Megan Riddle

Sarcoma derived from cultured mesenchymal stem cells

To study the biodistribution of MSCs, we labeled adult murine C57BL/6 MSCs with firefly luciferase and DsRed2 fluorescent protein using nonviral Sleeping Beauty transposons and coinfused labeled MSCs with bone marrow into irradiated allogeneic recipients. Using in vivo whole‐body imaging, luciferase signals were shown to be increased between weeks 3 and 12. Unexpectedly, some mice with the highest luciferase signals died and all surviving mice developed foci of sarcoma in their lungs. Two mice also developed sarcomas in their extremities. Common cytogenetic abnormalities were identified in tumor cells isolated from different animals. Original MSC cultures not labeled with transposons, as well as independently isolated cultured MSCs, were found to be cytogenetically abnormal. Moreover, primary MSCs derived from the bone marrow of both BALB/c and C57BL/6 mice showed cytogenetic aberrations after several passages in vitro, showing that transformation was not a strain‐specific nor rare event. Clonal evolution was observed in vivo, suggesting that the critical transformation event(s) occurred before infusion. Mapping of the transposition insertion sites did not identify an obvious transposon‐related genetic abnormality, and p53 was not overexpressed. Infusion of MSC‐derived sarcoma cells resulted in malignant lesions in secondary recipients. This new sarcoma cell line, S1, is unique in having a cytogenetic profile similar to human sarcoma and contains bioluminescent and fluorescent genes, making it useful for investigations of cellular biodistribution and tumor response to therapy in vivo. More importantly, our study indicates that sarcoma can evolve from MSC cultures.

Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa

BACKGROUND Recessive dystrophic epidermolysis bullosa is an incurable, often fatal mucocutaneous blistering disease caused by mutations in COL7A1, the gene encoding type VII collagen (C7). On the basis of preclinical data showing biochemical correction and prolonged survival in col7 −/− mice, we hypothesized that allogeneic marrow contains stem cells capable of ameliorating the manifestations of recessive dystrophic epidermolysis bullosa in humans. METHODS Between October 2007 and August 2009, we treated seven children who had recessive dystrophic epidermolysis bullosa with immunomyeloablative chemotherapy and allogeneic stem-cell transplantation. We assessed C7 expression by means of immunofluorescence staining and used transmission electron microscopy to visualize anchoring fibrils. We measured chimerism by means of competitive polymerase-chain-reaction assay, and documented blister formation and wound healing with the use of digital photography. RESULTS One patient died of cardiomyopathy before transplantation. Of the remaining six patients, one had severe regimen-related cutaneous toxicity, with all having improved wound healing and a reduction in blister formation between 30 and 130 days after transplantation. We observed increased C7 deposition at the dermal-epidermal junction in five of the six recipients, albeit without normalization of anchoring fibrils. Five recipients were alive 130 to 799 days after transplantation; one died at 183 days as a consequence of graft rejection and infection. The six recipients had substantial proportions of donor cells in the skin, and none had detectable anti-C7 antibodies. CONCLUSIONS Increased C7 deposition and a sustained presence of donor cells were found in the skin of children with recessive dystrophic epidermolysis bullosa after allogeneic bone marrow transplantation. Further studies are needed to assess the long-term risks and benefits of such therapy in patients with this disorder. (Funded by the National Institutes of Health; ClinicalTrials.gov number, NCT00478244.)

TALEN-based gene correction for epidermolysis bullosa.

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized by a functional deficit of type VII collagen protein due to gene defects in the type VII collagen gene (COL7A1). Gene augmentation therapies are promising, but run the risk of insertional mutagenesis. To abrogate this risk, we explored the possibility of using engineered transcription activator-like effector nucleases (TALEN) for precise genome editing. We report the ability of TALEN to induce site-specific double-stranded DNA breaks (DSBs) leading to homology-directed repair (HDR) from an exogenous donor template. This process resulted in COL7A1 gene mutation correction in primary fibroblasts that were subsequently reprogrammed into inducible pluripotent stem cells and showed normal protein expression and deposition in a teratoma-based skin model in vivo. Deep sequencing-based genome-wide screening established a safety profile showing on-target activity and three off-target (OT) loci that, importantly, were at least 10 kb from a coding sequence. This study provides proof-of-concept for TALEN-mediated in situ correction of an endogenous patient-specific gene mutation and used an unbiased screen for comprehensive TALEN target mapping that will cooperatively facilitate translational application.

