Showing papers by "Hossein Baharvand published in 2010"

Generation of Liver Disease-Specific Induced Pluripotent Stem Cells Along with Efficient Differentiation to Functional Hepatocyte-Like Cells

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.

Feeder- and serum-free establishment and expansion of human induced pluripotent stem cells

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.

Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging.

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.

Midterm outcomes of autologous cultivated limbal stem cell transplantation with or without penetrating keratoplasty.

Abstract: Purpose: To report the midterm outcomes of autologous limbal stem cell transplantation cultivated on amniotic membrane (AM) with or without subsequent penetrating keratoplasty (PKP) in patients with total unilateral limbal stem cell deficiency (LSCD). Methods: Eight eyes of 8 consecutive patients with unilateral total LSCD underwent autologous limbal stem cell transplantation cultivated on AM. Four eyes underwent subsequent optical PKP. Main outcome measures were corneal vascularization and transparency. Results: The patients were followed for 34.0 6 13.5 months (6‐48 months). Seven cases had a stable corneal epithelium with marked decrease in opacification and vascularization. Progressive sectorial conjunctivalization was evident in all cases with subsequent PKP at the last follow-up. Primary failure was observed in one case because of exposure. Conclusions: Transplantation of autologous stem cells cultivated on AM with or without subsequent PKP seems to be an effective way for visual rehabilitation in total LSCD. More work with more cases and longer follow-up are needed to optimize this procedure to provide and maintain an adequate supply of limbal stem cells in these patients.

An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells

Abstract: Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing-thawing process usually takes about 2 h excluding overnight incubation at -80 degrees C.

Progress and promise towards safe induced pluripotent stem cells for therapy.

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.

Enhanced Functions of Human Embryonic Stem Cell-derived Hepatocyte-like Cells on Three-dimensional Nanofibrillar Surfaces

Abstract: Human embryonic stem cell (hESC)-derived hepatocytes provide a promising unlimited resource for the treatment of liver disease. However, current protocols for the generation of mature and functional hepatocytes are inefficient. Therefore, in order to better differentiate and maintain the function of differentiating hESCs, we have hypothesized that hESCs undergo better differentiation into hepatocyte-like cells (HLCs) when induced on three-dimensional nanofibrillar surfaces. We have demonstrated that, during stepwise differentiation of induction, the markers of hepatic lineage expressed and finally lead to the generation of functional mature cells. In the presence of an ultraweb nanofiber, HLCs produced lower AFP, greater urea, glycogen storage, metabolic PROD activity, uptake of LDL and organic anion ICG, all of which are indicative of the differentiation of HLCs. These results show that topographically treated hESCs at the nano level have a distinct hepatic functionality profile which has implications for cell therapies.

In vitro maturation, apoptotic gene expression and incidence of numerical chromosomal abnormalities following cryotop vitrification of sheep cumulus-oocyte complexes

Abstract: Purpose The purpose of this study was to evaluate the effects of cryotop vitrification of sheep cumulus-oocyte complexes (COCs) on oocyte maturation, apoptotic gene expression and incidence of chromosomal abnormalities.

Novel method to obtain highly enriched cultures of adult rat Schwann cells.

Abstract: Schwann cells (SCs) can be used to repair both the peripheral and central nervous systems. Therefore, establishment of a procedure to obtain activated, highly proliferative SCs, in an appropriate time for clinical applications, is a prerequisite. Purification is complicated by contamination with fibroblasts which often become the predominant cell type in an in vitro SC culture. This study describes a novel and efficient method to enrich SCs by utilizing the differential detachment properties of the two cell types. In culture, cells were treated with two different media and the chelator, EGTA, which detached SCs faster than fibroblasts and allowed for easy isolation of SCs. Within seven days, high yields of SCs with a purity of greater than 99% were achieved. This was confirmed by immunostaining characterization and flow-cytometric analyses using an antibody against the p75 low affinity nerve growth factor receptor (p75LNGFR). The entire procedure was completed in approximately 21 days. This method has the advantage of being technically easier, faster, and more efficient than other previously described methods. An SC culture that was about 99% homogenous was achieved.

Intraepidermal injection of dissociated epidermal cell suspension improves vitiligo

Abstract: This study was initiated to evaluate the safety and effectiveness of intraepidermal injection of dissociated epidermal cells into the lesions of stable vitiligo patients. Autologous dissociated epidermal cell suspensions were injected intraepidermally into 10 stable vitiligo patients. None of the patients received adjuvant therapy. The response was evaluated as: marked (76-100%), moderate (51-75%), mild (26-50%) and minimal repigmentation (0-25%). Transmission electron microscopy was used to evaluate the transplanted cells and immunohistochemical staining with HMB-45 was performed to assess the repigmentation in vivo. In all cases, repigmentation started during the 4-week period after transplantation. Six months after transplantation, a marked repigmentation in four (40%), moderate repigmentation in two (20%) and mild repigmentation in two (20%) patients were observed. Two (20%) patients with white patches on their lids showed minimal repigmentation. No side effects were observed in any patients. Interestingly, repigmentation of gray hair in one patient, 4 months post transplantation was observed. Analysis of the ultrastructure of transplanted cells showed 1.5% of the cells had melanocyte morphology. HMB-45 positive cells were observed after cell transplantation. This method is an effective, simple and safe therapeutic option for stable vitiligo lesions.

