Hossein Baharvand

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

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.

Generation of new human embryonic stem cell lines with diploid and triploid karyotypes

Human pluripotent embryonic stem cells (hESC) have great promise for research into human developmental biology and the development of cell therapies for the treatment of diseases. To meet the increased demand for characterized hESC lines, we present the derivation and characterization of five hESC lines on mouse embryonic fibroblast cells. Our stem cell lines are characterized by morphology, long‐term expansion, and expression profiles of a number of specific markers, including TRA‐1‐60, TRA‐1‐81, alkaline phosphatase, connexin 43, OCT‐4, NANOG, CXCR4, NODAL, LEFTY2, THY‐1, TDGF1, PAX6, FOXD3, SOX2, EPHA2, FGF4, TAL1, AC133 and REX‐1. The pluripotency of the cell line was confirmed by spontaneous differentiation under in vitro conditions. Whereas all of the cell lines expressed all the characteristics of undifferentiated pluripotent hESC, two of the cell lines carried a triploid karyotype.

Differentiation of Bone Marrow-derived Mesenchymal Stem Cells into Hepatocyte-like Cells on Nanofibers and Their Transplantation into a Carbon Tetrachloride-Induced Liver Fibrosis Model

There are limited data available on the effect of a physicochemical microenvironment on mesenchymal stem cell (MSC) differentiation and repopulation of the liver. Therefore, in this study nanofibers have been used to better differentiate and maintain the function and engraftment of differentiating MSCs both in vitro and in vivo. Mouse MSCs were differentiated into early (day 18) and late (day 36) hepatocyte-like cells (HLCs) in the presence or absence of ultraweb nanofibers (nano+ and nano−) and their transplantation for recovery in mice with CCl4 induced hepatic fibrosis was investigated. In the nano+ group, hepatocyte markers-ALB and HNF4α- were elevated in a time-dependent manner; however, those were similar levels or slightly decreased in the nano− group from day 18 to 36. Ultrastructural studies of the differentiated cells revealed some similarities to hepatocytes. Urea production, secretion of albumin and α-fetoprotein, and metabolic activity of the CYP450 enzymes were significantly increased within in vitro differentiated HLCs on nanofibers at day 36. MSCs, early and late HLCs in both nano− and nano+ culture conditions that were transplanted by an intravenous route caused a decrease in liver fibrosis when engrafted in the recipient liver and were able to differentiate into functional hepatocytes (ALB+), except for late HLCs in the nano− group. Late HLCs transplanted in the nano+ group were more effective in rescuing liver failure, enhancing serum ALB, homing transplanted cells and undergoing functional engraftment than the other groups. These results showed that topographic properties of nanofibers enhance differentiation of HLCs from MSCs and maintain their function in long-term culture, which has implications for cell therapies.

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

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.

A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells

BACKGROUND 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. METHODS 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. RESULTS 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. CONCLUSIONS 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 embryonic stem cell-derived neural precursor transplants in collagen scaffolds promote recovery in injured rat spinal cord

BACKGROUND AIMS Several studies have reported functional improvement after transplantation of in vivo-derived neural progenitor cells (NPC) into injured spinal cord. However, the potential of human embryonic stem cell-derived NPC (hESC-NPC) as a tool for cell replacement of spinal cord injury (SCI) should be considered. METHODS We report on the generation of NPC as neural-like tubes in adherent and feeder-free hESC using a defined media supplemented with growth factors, and their transplantation in collagen scaffolds in adult rats subjected to midline lateral hemisection SCI. RESULTS hESC-NPC were highly expressed molecular features of NPC such as Nestin, Sox1 and Pax6. Furthermore, these cells exhibited the multipotential characteristic of differentiating into neurons and glials in vitro. Implantation of xenografted hESC-NPC into the spinal cord with collagen scaffold improved the recovery of hindlimb locomotor function and sensory responses in an adult rat model of SCI. Analysis of transplanted cells showed migration toward the spinal cord and both neural and glial differentiation in vivo. CONCLUSIONS These findings show that transplantation of hESC-NPC in collagen scaffolds into an injured spinal cord may provide a new approach to SCI.

Culture Condition Difference for Establishment of New Embryonic Stem Cell Lines from the C57BL/6 and BALB/c Mouse Strains

Abstract Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocysts. These cells are appropriate for creation of animal models of human genetic diseases, the study of gene function in vivo and differentiation into specific types as potential therapeutic agents for several human diseases. We describe here, the production of new ES cell lines from blastocysts recovered from the C57BL/6 and BALB/c mouse strains by changing the concentration of leukemia inhibitory factor (LIF) and primary culture conditions. The established cell lines were analyzed by simple karyotype, C banding, alkaline phosphatase activity, and Oct-4 expression as well as for the presence of the SRY gene. Two ES cell lines from C57BL/6 and three from the BALB/c were produced. The two C57BL/6 ES cell lines were established with either 1000 or 5000 IU LIF, whereas the BALB/c ES cell lines required 5000 IU LIF. Four of the ES cell lines had a normal karyotype. C banding and sex-determining region of Y chromosome–polymerase chain reaction showed that all cell lines had an XY sex chromosome composition. All five of the cell lines expressed alkaline phosphatase activity and Oct-4. One of the BALB/c ES cell lines, when injected into C57BL/6 blastocysts, produced high rates of chimerism as assessed by coat color, and the male chimera produced germ-line offspring when mated with BALB/c females. These results indicate that ES cells from inbred strains can be isolated using commercially available reagents and that the establishment of BALB/c ES cell lines may require different culture conditions to the 129 or C57BL/6 strains.SummaryHuman urokinase-type plasminogen activator (uPA) is a serine protease that converts plasminogen to plasmin. It is produced and secreted by a variety of different human cells in vivo and in vitro. We have studied human diploid kidney cell (HKC) cultures prepared from neonatal kidney tissue and cultures of purified populations of HKC to determine which cells synthesize and secrete uPA into the culture medium. Antibodies against cell specific antigens and uPA were used to correlate specific kidney cell types with uPA synthesis. In addition, secretion of uPA activity into growth and uPA production media was determined for each cell type and cultures containing a mixture of cell types. The results of these studies demonstrated that glomerular visceral epithelial and kidney tubular epithelial cells synthesize and secrete uPA into the culture medium.

