Directed differentiation of regulatory T cells from naive T cells and prevention of their inflammation-mediated instability using small molecules.

Regulatory T (Treg ) cell therapy is a promising approach for immune tolerance induction in autoimmunity conditions and cell/organ transplantations. Insufficient isolation yields and impurity during downstream processes and Treg instability after adoptive transfer in inflammatory conditions are major limitations to Treg therapy, and indicate the importance of seeking a valid, reliable method for de-novo generation of Tregs . In this research, we evaluated Treg -like cells obtained from different Treg differentiation protocols in terms of their yield, purity and activity. Differentiation was performed on naive CD4+ cells and a naive CD4+ /Treg co-culture by using three different protocols - ectopic expression of forkhead box protein P3 (E-FoxP3), soluble transforming growth factor ß (S-TGF) and small molecules [N-acetyl puromycin and SR1555 (N-Ac/SR)]. The results showed that a high yield of a homogeneous population of Treg -like cells could be achieved by the N-Ac/SR method under a T helper type 17 (Th17)-polarizing condition, particularly interleukin (IL)-6 and TGF-ß, when compared with the E-FoxP3 and S-TGF methods. Surprisingly, SR completely inhibited the differentiation of IL-17-producing cells and facilitated Treg generation in the inflammatory condition and had highly suppressive activity against T cell proliferation without Treg -specific demethylase region (TSDR) demethylation. For the first time, to our knowledge, we report the generation of efficient, pure Treg -like cells by using small molecules during in-vitro inflammatory conditions. Our results suggested that the N-Ac/SR method has several advantages for Treg generation when compared with the other methods, including a higher purity of Tregs , easier procedure, superior suppressive activity during the inflammatory condition and decreased cost.

Systematic selection of small molecules to promote differentiation of embryonic stem cells and experimental validation for generating cardiomyocytes.

Small molecules are being increasingly used for inducing the targeted differentiation of stem cells to different cell types. However, until now no systematic method for selecting suitable small molecules for this purpose has been presented. In this work, we propose an integrated and general bioinformatics- and cheminformatics-based approach for selecting small molecules which direct cellular differentiation in the desired way. The approach was successfully experimentally validated for differentiating stem cells into cardiomyocytes. All predicted compounds enhanced expression of cardiac progenitor (Gata4, Nkx2-5 and Mef2c) and mature cardiac markers (Actc1, myh6) significantly during and post-cardiac progenitor formation. The best-performing compound, Famotidine, increased the percentage of Myh6-positive cells from 33 to 56%, and enhanced the expression of Nkx2.5 and Tnnt2 cardiac progenitor and cardiac markers in protein level. The approach employed in the study is applicable to all other stem cell differentiation settings where gene expression data are available.

Enhanced remyelination following lysolecithin-induced demyelination in mice under treatment with fingolimod (FTY720).

Multiple sclerosis (MS) is a chronic, progressive demyelinating disorder which affects the central nervous system (CNS) and is recognized as the major cause of nervous system disability in young adults. Enhancing myelin repair by stimulating endogenous progenitors is a main goal in efforts for MS treatment. Fingolimod (FTY720) which is administrated as an oral medicine for relapsing-remitting MS has direct effects on neural cells. In this study, we hypothesized if daily treatment with FTY720 enhances endogenous myelin repair in a model of local demyelination induced by lysolecithin (LPC). We examined the response of inflammatory cells as well as resident OPCs and evaluated the number of newly produced myelinating cells in animals which were under daily treatment with FTY720. FTY720 at doses 0.3 and 1mg/kg decreased the inflammation score at the site of LPC injection and decreased the extent of demyelination. FTY720 especially at the lower dose increased the number of remyelinated axons and newly produced myelinating cells. These data indicate that repetitive treatment with FTY720, behind an anti-inflammatory effect, exerts beneficial effects on the process of endogenous repair of demyelinating insults.

ERK1/2 is a key regulator of Fndc5 and PGC1α expression during neural differentiation of mESCs.