Effect of poly-L-lysine coating on retinoic acid-loaded PLGA microspheres in the differentiation of carcinoma stem cells into neural cells

Abstract: In this study, PLGA microspheres were prepared using a water-in-oil-in-water emulsion/solvent evaporation technique. Some microspheres were coated with poly-L-lysine (an extracellular matrix (ECM) component), and then pluripotent P19 embryonic carcinoma cells were seeded on them. P19 cells attached onto the PLGA microspheres; subsequently, by adding retinoic acid (RA) to cell culture medium as a neurogenic inducer (RA was released from the microspheres), the cells differentiated into neural cells. Size and morphology of PLGA microspheres was characterized by scanning electron microscopy (SEM). Neurogenic differentiation was studied by immunofluorescent staining, real-time polymerase chain reaction (RT-PCR), and light microscopy. Histological assay showed that more cells attached onto microspheres coated with poly-L-lysine than the uncoated group. Immunofluoresent staining and RT-PCR analysis for s-Tubulin, Nestin and Pax6 genes indicated differentiation of P19 cells into neural cells on both coated and uncoated microspheres. It was found that a high surface area of microspheres improves cell attachment and expansion, which was significantly increased in those coated with poly-L-lysine. Finally, these results highlight the versatility of these sample scaffolds as a model system for nerve tissue engineering.

IVM and gene expression of sheep cumulus–oocyte complexes following different methods of vitrification

Abstract: To determine the optimal vitrification conditions for sheep cumulus-oocyte complexes (COC), good-quality isolated COC were randomly divided into non-vitrified control, conventional straw, cryotop and solid-surface vitrification groups. In the conventional and cryotop methods, the vitrified COC were respectively loaded by conventional straw and cryotop, whereas in the solid-surface group, the vitrified COC were loaded by cryotop and then cooled before plunging in liquid nitrogen. The results indicated that the mean percentage of viability of vitrified-warmed COC was higher in both cryotop groups than that of the conventional group (83.84+/-2.85 and 78.56+/-1.72 versus 63.43+/-1.48%, P<0.05). In the cryotop group, although the mean percentage of oocyte maturation was similar to that in the control group (48.81+/-3.09 versus 51.94+/-3.01%), it was significantly higher than the other vitrification groups (48.81+/-3.09 versus 36.60+/-1.69 and 6.09+/-2.51%, respectively, P<0.05). However, the expression of maturation genes (GDF9, BMP15) was retarded after vitrification. Among the vitrification groups, the cryotop group had better expression. BMPRII was also expressed highly in the control, whereas ALK5 was similar in all groups. In conclusion, direct cryotop, when compared with other vitrification methods, seemed to be safe and could increase the viability, post-warming maturation and maturation-gene expression rates of sheep COC.

Dehydroepiandrosterone Stimulates Neurogenesis in Mouse Embryonal Carcinoma Cell- and Human Embryonic Stem Cell-Derived Neural Progenitors and Induces Dopaminergic Neurons

Abstract: To evaluate the effect of dehydroepiandrosterone (DHEA) as a neurosteroid on the rate of neurogenesis, neural survival, and proliferation of pluripotent stem cell-derived neurons, we have added DHEA to mouse P19 embryonal carcinoma cell- and human embryonic stem cell-derived neural progenitors (ECC- and ESC-NPs). In ECC-derived NPs, flow cytometric analysis of nestin and Tuj1-positive cells revealed that the percentages of these cells increased significantly for the markers following DHEA treatment of the cells. Moreover, the percentages of tyrosine hydroxylase (TH)-positive cells, the marker of dopaminergic neurons, significantly increased in the presence of DHEA. The expression of neural-specific genes such as Mash1, Pax6, Tuj1, and TH was also detected by RT-PCR analysis. BrdU incorporation and estrogen receptor (EsR) were found to be increased after DHEA induction. Moreover, apoptosis was significantly decreased after DHEA treatment. DHEA effect was also confirmed on human ESC-NPs by the enhancement of Tuj1- and TH-immunofluorescent-positive cells and TH and Nurr1 transcripts, as detected by quantitative RT-PCR. In conclusion, these results have presented evidence that DHEA was able to induce neurogenesis in mouse ECC and human ESC-NPs. This observation was related to the division of NPs and the reduction of apoptosis. Moreover, DHEA has dopaminergic potential in the cells of both orders. This provides a better insight into the differentiation and maintenance of neural cells and treatment of a wide variety of neurological diseases such as Alzheimer's and Parkinson's by stem cells.

