendous opportunity for the creation of autologous cellular therapies, in which gene correction or the avoidance of immune response issues are desirable. In addition, iPS cells avoid the ethical concerns raised by the sourcing of human embryonic stem cells (hESCs) from embryos. iPS cells share many characteristics with hESCs and it is anticipated that existing experience with hESCs will translate to rapid progress in moving iPS cell-derived products toward clinical trials. While the potential clinical value for these products is considerable, the nature of current manufacturing paradigms for autologous iPS cell products raises considerable regulatory concerns. Here, the regulatory challenges posed by autologous iPS cell-derived products are examined. We conclude that there will be considerable regulatory concerns primarily relating to reproducibility of the manufacturing process and safety testing within clinically limited time constraints. Demonstrating safety of the final cell product in an autologous setting will be the single greatest obstacle to progressing autologous iPS cell-based therapies into the clinic.

Translational potential of patient‐specific human pluripotent stem cells

Pluripotent stem cells are defined by two features: self‐renewal and the ability to differentiate into any cell type. Multiple methods now exist to reprogram differentiated cells from an adult animal to a state of pluripotency. In turn, it is possible to differentiate these reprogrammed cells into specific tissue progenitors in vitro. Combined, these advances provide the basis for development of patient‐specific cell replacement therapies, which may address the donor incompatibility and availability problems pervasive in modern tissue transplantation. We are generating patient‐specific pluripotent cell lines using “direct” reprogramming, wherein fibroblasts are transduced with defined factors and cultured to yield “induced pluripotent stem” (iPS) cells. We have derived iPS cell lines from the somatic cells of patients with bone marrow failure syndromes including dyskeratosis congenita and Pearson syndrome, which are caused by telomerase gene mutations and mitochondrial DNA deletions, respectively. Unexpectedly, we find that the underlying genetic lesions in these cells can be compensated or corrected during propagation in the pluripotent state. These results demonstrate the value of patient‐specific iPS cells as disease models, and further the concept of autologous iPS based cellular therapy for genetic hematologic disorders.

Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.

Abstract 497Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures:Daley:MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

Stem cell sources to treat diabetes

We review progress towards the goal of utilizing stem cells as a source of engineered pancreatic beta-cells for therapy of diabetes. Protocols for the in vitro differentiation of embryonic stem (ES) cells based on normal developmental cues have generated beta-like cells that produce high levels of insulin, albeit at low efficiency and without full responsiveness to extracellular levels of glucose. Induced pluripotent stem (iPS) cells also can yield insulin-producing cells following similar approaches. An important recent report shows that when transplanted into mice, human ES-derived cells with a phenotype corresponding to pancreatic endoderm matured to yield cells capable of maintaining near-normal regulation of blood sugar [Kroon et al., 2008]. Major hurdles that must be overcome to enable the broad clinical translation of these advances include teratoma formation by ES and iPS cells, and the need for immunosuppressive drugs. Classes of stem cells that can be expanded extensively in culture but do not form teratomas, such as amniotic fluid-derived stem cells and hepatic stem cells, offer possible alternatives for the production of beta-like cells, but further evidence is required to document this potential. Generation of autologous iPS cells should prevent transplant rejection, but may prove prohibitively expensive. Banking strategies to identify small numbers of stem cell lines homozygous for major histocompatibility loci have been proposed to enable beneficial genetic matching that would decrease the need for immunosuppression.