Program death-1 signaling and regulatory T cells collaborate to resist the function of adoptively transferred cytotoxic T lymphocytes in advanced acute myeloid leukemia

Tumor-induced immune defects can weaken host immune response and permit tumor cell growth. In a systemic model of murine acute myeloid leukemia (AML), tumor progression resulted in increased regulatory T cells (Treg) and elevation of program death-1 (PD-1) expression on CD8(+) cytotoxic T cells (CTLs) at the tumor site. PD-1 knockout mice were more resistant to AML despite the presence of similar percentage of Tregs compared with wild type. In vitro, intact Treg suppression of CD8(+) T-cell responses was dependent on PD-1 expression by T cells and Tregs and PD-L1 expression by antigen-presenting cells. In vivo, the function of adoptively transferred AML-reactive CTLs was reduced by AML-associated Tregs. Anti-PD-L1 monoclonal antibody treatment increased the proliferation and function of CTLs at tumor sites, reduced AML tumor burden, and resulted in long-term survivors. Treg depletion followed by PD-1/PD-L1 blockade showed superior efficacy for eradication of established AML. These data demonstrated that interaction between PD-1 and PD-L1 can facilitate Treg-induced suppression of T-effector cells and dampen the antitumor immune response. PD-1/PD-L1 blockade coupled with Treg depletion represents an important new approach that can be readily translated into the clinic to improve the therapeutic efficacy of adoptive AML-reactive CTLs in advanced AML disease.

Amelioration of epidermolysis bullosa by transfer of wild-type bone marrow cells

The recessive dystrophic form of epidermolysis bullosa (RDEB) is a disorder of incurable skin fragility and blistering caused by mutations in the type VII collagen gene (Col7a1). The absence of type VII collagen production leads to the loss of adhesion at the basement membrane zone due to the absence of anchoring fibrils, which are composed of type VII collagen. We report that wild-type, congenic bone marrow cells homed to damaged skin, produced type VII collagen protein and anchoring fibrils, ameliorated skin fragility, and reduced lethality in the murine model of RDEB generated by targeted Col7a1 disruption. These data provide the first evidence that a population of marrow cells can correct the basement membrane zone defect found in mice with RDEB and offer a potentially valuable approach for treatment of human RDEB and other extracellular matrix disorders.

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.

Depletion of endogenous tumor-associated regulatory T cells improves the efficacy of adoptive cytotoxic T cell immunotherapy in murine acute myeloid leukemia

Tumor-induced immune suppression can permit tumor cells to escape host immune resistance. To elucidate host factors contributing to the poor response of adoptively transferred tumor-reactive cytotoxic T lymphocytes (CTLs), we used a systemic model of murine acute myeloid leukemia (AML). AML progression resulted in a progressive regulatory T-cell (Treg) accumulation in disease sites. The adoptive transfer of in vitro-generated, potently lytic anti-AML-reactive CTLs failed to reduce disease burden or extend survival. Compared with non-AML-bearing hosts, transferred CTLs had reduced proliferation in AML sites of metastases. Treg depletion by a brief course of interleukin-2 diphtheria toxin (IL-2DT) transiently reduced AML disease burden but did not permit long-term survival. In contrast, IL-2DT prevented anti-AML CTL hypoproliferation, increased the number of transferred CTLs at AML disease sites, reduced AML tumor burden, and resulted in long-term survivors that sustained an anti-AML memory response. These data demonstrated that Tregs present at AML disease sites suppress adoptively transferred CTL proliferation, limiting their in vivo expansion, and Treg depletion before CTL transfer can result in therapeutic efficacy in settings of substantial pre-existing tumor burden in which antitumor reactive CTL infusion alone has proven ineffective.

Patient-Specific Naturally Gene-Reverted Induced Pluripotent Stem Cells in Recessive Dystrophic Epidermolysis Bullosa

Spontaneous reversion of disease-causing mutations has been observed in some genetic disorders. In our clinical observations of severe generalized recessive dystrophic epidermolysis bullosa (RDEB), a currently incurable blistering genodermatosis caused by loss-of-function mutations in COL7A1 that results in a deficit of type VII collagen (C7), we have observed patches of healthy-appearing skin on some individuals. When biopsied, this skin revealed somatic mosaicism resulting from the self-correction of C7 deficiency. We believe this source of cells could represent an opportunity for translational “natural” gene therapy. We show that revertant RDEB keratinocytes expressing functional C7 can be reprogrammed into induced pluripotent stem cells (iPSCs) and that self-corrected RDEB iPSCs can be induced to differentiate into either epidermal or hematopoietic cell populations. Our results give proof in principle that an inexhaustible supply of functional patient-specific revertant cells can be obtained—potentially relevant to local wound therapy and systemic hematopoietic cell transplantation. This technology may also avoid some of the major limitations of other cell therapy strategies, e.g., immune rejection and insertional mutagenesis, which are associated with viral- and nonviral- mediated gene therapy. We believe this approach should be the starting point for autologous cellular therapies using “natural” gene therapy in RDEB and other diseases.

