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 transposonrelated 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. STEM CELLS 2007;25:371-379

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.)

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.

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 anti-gen-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. (Blood. 2010;116(14):2484-2493)

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. IntroductionEpidermolysis bullosa represents a family of severe, lifethreatening skin disorders resulting from mutations in genes encoding protein components of the cutaneous basement membrane zone. Although some forms, such as the junctional type, are lethal in the neonatal period, others, such as the dystrophic forms, lead to years of painful skin blistering and mutilating scarring. The most severe form of dystrophic epidermolysis bullosa (the Hallopeau-Siemens type) is caused by recessive mutations in the type VII collagen gene (Col7A1). 1 The recessive dystrophic form of epidermolysis bullosa (RDEB) is characterized by severely diminished type VII collagen (col7) production. 2 The homotrimeric col7 protein is synthesized by fibroblasts and keratinocytes and represents the key component of anchoring fibrils that connect cutaneous basement membrane to the dermal matrix. 3 Severe attenuation of anchoring fibrils in RDEB results in impaired dermal-epidermal cohesion and diminished adhesion of gastrointestinal mucosa at the basement membrane zone. Compromised integrity of the stratifying squamous epithelia leads to increased cutaneous and mucosal sensitivity to mechanical stress and stigmatizing, and to an eventually lethal, clinical phenotype. Children with RDEB develop painful skin and mucosal blistering, mutilating scarring, alopecia, corneal erosions, tooth decay, esophageal strictures, anemia, joint contractures, small epidermal inclusion cysts (milia), nail dystrophy, and fusion of fingers and toes (pseudosyndactyly or "mitten" deformity) by the age of 6 to 8 years. As a result of extreme skin fragility, aberrant tissue repair, and chronic inflammation, RDEB patients develop squamous cell carcinomas in the third decade of life. 4 At this time, there is no therapeutic intervention with proven curative benefit. Palliative measures include complex bandaging of most of the body surface (to protect the skin from the slightest friction, and to prevent infection and excessive loss of body fluid), surgical debridement and analgesia, and nutritional support (using liquid or pureed food by mouth or via percutaneous gastric feeding tube) and analgesia. Faced wit...

IntroductionAcute myeloid leukemia (AML) with unfavorable cytogenetics has a poor outcome, even when treated with aggressive chemotherapy. 1,2 With chemoradiotherapy and hematopoietic stem cell transplantation, a graft-versus-leukemia effect can be observed even in patients with unfavorable cytogenetics. 3 Although donor lymphocyte infusion (DLI) given as adoptive immunotherapy after hematopoietic stem cell transplantation has improved the outcomes of certain types of leukemia, 4-6 for patients with AML, DLI has been less effective likely due at least in part to its rapid tumor progression. 7,8 As a result of the anti-AML effects of DLI and with the observation that antitumor-specific cytotoxic T cells (CTLs) can be generated in vitro from cancer patients, adoptive CTL therapy has been proposed for decades as cancer treatment. [9][10][11][12][13][14] However, the adoptive transfer of anti-AML-reactive CTLs alone has not solved the problem of AML disease recurrence. 15 In patients with chronic myelogenous leukemia, a complete remission was achieved in a patient with accelerated-phase disease after adoptive treatment with leukemia-reactive CTLs. 16 In rodents with minimal disease, CTL adoptive transfer also has not been uniformly curative despite the early transfer of large numbers of anti-AML-reactive CTLs. 17,18 Enhancing CTL function in vivo via the administration of supportive cytokine therapy such as interleukin-2 (IL-2), 20 and interferons 21 can improve antitumor efficacy but has been associated with substantial side effects. Therefore, recent studies have focused on eliminating the suppressive factors in the tumor environment to circumvent CTLs from inhibition. 22,23 T-regulatory cells (Tregs) are important regulators of immune responses in transplantation, 24,25 allergy, 26,27 and autoimmune disease. 28,29 In AML patients, the frequency of Tregs was noted to be significantly higher compared with healthy persons, likely due to increased proliferation. 30 Human AML cells have been noted to favor the conversion of CD4 ϩ 25 Ϫ T cells into Tregs via modulation of tryptophan catabolism. 31 Tregs that have been recruited to, or converted either before migration into or within the tumor environment, can have a profound inhibition on T cell-mediated immune response. 32 Multiple mechanisms have been defined to be responsible for the suppression, including secretion of transforming growth factor-␤ 33,34 and IL-10, 33,34 as well as inhibition of dendritic cell (DC) maturation. 33,35 Despite the great potential for Treg depletion in cancer therapies, the efficacy has been limited to prophylactic settings where depletion of Tregs is given before the establishment of tumor. 36,37 The present studies were undertaken to determine whether endogenous Tregs present at the site of AML dissemination constrained the antileukemia efficacy of anti-AML CTL adoptive transfer in a rodent model as a prelude to future clinical trials. We observed that AML progression correlated with increased Tregs at the sites of AML disease. Incr...