Derivation of new human embryonic stem cell lines from preimplantation genetic screening and diagnosis-analyzed embryos

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.

Epigenetic analysis of human embryonic carcinoma cells during retinoic acid-induced neural differentiation.

Abstract: Differentiation of stem cells from a pluripotent to a committed state involves global changes in genome expression patterns, critically determined by chromatin structure and interactions of chromat...

Human-induced pluripotent stem cells: derivation, propagation, and freezing in serum- and feeder layer-free culture conditions.

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.

Proteome analysis of brain in murine experimental autoimmune encephalomyelitis.

Abstract: Multiple sclerosis is considered a prototype inflammatory autoimmune disorder of the CNS. Experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein is one of the best-characterized animal models of multiple sclerosis. Comprehensive understanding of gene expression in EAE can help identify genes that are important in drug response and pathogenesis. We applied a 2-DE-based proteomics approach to analyze the protein expression pattern of the brain in healthy and EAE samples. Of more than 1000 protein spots we analyzed, 70 showed reproducible and significant changes in EAE compared to controls. Of these, 42 protein spots could be identified using MALDI TOF-TOF-MS. They included mitochondrial and structural proteins as well as proteins involved in ionic and neurotransmitter release, blood barriers, apoptosis, and signal transduction. The possible role of these proteins in the responses of mice to animal models of multiple sclerosis is discussed.

Assessment of heat shock protein (HSP60, HSP72, HSP90, and HSC70) expression in cultured limbal stem cells following air lifting

Abstract: Objectives The aim of this study is to create an ex vivo model to examine the expression of major heat-shock protein (HSP) families; HSP60, HSP72, and HSP90, and heat-shock cognate 70 (HCS70) at the mRNA and protein level in differentiating corneal cells from limbal stem cells (LSC) following air exposure.

Nuclear Proteome Analysis of Monkey Embryonic Stem Cells During Differentiation

Abstract: The nuclear proteome enables, manages, and regulates the genome by the collective actions and interactions of proteins found in the nucleus. We applied a proteomic approach to analyze a nuclear proteome during embryonic stem cell (ESC) proliferation, and 3 and 9 days after initiation of differentiation. The nuclei were isolated from cells and their proteins were separated using 2-DE. Out of about 560 protein spots reproducible detected on any give gel, 49 differentially expressed proteins were identified by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI TOF/TOF) mass spectrometry. Of them, several nuclear located proteins involved in chromatin remodeling, transcription regulation, apoptosis, cell proliferation, and differentiation were identified including CTBP1, MM-1, RUVBL1, HCC-1, SGTA, SUMO2, and Galectin-1. Functional interaction analysis of differentially expressed proteins revealed that most of nuclear proteins had a direct interaction with c-Myc and p53.

Cell-Based Regenerative Therapy as an Alternative to Liver Transplantation for End-Stage Liver Disease: Experience from Iran

Abstract: Several types of cells including mature hepatocytes, adult liver progenitor cells and human embryonic stem cells, fetal liver progenitor cells, bone marrow derived hematopoietic or mesenchymal stem cells, and umbilical cord blood cells—both in rodents and humans—have been reported to be capable of self-replication, giving rise to daughter hepatocytes, both in vivo and in vitro. They have been shown to be able to repopulate liver in both animal models of liver injury and in patients with liver disease and to improve liver function. Human embryonic stem cell therapy seems to be a great promise for the treatment of liver cirrhosis, but there is no human clinical application due to ethical concerns or difficulties in harvesting or safely and efficiently expanding sufficient quantities. In contrast, adult bone marrow-derived hematopoietic or mesenchymal stem cells, which can be easily and safely harvested, have been used in clinical trials to treat several chronic diseases including chronic liver disease. Cell therapy offers exciting promise for future treatment of cirrhosis and metabolic liver diseases, but significant technical hurdles remain that will only be overcome through years of intensive research. There is also serious concern about the long-term safety of stem cell therapy and the possibility of tumor development. Herein, we present our experience with cell therapy in treatment of chronic liver disease in Iran.

Sorting analysis of mouse peroxisomal protein by in vitro studies

Abstract: The recently cloned peroxisomal matrix protein (PeP), contains two hydrophobic domains at 12-31 and 152169 amino acid residues and a fibronectin type III (FnIII) domain between residues 31-114. To understand the importance of the above mentioned domains in peroxisome sorting of PeP, site-directed mutagenesis was performed to delete these domains separately. Amplified mutants of PEP cDNA were constructed downstream of enhanced green fluorescent protein (EGFP) cDNA for transfection into the chinese hamster ovary (CHO) K1 and P19 cell lines. Upon their transfections, numerous green fluorescent punctuate structures, superimposable on those punctuates stained with the anti-catalase antibody, appeared in the cells. Thus, the aforementioned domains do not exert the targeting signal activity for the peroxisomal sorting of PeP.