Cardiac Functional and Histopathologic Findings in Humans and Mice with Mucopolysaccharidosis Type I: Implications for Assessment of Therapeutic Interventions in Hurler Syndrome

Hurler syndrome (mucopolysaccharidosis type I [MPS I]) is a uniformly lethal autosomal recessive storage disease caused by absence of the enzyme α-l-iduronidase (IDUA), which is involved in lysosomal degradation of sulfated glycosaminoglycans (GAGs). Cardiomyopathy and valvar insufficiency occur as GAGs accumulate in the myocardium, spongiosa of cardiac valves, and myointima of coronary arteries. Here we report the functional, biochemical, and morphologic cardiac findings in the MPS I mouse. We compare the cardiac functional and histopathological findings in the mouse to human MPS I. In MPS I mice, we have noted aortic insufficiency, increased left ventricular size, and decreased ventricular function. Aortic and mitral valves are thickened and the aortic root is dilated. However, murine MPS I is not identical to human MPS I. Myointimal proliferation of epicardial coronary arteries is unique to human MPS I, whereas dilation of aortic root appears unique to murine MPS I. Despite the differences between murine and human MPS I, the murine model provides reliable in vivo outcome parameters, such as thickened and insufficient aortic valves and depressed cardiac function that can be followed to assess the impact of therapeutic interventions in preclinical studies in Hurler syndrome.

Keratinocytes from Induced Pluripotent Stem Cells in Junctional Epidermolysis Bullosa