Patient-Specific Induced Pluripotent Stem Cells in Hurler Syndrome

Hurler syndrome (HS; mucopolysaccharidosis type I) is caused by severe mutations in the iduronidase (IDUA) gene, leading to multi-organ system dysfunction due to the toxic accumulation of glycosaminoglycans. Although allogeneic hematopoietic cell transplantation (HCT) has been shown to provide the IDUA protein and to reverse many of the manifestations of HS, allogeneic HCT is associated with significant morbidity and mortality. We hypothesized that an advantageous alternative strategy may be to induce gene-corrected autologous pluripotent cells to become hematopoietic stem cells, which then provide the missing IDUA enzyme. Because patient-specific embryonic stem cell isolation is not practical, recent strategies have been developed that reprogram adult cells to acquire pluripotency. Such induced pluripotent stem (iPS) cells can be created from fibroblasts or mesenchymal stromal cells (MSCs). As a first step in testing of iPS cells for gene-corrected HS treatment, we isolated host MSCs from the bone chips of a 9-year-old boy with HS who had undergone spinal surgery 8 years after successful allogeneic HCT. HS-MSCs expressed no IDUA, confirming a lack of contamination from either donor-derived hematopoietic cells or MSCs. To create HS-iPS cells, HS-MSCs were transduced with viral vectors carrying reprogramming transcription factors (OCT4, SOX2, KLF4, and c-MYC) that are typically associated with pluripotency and expressed at high levels in embryonic but not adult stem cells. Transduced cells were cultured on supportive stroma of irradiated mouse embryo fibroblasts. Within several weeks, colonies of iPS cells emerged from the two-dimensional culture. When compared to MSCs, the HS-iPS cells showed persistent mRNA expression of OCT3/4 and SOX2 and transient mRNA expression of c-MYC and KLF4, which is expected to occur in the wild-type iPS cells. HS-iPS cells expressed protein markers characteristic of reprogrammed immature cells: OCT3/4, NANOG, stage-specific embryonic antigens (SSEA) 3 and 4, tumor rejection antigens (TRA) 1–60 and 1–81, and alkaline phosphatase. HS-iPS cells had normal male karyotype as determined by chromosomal G-banding. As a second step in creating gene-corrected HS-iPS cells, we employed the non-viral Sleeping Beauty (SB) transposon system (because of the less random pattern of genome integration when compared to viral vectors). Human HS-iPS cells were co-nucleofected with an SB transposon that harbored the human IDUA gene and an expression cassette of the green fluorescent protein (GFP) along with an SB transposase plasmid that provides the enzymatic machinery necessary for integration into TA dinucleotide sites within the genome. Two weeks after nucleofection 10%-15% of HS-iPS cells expressed GFP. Total glycosaminoglycans (a hallmark of the biochemical defect in HS) in unsorted cultures were decreased to wild-type levels. IDUA expression in unsorted cultures was approximately 10% of wild-type IDUA levels, which is within the range sufficient for phenotypic rescue in HS patients after allogeneic HCT. Experiments are ongoing, and data will be presented in regards to: a) map transposon insertions in the genome to prove stable transgenesis by transposition; b) characterization of the differentiation potential of the corrected HS-iPS cells into various mesodermal lineages relevant to rescue of the clinical phenotype associated with HS (hematopoietic, chondrogenic, and osteogenic); c) assessment of development and consequences of cellular pathology in numerous tissue types affected by IDUA deficiency. To our knowledge these are the first data to report that autologous iPS cells can be obtained from HS patients. In summary, HS-iPS cells present an opportunity to use the hematopoietic progeny of gene-corrected autologous cells clinically in a manner that may preclude the immunologic complications of allogeneic transplantation.

Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin

It has recently been demonstrated that mouse and human fibroblasts can be reprogrammed into an embryonic stem cell-like state by introducing combinations of four transcription factors. However, the therapeutic potential of such induced pluripotent stem (iPS) cells remained undefined. By using a humanized sickle cell anemia mouse model, we show that mice can be rescued after transplantation with hematopoietic progenitors obtained in vitro from autologous iPS cells. This was achieved after correction of the human sickle hemoglobin allele by gene-specific targeting. Our results provide proof of principle for using transcription factor-induced reprogramming combined with gene and cell therapy for disease treatment in mice. The problems associated with using retroviruses and oncogenes for reprogramming need to be resolved before iPS cells can be considered for human therapy.