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 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 to both hematopoietic and non-hematopoietic 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 iPS cells. Importantly, human wild-type and RDEB iPS cells differentiated in vivo into structures resembling 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 the skin and mucosal erosions that typify this genodermatosis.

Protein cysteine thiols can be divided into four groups based on their reactivities: those that form permanent structural disulfide bonds, those that coordinate with metals, those that remain in the reduced state, and those that are susceptible to reversible oxidation. Physicochemical parameters of oxidationsusceptible protein thiols were organized into a database named the Balanced Oxidation Susceptible Cysteine Thiol Database (BALOSCTdb). BALOSCTdb contains 161 cysteine thiols that undergo reversible oxidation and 161 cysteine thiols that are not susceptible to oxidation. Each cysteine was represented by a set of 12 parameters, one of which was a label (1/0) to indicate whether its thiol moiety is susceptible to oxidation. A computer program (the C4.5 decision tree classifier re-implemented as the J48 classifier) segregated cysteines into oxidation-susceptible and oxidation-non-susceptible classes. The classifier selected three parameters critical for prediction of thiol oxidation susceptibility: (1) distance to the nearest cysteine sulfur atom, (2) solvent accessibility, and (3) pKa. The classifier was optimized to correctly predict 136 of the 161 cysteine thiols susceptible to oxidation. Leave-one-out cross-validation analysis showed that the percent of correctly classified cysteines was 80.1% and that 16.1% of the oxidation-susceptible cysteine thiols were incorrectly classified. The algorithm developed from these parameters, named the Cysteine Oxidation Prediction Algorithm (COPA), is presented here. COPA prediction of oxidation-susceptible sites can be utilized to locate protein cysteines susceptible to redox-mediated regulation and identify possible enzyme catalytic sites with reactive cysteine thiols.Keywords: cysteine; thiol; oxidation; prediction; C4.5; J48; redox; classifier; decision tree Protein cysteine thiols can be divided into four broad categories: those that form permanent structural disulfide bonds, those that coordinate metals, those that are permanently in the reduced state, and those that are reversibly oxidized. Permanent structural disulfide bonds are formed during the folding process by oxidizing enzymes (for example, DsbA in bacteria and protein disulfide isomerase in eukaryotes) (Kadokura et al. 2003;Maattanen et al. 2006). Permanent structural disulfide bonds are typically observed in oxidizing environments such as extracellular spaces and the endoplasmic reticulum. Protein cysteine thiols can be coordinated to metal ions, typically iron, copper, or zinc. Metal coordinated thiols are found in oxidizing environments and in the cytosolic compartment of the cell. The remaining protein cysteine thiols in the cytosol are either permanently reduced or are susceptible to reversible oxidation (Thomas et al. 1995). The reversibly oxidized protein thiols (ROPTs) in the cytosol are often required for enzyme catalysis or for regulation of protein activity (Finkel 2003;Linke and Jakob 2003).Reprint requests to: Jamil Momand, Department of Chemistry and Biochemistry, California State Univ...