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PURPOSE: The presence of multipotent human limbal stromal cells resembling mesenchymal stromal cells (MSC) provides new insights to the characteristic of these cells and its therapeutic potential. However,little is known about the expression of stage-specific embryonic antigen 4 (SSEA-4) and the embryonic stem cell (ESC)-like properties of these cells. We studied the expression of SSEA-4 surface protein and the various ESC and MSC markers in the ex vivo cultured limbal stromal cells. The phenotypes and multipotent differentiation potential of these cells were also evaluated.backslashnbackslashnMETHODS: Limbal stromal cells were derived from corneoscleral rims. The SSEA-4(+) and SSEA-4(-) limbal stromal cells were sorted by fluorescence-activated cells sorting (FACS). Isolated cells were expanded and reanalyzed for their expression of SSEA-4. Expression of MSC and ESC markers on these cells were also analyzed by FACS. In addition,expression of limbal epithelial and corneal stromal proteins such as ATP-binding cassette sub-family G member 2 (ABCG2),tumour protein p63 (p63),paired box 6 (Pax6),cytokeratin 3 (AE5),cytokeratin 10,and keratocan sulfate were evaluated either by immunofluorecence staining or reverse transcription polymerase chain reaction. Appropriate induction medium was used to differentiate these cells into adipocytes,osteocytes,and chondrocytes.backslashnbackslashnRESULTS: Expanded limbal stromal cells expressed the majority of mesenchymal markers. These cells were negative for ABCG2,p63,Pax6,AE-5,and keratocan sulfate. After passaged,a subpopulation of these cells showed low expression of SSEA-4 but were negative for other important ESC surface markers such as Tra-1-60,Tra-1-81,and transcription factors like octamer-binding transcription factor 4 (Oct4),SRY(sex determining region Y)-box 2 (Sox2),and Nanog. Early passaged cells when induced were able to differentiate into adipocytes,osteocytes and chondrocytes.backslashnbackslashnCONCLUSIONS: The expanded limbal stromal cells showed features of multipotent MSC. Our study confirmed the expression of SSEA-4 by a subpopulation of cultured limbal stromal cells. However,despite the expression of SSEA-4,these cells did not express any other markers of ESC. Therefore,we conclude that the cells did not show properties of ESC. View Publication

Immunosuppressants are powerful drugs,capable of triggering severe adverse effects. Hence,there is tremendous interest in replacing them with less-toxic agents. Adoptive therapy with CD25(+)CD4(+) T regulatory cells (Tregs) holds promise as an alternative to immunosuppressants. Tregs have been described as the most potent immunosuppressive cells in the human body. In a number of experimental models,they have been found to quench autoimmune diseases,maintain allogeneic transplants,and prevent allergic diseases. A major stumbling block in their clinical application is related to Treg phenotype and the very limited number of these cells in the periphery,not exceeding 1-5% of total CD4(+) T cells. Recent progress in multicolor flow cytometry and cell sorting as well as cellular immunology has found ways of overcoming these obstacles,and has opened the doors to the clinical application of Tregs. In the review,we describe Treg sorting and expansion techniques that have been developed in recent years. In the experience of our laboratory,as well as some published reports,Treg adoptive therapy is a promising tool in immunosuppressive therapy,and should be considered for clinical trials. View Publication

Adenosine deaminase (ADA) deficiency is caused by a purine metabolic dysfunction,leading to severe combined immunodeficiency (SCID) and multiple organ damage. To investigate the efficacy of ex vivo gene therapy with self-inactivating lentiviral vectors (LVs) in correcting this complex phenotype,we used an ADA(-/-) mouse model characterized by early postnatal lethality. LV-mediated ADA gene transfer into bone marrow cells combined with low-dose irradiation rescued mice from lethality and restored their growth,as did transplantation of wild-type bone marrow. Mixed chimerism with multilineage engraftment of transduced cells was detected in the long term in animals that underwent transplantation. ADA activity was normalized in lymphocytes and partially corrected in red blood cells (RBCs),resulting in full metabolic detoxification and prevention of severe pulmonary insufficiency. Moreover,gene therapy restored normal lymphoid differentiation and immune functions,including antigen-specific antibody production. Similar degrees of detoxification and immune reconstitution were obtained in mice treated early after birth or after 1 month of enzyme-replacement therapy,mimicking 2 potential applications for ADA-SCID. Overall,this study demonstrates the efficacy of LV gene transfer in correcting both the immunological and metabolic phenotypes of ADA-SCID and supports the future clinical use of this approach. View Publication

