Publications (12)37.12 Total impact
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Article: Disease-Corrected Hepatocyte-Like Cells from Familial Hypercholesterolemia-Induced Pluripotent Stem Cells.
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ABSTRACT: The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.Molecular Biotechnology 12/2012; · 2.17 Impact Factor -
Article: A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells.
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ABSTRACT: We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum- and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these disease-specific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases.Stem cells and development 12/2011; 21(12):2262-72. · 4.15 Impact Factor -
Article: Long-term maintenance of undifferentiated human embryonic and induced pluripotent stem cells in suspension.
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ABSTRACT: Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.Stem cells and development 01/2011; 20(11):1911-23. · 4.15 Impact Factor -
Article: Generation of liver disease-specific induced pluripotent stem cells along with efficient differentiation to functional hepatocyte-like cells.
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ABSTRACT: The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka's factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.Stem cell reviews 12/2010; 6(4):622-32. · 5.08 Impact Factor -
Article: Human cardiomyocytes with long-QT syndrome in dish.
Archives of Iranian medicine 11/2010; 13(6):573-5. · 0.97 Impact Factor -
Article: Derivation of new human embryonic stem cell lines from preimplantation genetic screening and diagnosis-analyzed embryos.
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ABSTRACT: In this study, we focused on the derivation of human embryonic stem cell (hESC) from preimplantation genetic screening (PGS)-analyzed and preimplantation genetic diagnosis (PGD)-analyzed embryos. Out of 62 fresh PGD/PGS-analyzed embryos, 22 embryos reached the blastocyst stage. From 12 outgrowth blastocysts, we derived four hESC lines onto a feeder layer. Surprisingly, karyotype analysis showed that hESC lines derived from aneuploid embryos had diploid female karyotype. One hESC line was found to carry a balanced Robertsonian translocation. All the cell lines showed hESC markers and had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. The established lines had clonal propagation with 22-31% efficiency in the presence of ROCK inhibitor. These results further indicate that hESC lines can be derived from PGD/PGS-analyzed embryos that are destined to be discarded and can serve as an alternative source for normal euploid lines.In Vitro Cellular & Developmental Biology - Animal 02/2010; 46(3-4):395-402. · 1.31 Impact Factor -
Article: Progress and promise towards safe induced pluripotent stem cells for therapy.
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ABSTRACT: The recent generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an invaluable resource for drug or toxicology screening, medical research, and patient-specific cell therapy. However, there are currently a number of obstacles including virus integration and the genetic alteration of iPSCs that will need to be overcome before these cells may be considered safe for clinical applications. Here, we highlight the potential and challenges of iPSC research and review advances in reprogramming methods that have rapidly moved the field closer to realizing the goal of generating safe iPSCs for transplantation.Stem cell reviews 02/2010; 6(2):297-306. · 5.08 Impact Factor -
Article: Human-induced pluripotent stem cells: derivation, propagation, and freezing in serum- and feeder layer-free culture conditions.
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ABSTRACT: The recent discovery of genomic reprogramming of human somatic cells to an embryonic stem (ES) cell-like pluripotent state provides a unique opportunity for stem cell research. The reprogrammed cells, named as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells and represent one of the most promising sources of patient-specific cells for use in disease model, development of pharmacology and toxicology, screening teratogens, and regenerative medicine. Here we describe the detailed methods for the generation of undifferentiated human iPS (hiPS) cells in feeder layer- and serum-free conditions. This system eliminates direct contact of stem cells with MEFs and reduces use of unknown serum factors that may have undesired activities and enables consistency in large-scale and long-term expansion of undifferentiated hiPS cells. Our findings greatly simplify the method for induction of pluripotency and bring it one step closer to clinical applications. Moreover, the established hiPS cells showed chromosomal stability during long-term culture.Methods in molecular biology (Clifton, N.J.) 01/2010; 584:425-43. -
Article: Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging.
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ABSTRACT: Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 microM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (alphaV, alpha6, and beta1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells.Stem cell reviews 12/2009; 6(1):96-107. · 5.08 Impact Factor -
Article: Generation of human induced pluripotent stem cells from a Bombay individual: moving towards "universal-donor" red blood cells.
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ABSTRACT: Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, alpha-globulin, and gamma-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.Biochemical and Biophysical Research Communications 11/2009; 391(1):329-34. · 2.48 Impact Factor -
Article: Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells.
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ABSTRACT: Although human induced pluripotent stem cells (hiPSCs) hold great promise as a source of differentiated cells for vast therapeutic implications, many obstacles still need to be surmounted before this can become a reality. One obstacle, a robust feeder- and serum-free system to generate and expand hiPSCs in culture is still unavailable. Here, for the first time, we describe a novel establishment and maintenance culture technique that uses human dermal fibroblasts to generate hiPSCs by introducing four factors, Klf4, Oct4, Sox2, and c-Myc under serum- and feeder-independent conditions. We have used a serum replacement product, conditioned medium (CM), or feeder-free medium (FFM) supplemented with high elevated basic-fibroblast growth factor in the absence or presence of Matrigel. Our FFM system in the presence of Matrigel enhanced the efficiency of alkaline phosphatase-positive colonies at a frequency at least 10-fold greater than the conventional method on feeder cells. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, ultrastructure, and in vitro differentiation. Such hiPSCs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues have been overcome.The International journal of developmental biology 10/2009; 54(5):877-86. · 2.16 Impact Factor -
Article: A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells.
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ABSTRACT: An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation. We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632. Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.Human Reproduction 08/2009; 24(10):2468-76. · 4.47 Impact Factor
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Institutions
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2011
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Academic Center for Education, Culture and Research
Tehrān, Ostan-e Tehran, Iran
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