Keratinocytes and dermal fibroblasts express adhesive proteins that ensure the epidermis remains attached to the skin basement membrane and to the papillary dermis. Congenital deficiency of any of at least 15 such proteins results in a blistering condition, termed epidermolysis bullosa (EB)(Fine et al., 2008). The most severe form of EB is the Herlitz variant of junctional EB (JEB-H) caused by loss-of-function mutations in one of the three genes (LAMA3, LAMB3, and LAMC2) encoding one of three chains of the heterotrimeric protein laminin 332 (LM-332)(Kiritsi et al., 2011). LM-332 is secreted by keratinocytes and interacts with integrin receptors α3β1 and α6β4 to form focal adhesions and stable anchoring contacts in the dermalepidermal junction (DEJ). Children with this autosomal recessive genodermatosis develop generalized skin blistering; extensive mucosal erosions in the upper respiratory, gastrointestinal, and genitourinary tracts; infections; and, despite supportive measures, typically die within the first year of life. Even though it has been largely accepted that JEB-H is untreatable(Yuen et al., 2012), evidence from a gene therapy trial for the less severe (non-Herlitz) form of JEB(Mavilio et al., 2006), and from LAMB3 gene correction of human JEB-H cells –(Robbins et al., 2001; Sakai et al., 2010), suggests novel treatment options. Prominent among these, the novel technology of reprogramming skin cells into pluripotent stem cells (iPSCs), already applied to EB (Bilousova et al., 2011; Itoh et al., 2011; Tolar et al., 2010) by a combination of specific transcription factors has the dual potential of generating patient-specific, highly proliferative cells for gene-correction strategies and of providing a tool for better understanding the biology of JEB-H. We hypothesized that such iPSCs can be derived from JEB-H individuals. Thus, in principle, an inexhaustible supply of patient-specific stem cells can be generated for local wound therapy and for systemic administration aimed at reaching both skin and internal mucosal membranes. To investigate this, we obtained skin biopsies from two individuals with JEB-H, who carried mutations in the LAMB3 gene: patient 1 P1; c.1365_1366del (p.Asn456ArgfsX7); c.2207C>A (p.Ser736X(Varki et al., 2006)) and patient 2 (P2; c.1903 C>T (p.Arg635X); c.1117C>T (p.Gln373X). Samples were obtained with written informed consent and following a protocol approved by the University of Minnesota Institutional Review Board and with adherence to the Helsinki Guidelines. All mutations create premature stop codons in the open reading frame, with expected nonsense-mediated decay of the mRNA or truncation of the protein product. The children experienced extensive areas of mucocutaneous lesions from birth, hoarseness and stridor, numerous infections, and progressive severe malnutrition. Examination of skin sections revealed the absence of laminin β3 chain at the DEJ. Electron microscopy examination showed infrequent and underdeveloped hemidesmosomes. Collectively these molecular, clinical, biochemical, and ultrastructural features were consistent with the diagnosis of JEB-H. To derive JEB-iPSCs, we transduced skin fibroblasts with the four transcription factors OCT4, SOX2, KLF4, and c-MYC, which are known to induce pluripotency in somatic cells(Tolar et al., 2010). Within three weeks of culture, the patient-specific JEB-iPSCs emerged as raised clusters of cells (Figure 1 a–c). To document the embryonic stem cell-like cellular state, we examined their mRNA and protein expression patterns. When compared with the parental fibroblasts, the JEB-iPSCs expressed the genes coding for nuclear, cytoplasmic, and cell surface proteins (e.g., TRA-1-60, TRA-1-81, stage-specific embryonic antigens 3 and 4, Lin28, Rex1, ABCG2 and DNMT3b, OCT4, and NANOG) in a pattern consistent with embryonic stem cell and iPSC phenotype (Figure 1 d–m). In support of the known activation of endogenous expression of stem cell genes by exogenous reprogramming factors, the maintenance of pluripotency became independent of the original exogenous reprogramming factors (data not shown). As expected in fully reprogrammed iPSCs, the epigenetic profiles showed that endogenous OCT4 and NANOG promoters were largely demethylated (Supplementary Figure 1). The JEB-iPSC lines were maintained for more than 20 passages, and they showed no evidence of genomic instability as evidenced by cytogenetic analysis (Supplementary Figure 2). To exclude the possibilities of cell contamination or the mosaicism observed in JEB(Pasmooij et al., 2007), we verified the authenticity of the JEB-iPSCs by genomic finger-typing with competitive polymerase chain reaction of a variable number of tandem repeat polymorphisms and by sequencing of LAMB3 gene mutations in the JEB-iPSCs (data not shown). To show that JEB-iPSCs are capable of differentiating into cells of endodermal, mesodermal, and ectodermal origin, we injected them into immune-deficient mice lacking T cells, B cells, and natural killer cells, and having a macrophage defect that makes them reliable recipients of human cells. In 6–8 weeks, cystic teratomas formed and cells derived from all three embryonic layers were seen (Figure 1 n). In aggregate, these data show that fully reprogrammed iPSCs can be derived from skin cells of JEB-H individuals. Figure 1 Expression profile of JEB-iPSCs JEB-iPSCs can provide means for drug screening and to model cellular interactions among various mucocutaneous cell types derived from the same individual. To our knowledge, this is a previously unreported use of iPSCs as a cellular tool to study the skin pathology in JEB. We showed first that skin-like structures formed in the process of in vivo JEB-iPSC differentiation (Supplementary Figure 3). In contrast to wild-type iPSCs, the skin-like structures arising from the JEB-iPSCs expressed no detectable laminin β3, but expressed collagen type VII, the DEJ protein deficient in distinct, dystrophic forms of EB (Figure 2 a–d). Next, to substantiate the proof-of-concept that skin cell cultures can be derived from JEB-iPSCs, we differentiated JEB-iPSCs into keratinocytes (Supplementary Figures 4 and 5). Lastly, to demonstrate that this operating procedure can serve as a platform for gene correction of these highly proliferative cells, we transduced the JEB-iPSCs with LAMB3 gene. After transduction, the JEB-iPSC-derived cells expressed and secreted laminin β3 protein (Figure 2 e). Figure 2 Skin cells derived from JEB-iPSCs In summary, we have shown that the LAMB3 defect does not preclude reprogramming into pluripotency, as has been observed in other genetic diseases(Raya et al., 2009). We have also shown that the JEB-iPSCs—in addition to establishing a reliable stem cell source for gene therapy interventions in JEB-H—can be used in the study of early human skin formation and compared to LM-332 in early development. With the ultimate clinical application of iPSC technology in mind, it is worth noting that strategies exist for genome-nonintegrating reprogramming, for depletion of tumor-inducing cells from differentiated iPSC cultures, and—as an JEB-H individual can develop anti-LM-332 antibody—for induction of immunological tolerance to disease-correcting transgenes(Vailly et al., 1998; Wu and Hochedlinger, 2011). Thus, gene-corrected JEB-iPSCs can inform medical advances in this severe and lethal blistering disease, as well as additional extracellular matrix disorders of the skin and other tissues(McGowan and Marinkovich, 2000).