Because ex vivo rapamycin generates murine Th2 cells that prevent Graft-versus-host disease more potently than control Th2 cells,we hypothesized that rapamycin would generate Th2/Tc2 cells (Th2/Tc2.R cells) that abrogate fully MHC-disparate hemopoietic stem cell rejection more effectively than control Th2/Tc2 cells. In a B6-into-BALB/c graft rejection model,donor Th2/Tc2.R cells were indeed enriched in their capacity to prevent rejection; importantly,highly purified CD4+ Th2.R cells were also highly efficacious for preventing rejection. Rapamycin-generated Th2/Tc2 cells were less likely to die after adoptive transfer,accumulated in vivo at advanced proliferative cycles,and were present in 10-fold higher numbers than control Th2/Tc2 cells. Th2.R cells had a multifaceted,apoptosis-resistant phenotype,including: 1) reduced apoptosis after staurosporine addition,serum starvation,or CD3/CD28 costimulation; 2) reduced activation of caspases 3 and 9; and 3) increased anti-apoptotic Bcl-xL expression and reduced proapoptotic Bim and Bid expression. Using host-versus-graft reactivity as an immune correlate of graft rejection,we found that the in vivo efficacy of Th2/Tc2.R cells 1) did not require Th2/Tc2.R cell expression of IL-4,IL-10,perforin,or Fas ligand; 2) could not be reversed by IL-2,IL-7,or IL-15 posttransplant therapy; and 3) was intact after therapy with Th2.R cells relatively devoid of Foxp3 expression. We conclude that ex vivo rapamycin generates Th2 cells that are resistant to apoptosis,persist in vivo,and effectively prevent rejection by a mechanism that may be distinct from previously described graft-facilitating T cells. View Publication

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling,we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor,p21Cip1/Waf1 (p21),have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling,we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control,manipulated cells. Further,we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore,lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion. View Publication

The generation of patient-specific induced pluripotent stem (iPS) cells provides an invaluable resource for cell therapy,in vitro modeling of human disease,and drug screening. To date,most human iPS cells have been generated with integrating retro- and lenti-viruses and are limited in their potential utility because residual transgene expression may alter their differentiation potential or induce malignant transformation. Alternatively,transgene-free methods using adenovirus and protein transduction are limited by low efficiency. This unit describes a protocol for the generation of transgene-free human induced pluripotent stem cells using retroviral transfection of a single vector,which includes the coding sequences of human OCT4,SOX2,KLF4,and cMYC linked with picornaviral 2A plasmids. Moreover,after reprogramming has been achieved,this cassette can be removed using mRNA transfection of Cre recombinase. The method described herein to excise reprogramming factors with ease and efficiency facilitates the experimental generation and use of transgene-free human iPS cells. View Publication

Integrating viruses represent robust tools for cellular reprogramming; however,the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here,we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage,we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple,rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling,tissue engineering and cell replacement therapies. View Publication

BACKGROUND Recently,accumulating evidence has shown that exosomes,the naturally secreted nanocarriers of cells,can exert therapeutic effects in various disease models in the absence of parent cells. However,application of exosomes in bone defect repair and regeneration has been rarely reported,and little is known regarding their underlying mechanisms. METHODS Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects,and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation,migration,and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting,scratch assays,and qRT-PCR,respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair. RESULTS We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition,the internalization of exosomes into hBMSCs could profoundly enhance the proliferation,migration,and osteogenic differentiation of hBMSCs. Furthermore,gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs. CONCLUSIONS We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs,which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These results indicate that naturally secreted nanocarriers-exosomes can be used as a bioactive material to improve the bioactivity of the biomaterials,and that hiPS-MSC-Exos combined with β-TCP scaffolds can be potentially used for repairing bone defects. View Publication

Umbilical cord blood (UCB) is increasingly being used for human hematopoietic stem cell (HSC) transplantation in children but often requires pooling multiple cords to obtain sufficient numbers for transplantation in adults. To overcome this limitation,we have used an ex vivo two-week culture system to expand the number of hematopoietic CD34(+) cells in cord blood. To assess the in vivo function of these expanded CD34(+) cells,cultured human UCB containing 1 x 10(6) CD34(+) cells were transplanted into conditioned NOD-scid IL2rgamma(null) mice. The expanded CD34(+) cells displayed short- and long-term repopulating cell activity. The cultured human cells differentiated into myeloid,B-lymphoid,and erythroid lineages,but not T lymphocytes. Administration of human recombinant TNFalpha to recipient mice immediately prior to transplantation promoted human thymocyte and T-cell development. These T cells proliferated vigorously in response to TCR cross-linking by anti-CD3 antibody. Engrafted TNFalpha-treated mice generated antibodies in response to T-dependent and T-independent immunization,which was enhanced when mice were co-treated with the B cell cytokine BLyS. Ex vivo expanded CD34(+) human UCB cells have the capacity to generate multiple hematopoietic lineages and a functional human immune system upon transplantation into TNFalpha-treated NOD-scid IL2rgamma(null) mice. View Publication

Recent reports have documented the differentiation of human pluripotent stem cells toward the skeletal myogenic lineage using transgene- and cell purification-free approaches. Although these protocols generate myocytes,they have not demonstrated scalability,safety,and in vivo engraftment,which are key aspects for their future clinical application. Here we recapitulate one prominent protocol,and show that it gives rise to a heterogeneous cell population containing myocytes and other cell types. Upon transplantation,the majority of human donor cells could not contribute to myofiber formation. As a proof-of-principle,we incorporated the inducible PAX7 lentiviral system into this protocol,which then enabled scalable expansion of a homogeneous population of skeletal myogenic progenitors capable of forming myofibers in vivo. Our findings demonstrate the methods for scalable expansion of PAX7(+) myogenic progenitors and their purification are critical for practical application to cell replacement treatment of muscle degenerative diseases. View Publication