Fibronectin type III domain containing 5 (Fndc5) has already been distinguished to be involved in neural differentiation. However, cellular events of Fndc5 function are still ambiguous in the nervous system. One approach to shed light on duty of this protein in the nervous system is to find its cross-talks with various signaling pathways with defined characteristics and roles. Identification of the underlying molecular mechanism which controls Fndc5 expression and switches its activity up and down enables us to find out the Fndc5 functional map in the nervous system and other human body systems. Retinoic acid (RA) is a bio-small molecule which exerts its role as a neural inducer in the neurodevelopmental process of neural tube. RA up-regulates the expression of various genes involved in neural differentiation process via two distinct pathways: the genomic and the non-genomic. Our previous study has revealed that RA induces Fndc5 expression during neural differentiation process. In this study we have evaluated our hypothesis about the non-genomic up regulation of Fndc5 expression by RA. Interestingly we have identified that there is an association between ERK signaling pathway and Fndc5 expression. Furthermore, inhibition of this pathway by PD0325901 dramatically reduced Fndc5 mRNA level, while activating the pathway up-regulated Fndc5 transcription. In addition, it has been proven that ERK1/2 modulation via RA has more significant controlling effect on Fndc5 promoter rather than bFGF. This led us to conclude that RA enhances Fndc5 expression through a non-genomic pathway via the ERK signaling pathway.

Long-term and efficient expression of human ß-globin gene in a hematopoietic cell line using a new site-specific integrating non-viral system.

Targeted integration of a therapeutic gene at specific loci in safe genomic regions by a non-viral vector can restore the function of the damaged gene. This approach also minimizes the potential genotoxic effects of transferred DNA. In this study, we have developed a non-viral vector that functions according to site-specific recombination (SSR). The vector contained a bacterial backbone and puromycin resistance gene (pur(r)), a ß-globin expressing cassette and an attB recombination site. We used phiC31 integrase to insert a copy of the vector into specific genomic locations of a human hematopoietic cell line. Site-specific integration of the vector with one or two copies in the transcriptionally active regions of the genome was confirmed. After genomic integration, we used Cre recombinase to remove the bacterial backbone and pur(r). This removal was verified by negative selection and genomic PCR screening. Following deletion of these sequences, the stable ß-chain expression was continued for several months in the absence of selective pressure. Consequently, this vector may potentially be a powerful tool for ex vivo correction of ß-globinopathies such as ß-thalassemia through successful genomic integration of a functional copy of the globin gene into the patient's target cells.

Inducible VEGF expression by human embryonic stem cell-derived mesenchymal stromal cells reduces the minimal islet mass required to reverse diabetes.

Islet transplantation has been hampered by loss of function due to poor revascularization. We hypothesize that co-transplantation of islets with human embryonic stem cell-derived mesenchymal stromal cells that conditionally overexpress VEGF (hESC-MSC:VEGF) may augment islet revascularization and reduce the minimal islet mass required to reverse diabetes in mice. HESC-MSCs were transduced by recombinant lentiviruses that allowed conditional (Dox-regulated) overexpression of VEGF. HESC-MSC: VEGF were characterized by tube formation assay. After co-transplantation of hESC-MSC:VEGF with murine islets in collagen-fibrin hydrogel in the omental pouch of diabetic nude mice, we measured blood glucose, body weight, glucose tolerance and serum C-peptide. As control, islets were transplanted alone or with non-transduced hESC-MSCs. Next, we compared functional parameters of 400 islets alone versus 200 islets co-transplanted with hESC-MSC:VEGF. As control, 200 islets were transplanted alone. Metabolic function of islets transplanted with hESC-MSC:VEGF significantly improved, accompanied by superior graft revascularization, compared with control groups. Transplantation of 200 islets with hESC-MSC:VEGF showed superior function over 400 islets alone. We conclude that co-transplantation of islets with VEGF-expressing hESC-MSCs allowed for at least a 50% reduction in minimal islet mass required to reverse diabetes in mice. This approach may contribute to alleviate the need for multiple donor organs per patient.

Fndc5 knockdown significantly decreased neural differentiation rate of mouse embryonic stem cells.

Fibronectin type III domain-containing 5 protein (Fndc5) or peroxisomal protein, is a type I membrane protein that has 209 amino acid residues. Previous studies by our group have shown an increase in its expression after retinoic acid treatment of mouse embryonic stem cells (mESCs) during the process of neural differentiation, leading us to conclude that it might be involved in neurogenesis. In the present study, we have constructed an inducible short hairpin RNA (shRNA) vector that is expressed under induction by doxycycline. Next, we generated a stably transformed mESCs line that expressed shRNA against the Fndc5 gene. The knockdown of Fndc5 was performed in two stages of mESC neural differentiation during and post-neural progenitor (NP) formation. Our results indicated that in the process of NPs formation, decreased Fndc5 expression significantly reduced expression of NPs and mature neuronal markers which modulated neuronal differentiation. Decreased Fndc5 expression during the post-NPs formation stage also caused significant reduction in the levels of mature neuronal markers. Fndc5 knockdown during both stages significantly affected both neuronal and astrocytes maturation. We have concluded that Fndc5 expression is required for the appropriate neural differentiation of mESCs. These data confirm the importance of Fndc5 in the generation and development of the nervous system.