Safety Analysis and Improved Cardiac Function Following Local Autologous Transplantation of CD133 + Enriched Bone Marrow Cells After Myocardial Infarction

The CD133(+) bone marrow cell (BMC) population includes primitive multipotent stem cells which induce neoangiogenesis. Studies suggested transplantation of these cells to infarcted myocardium can have a favorable impact on tissue perfusion and contractile performance. We assessed the feasibility, safety and functional outcomes of autologus CD133(+) BMC transplantation during coronary artery bypass grafting (CABG) in patients with recent myocardial infarction. In a prospective, nonrandomized, open-label study, 27 patients with recent myocardial infarction underwent CABG and intramyocardial injection of autologous bone marrow-derived CD133(+) cells (18 patients, BMC group) or CABG alone (9 patients, control group). At 6 months after CABG, the Wall Motion Score Index (WMSI) was significantly reduced for akinetic/dyskinetic segments treated with CD133(+) cells compared with the control group (P<0.006). Likewise, comparison between baseline and follow up results of dobutamine stress echocardiography and myocardial perfusion scintigraphy showed improvement of myocardial viability and local perfusion of the infarcted zone of the BMC group compared with the control group. No complications related to CD133(+) cell transplantation were noted, either procedurally or during postoperative at a mean of 14 months follow up. In patients with recent myocardial infarction, transplantation of CD133(+) cells to the peri-infarct zone during CABG surgery is feasible and safe, with no evidence of early or late adverse events. Moreover, these cells might restore tissue viability and improve perfusion of the infarcted myocardium, suggesting that they may induce myogenesis as well as angiogenesis.

Induced in vitro differentiation of neural-like cells from human exfoliated deciduous teeth-derived stem cells

Stem cells from human exfoliated deciduous teeth (SHED) are highly proliferative, clonogenic and multipotent stem cells with a neural crest cell origin. Additionally, they can be collected with minimal invasiveness in comparison with other sources of mesenchymal stem cells (MSCs). Therefore, SHED could be a desirable option for potential therapeutic applications. In this study, SHEDs were established from enzyme-disaggregated deciduous dental pulp obtained from 6 to 9 year-old children. The cells had typical fibroblastoid morphology and expressed antigens characteristic of MSCs, STRO1, CD146, CD45, CD90, CD106 and CD166, but not the hematopoietic and endothelial markers, CD34 and CD31, as assessed by FACS analysis. Differentiation assessment revealed a strong osteogenic and adipogenic potential of SHEDs. In order to further evaluate the in vitro differentiation potential of SHED into neural cells, a simple short time growth factor-mediated induction was used. Immunofluorescence staining and flow cytometric analysis revealed that SHED rapidly expressed nestin and b-III tubulin, and later expressed intermediate neural markers. In addition, the intensity and percentages of nestin and b-III tubulin and mature neural markers (PSA-NCAM, NeuN, Tau, TH, or GFAP) increased significantly following treatment. Moreover, RT-PCR and Western blot analyses showed that the neural markers were strongly up-regulated after induction. In conclusion, these results provide evidence that SHED can differentiate into neural cells by the expression of a comprehensive set of genes and proteins that define neural-like cells in vitro. SHED cells might be considered as new candidates for the autologous transplantation of a wide variety of neurological diseases and neurotraumatic injuries.

Transplantation of primed or unprimed mouse embryonic stem cell-derived neural precursor cells improves cognitive function in Alzheimerian rats.

Alzheimers disease (AD) is a neurodegenerative disorder that is characterized by progressive and irreversible decline of memory. Neuropathological features include the progressive degeneration of cholinergic neurons in the forebrain cholinergic projection system especially nucleus basalis of Meynert (nbM). New cell therapeutic approaches for the replacement of degenerated cells are being researched. The aim of this study was to investigate the production of cholinergic neurons from mouse embryonic stem cells (ESCs) and potential for utilizing ESC-derived neuronal precursor cells (NPCs) and primed NPCs (PNPCs) for cell restorative therapy in a rodent model of AD. NPCs were produced by growth factor-mediated selection under serum-free conditions and differentiated better into cholinergic neurons when NPCs primed with Shh (approximately 22%) in comparison with different cholinergic promoting factors. Behavioral assessment of unilateral nbM ibotenic acid-lesioned rats by Morris water maze and spatial probe test revealed a significant behavioral improvement in memory deficits following transplantation with NPCs and/or PNPCs. Immunohistochemical analysis revealed that the majority (approximately 70%) of the NPCs and/or PNPCs retained neuronal phenotype and approximately 40% of them had a cholinergic cell phenotype following transplantation with no tumor formation, indicating that these may be safe for transplantation. This experimental study has important implications as it suggests that the transplantation of mouse ESC-derived NPCs and/or following commitment to a cholinergic cell phenotype can promote behavioral recovery in a rodent model of AD.