Functional expression of potassium channels in cardiomyocytes derived from embryonic stem cells.

Royan B(1) stem cell can be differentiated to specialized cell types including cardiomyocytes. This developmental change is accompanied with expression of various K(+) channel types. The aim of this study was to detect functional expression of K(+) currents from stem cell stage and one week and two weeks after differentiation into cardiomyocyte. Mouse stem cell derived cardiomyocytes (ES-cardiomyocytes) were isolated to single cell suspension for K(+) current recording using whole cell patch-clamp technique. The predominant depolarizing current in ES-cardiomyocytes was a tetraethylammonium (TEA) (10 mM) sensitive current which was partially blocked by nifedipine (1 µM) and attenuated by increasing concentration of EGTA (10 mM) in the pipette solution. Pharmacology and electrophysiological properties of this oscillatory sustained current very well matched with characteristics of Ca(2+) activated K(+) current. In addition there was another kind of sustained outward K(+) current which was resistance to TEA but was inhibited by 3,4-diaminopyridine. The characteristic features of this current indicate that this current was due to activation of delayed rectifier K(+) channels. RT-PCR study also confirmed expression of these two types of K(+) channels in ES-cardiomyocytes. Therefore, present study shows functional expression of two types of K(+) ionic current in ES-cardiomyocytes.

Effects of different feeder layers on short-term culture of prepubertal bovine testicular germ cells in-vitro.

Spermatogonial stem cells (SSCs) are exceptional adult stem cells that transfer genes to new generations. This behavior makes them unique cells for the production of transgenic farm animals. However, this goal has been hampered by their spontaneous differentiation during in vitro culture. Therefore, the objective of this study was the evaluation of the effects of different feeders on in vitro short-term culture of prepubertal bovine testicular germ cells. The isolated cell suspensions containing SSCs were enriched by Bovine serum albumin (BSA) and gelatin and were cultured in the presence of Glial-derived neurotrophic factor (GDNF), Epidermal Growth Factor (EGF) and basic Fibroblastic Growth Factor (bFGF). After 7 d of culture, colonies were harvested and cultured on four different feeders, including SIM mouse embryo-derived thioguanine and ouabain resistant (STO), mouse embryonic fibroblast, bovine Sertoli cells (BSC) and on a laminin-coated plate. The number and area of colonies were measured at seven, 11 and 14 d post-culture. The expression of germ cells markers was detected using immunofluorescence and flow cytometry analyses on day 7, and quantitative real-time PCR at 14 d post-culture. Immunocytochemical staining revealed that colonies were positive for Dolichos biflorus agglutinin (DBA), Thy-1, Oct-4, c-ret, α6-integrin, ß1-integrin and negative for c-kit. In addition, the number and area of those colonies formed on the STO feeder were significantly greater than the other groups. Relative expressions of Thy-1 in the STO and in BSC groups were significantly higher than other groups but expression of Oct-4 was highest in the laminin group compared to other groups. In conclusion, STO might be a suitable feeder layer for in vitro propagation of bovine testicular germ cells.

Short-term in-vitro culture of goat enriched spermatogonial stem cells using different serum concentrations.

PURPOSE: To investigate the effect of serum supplementing on short-term culture, fate determination and gene expression of goat spermatogonial stem cells (SSCs). METHODS: Crude testicular cells were plated over Datura-Stramonium Agglutinin (DSA) for 1 h, and non-adhering cells were cultured in the presence of different serum concentrations (1, 5, 10, and 15%) for 7 days in a highly enriched medium initially developed in mice. Colonies developed in each group were used for the assessment of morphology, immunocytochemistry, and gene expression. RESULTS: Brief incubation of testicular cells with DSA resulted in a significant increase in the number of cells that expressed the germ cell marker (VASA). The expression of THY1, a specific marker of undifferentiated spermatogonia, was significantly higher in colonies developed in the presence of 1% rather than 5, 10 and 15% serum. CONCLUSION: Goat SSCs could proliferate and maintain in SSC culture media for 1 week at serum concentrations as low as 1%, while higher concentrations had detrimental effects on SSC culture/expansion.

Cell-based regenerative therapy as an alternative to liver transplantation for end-stage liver disease: experience from iran.

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.

Separation of X- and Y-bearing human spermatozoa by sperm isolation medium gradients evaluated by FISH.

An eight-layer discontinuous sperm isolation medium (PureSperm gradient) was evaluated in separation of human spermatozoa according to sex chromosomes by fluorescence in-situ hybridization (FISH). This study was carried out on spare samples from normozoospermic and oligozoospermic patients referred to the Royan Institute for infertility treatment. Semen analysis was assessed according to World Health Organization criteria. X- and Y-bearing spermatozoa were simultaneously identified in the neat semen (control) and sperm isolation medium fractions from the same samples using FISH and chromosome specific DNA probes. At least 1000 spermatozoa were scored for each sample. The proportions of X- and Y-bearing spermatozoa were determined by presence of red or green fluorescent signals. Before separation, there was no significant difference in the percentage of spermatozoa with the specific signals of X and Y chromosomes. After separation, in both normal and oligozoospermic patients, the percentage of X-bearing spermatozoa in the bottom layer slightly exceeded that in the top layer (P = 0.001). In both the normal and oligozoospermic groups, the difference between the frequencies of Y-bearing spermatozoa in the top and bottom layers was significant (P = 0.001). It seemed that eight-step discontinuous gradient was not a reliable method for the separation X- and Y-bearing spermatozoa for clinical purposes.

Assessment of potassium current in Royan B(1) stem cell derived cardiomyocytes by patch-clamp technique.

Embryonic stem cells are capable of differentiating to variety of cell tissues including cardiomyocytes. This developmental change is accompanied with a great deal of ion channel expression and functions. Mouse stem cell derived cardiomyocytes were prepared and separated to yield isolated single cell suspension for cell current recording. In the present study some properties of the K(+)-current in Royan B(1) stem cell derived cardiomyocytes were investigated using whole cell patch-clamp technique. When the holding potential was - 60 mV, in some cells a major outward current was elicited by square depolarizing pulses from -60 mV to +50 mV. This outward current was sustained for the duration of 300 ms test pulse. The sustained outward K(+) current was inhibited by tetraethylammonium (10 mM) indicating the activity of Ca(2+) activated K(+) channel in these cells. In some of the cells with 0.2 mM 3,ethylene glycol-bis (ß-aminoethyl ether) N,N,N(`),N(`)-tetraacetic acid in the pipette, only a very small outward current was recorded which suggests that in these cells the voltage activated K(+) channels is either absent or if existed it is not fully functional. Other cells were in far between, indicating that voltage activated K(+) channels are developing in these cells but it is not yet fully functional. In conclusion, we have identified functional large conductance Ca(2+) activated K(+) channel in Royan B(1) stem cell derived cardiomyocytes.

Embryonic stem cell sphere: a controlled method for production of mouse embryonic stem cell aggregates for differentiation.

OBJECTIVES: Embryonic stem cells (ESCs) are of significant interest as a renewable source of nonproliferating cells. Differentiation of ESCs is initiated by the formation of embryoid bodies (EBs). Standard methods of EB formation are limited in their production capacity, in any variations in EB size and formation of EBs through frequent passages. Here we have reported the utility of a microencapsulation technique for overcoming these limitations by mass production of mouse ESCs in alginate beads called ESC spheres. METHODS: The mouse ESCs were encapsulated in 1.2% alginate solution and cocultured on a feeder layer. The cells were evaluated by flow cytometry, in vitro differentiation, immunofluorescence, and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Analysis of encapsulated ESC spheres by flow cytometry showed similar percentages of Oct-4 and stage-specific embryonic antigen-1 (SSEA-1) expression in comparison with routine culture of ESCs. Moreover, the ESC spheres maintained a pluripotency potential which was comparable with ESCs cultured on feeder cells directly, as demonstrated by immunofluorescence and RT-PCR. CONCLUSIONS: The results demonstrated that alginate encapsulation as a simple bioreactor, provides a scalable system for mass undifferentiated ESC sphere production with similar sizes and without the need for frequent passages for differentiation and clinical and pharmaceutical applications.