Ali Farrokhi

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.

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.

Inhibition of TGFβ Signaling Promotes Ground State Pluripotency

Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFβ signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. Global transcriptome analysis of the ES cells highlights augmented BMP4 signaling pathway. The crucial role of the BMP4 pathway in maintaining the R2i ground state pluripotency is demonstrated by BMP4 receptor suppression, resulting in differentiation and cell death. In conclusion, by inhibiting TGFβ and FGF signaling pathways, we introduce a novel defined approach to efficiently establish the ground state pluripotency.

Simultaneous Suppression of TGF-β and ERK Signaling Contributes to the Highly Efficient and Reproducible Generation of Mouse Embryonic Stem Cells from Previously Considered Refractory and Non-permissive Strains

Mouse embryonic stem cells (ESCs) are pluripotent stem cell lines derived from pre-implantation embryos. The efficiency of mESC generation is affected by genetic variation in mice; that is, some mouse strains are refractory or non-permissive to ESC establishment. Developing an efficient method to derive mESCs from strains of various genetic backgrounds should be valuable for establishment of ESCs in various mammalian species. In the present study, we identified dual inhibition of TGF-β and ERK1/2, by SB431542 and PD0325901, respectively led to the highly efficient and reproducible generation of mESC lines from NMRI, C57BL/6, BALB/c, DBA/2, and FVB/N strains, which previously considered refractory or non-permissive for ESC establishment. These mESCs expressed pluripotency markers and retained the capacity to differentiate into derivatives of all three germ layers. The evaluated lines exhibited high rates of chimerism when reintroduced into blastocysts. To our knowledge, this is the first report of efficient (100%) mESC lines generation from different genetic backgrounds. The application of these two inhibitors will not only solve the problems of mESC derivation but also clarifies new signaling pathways in pluripotent mESCs.

Expression of maturation genes and their receptors during in vitro maturation of sheep COCs in the presence and absence of somatic cells of cumulus origin.

The purpose of this study was to investigate the effects of somatic cells of cumulus origin (sCC) on gene expression and maturation of cumulus oocyte complexes (COCs) in vitro. Good quality (i.e., healthy-looking) isolated sheep COCs were randomly divided into two treatment groups: control (COC with no sCC) and coculture (COC with sCC). Nuclear maturation statuses of oocytes were assessed after 27 hours of in vitro culture. Moreover, the expression levels of growth differentiation factor 9 (GDF9), bone morphogenetic protein (BMP)15, BMP6, bone morphogenetic protein receptor II (BMPRII), activin like kinase 5 (ALK5) (transforming growth factor β receptor 1: TGFβR1), ALK6 (BMPR1b), activin A receptor, type IIB (ActRIIB), and ALK3 (BMPR1a), as well as hyaluronan synthase 2 (HAS2) and prostaglandin endoperoxide synthase 2 (Ptgs2) in the COCs were assessed in both treatment groups after 3 h and 27 h of culture. The results showed that the proportion of metaphase II (MII) stage oocytes was significantly higher in the coculture group compared with the controls (77.21%±1.17 vs. 67.49%±1.80; P<0.05). The relative expressions of BMPRII, ALK6, and ActRIIB in control group and GDF9 and ActRIIB in coculture group showed significant differences during culture as assessed by real time polymerase chain reaction (P<0.05). The mean expression levels of BMPRII, ALK5, ALK6, and ActRIIB mRNA were decreased in the coculture group compared with those in the control group after 27 h of culture (P<0.05). In conclusion, we propose that in vitro maturation of sheep COCs alone disrupted the normal gene expression levels of both TGFβ ligands and receptors, and also reduced the maturation rate. Coculture with sCC enhanced the maturation rate of oocytes concomitantly with reduced gene expression levels of a number of TGFβ ligands and receptors.

Differential effect of activin on mouse embryonic stem cell differentiation in insulin‐secreting cells under nestin‐positive selection and spontaneous differentiation protocols

Parallel to the importance of the development of cell therapies to treat diabetes is the production of sufficient numbers of pancreatic endocrine cells that function like primary islets. To increase the efficiency of endocrine pancreatic‐like cell differentiation from mouse embryonic stem cells (ESCs), we applied activin‐B to nestin‐positive selection (protocol 1) and spontaneous differentiation (protocol 2) in different groups including: [A] activin‐B, or [B] basic fibroblast growth factor (bFGF), and/or [C] activin‐B + bFGF. The differentiated cells expressed most pancreatic‐related genes. The number of insulin‐ and C peptide‐positive cells, as well as dithizone‐positive clusters in group A of protocol 1 was higher than in the other groups. Significant insulin concentrations in protocol 1 were produced when glucose was added to the medium, in comparison with protocol 2. Moreover, insulin release was increased significantly in group A of protocol 1 even with lower glucose. In conclusion, Addition of activin‐B in a nestin‐positive selection protocol increased the insulin‐secreting cells in comparison with the same protocol with bFGF and/or spontaneous differentiation in presence of bFGF and/or activin‐B alone. However, improvements of the current method are required to generate a sufficient source of true beta‐cells for the treatment of diabetes mellitus.

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

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.

Effect of ovarian tissue vitrification method on mice preantral follicular development and gene expression

Vitrification is considered a viable method for cryopreservation of ovarian tissue and selection of methods that minimize follicular damage is important. The objective of the present study was to evaluate the effects of two vitrification methods on ovarian tissue morphology, preantral follicles survival rate during in vitro culture, and relative expression of genes associated with oocyte maturation and cumulus expansion. Ovaries from 12-day-old mice were vitrified in media containing ethylene glycol, dimethyl sulphoxide, and sucrose. Before plunging in liquid nitrogen, ovaries were first loaded into an acupuncture needle (needle immersion vitrification [NIV]) or placed on a cold steel surface for 10 to 20 seconds (solid surface vitrification [SSV]). The integrity of the ovarian tissue was well-preserved after vitrification and was similar controls. Follicle viability in the SSV group was lower (P < 0.05) than in the control group after 6 days of culture and the NIV group after 10 day of culture. Follicle viability after 12 day of culture was 92.8%, 82.1%, and 58.4% in control, NIV, and SSV groups, respectively. Bmp15, Gdf9, BmprII, Alk6, Alk5, Has2, and Ptgs2 gene expression patterns were similar among groups. However, the level of gene expression in the vitrification groups during Days 6 to 10 were higher compared with the control group. In conclusion, ovarian tissue morphologic integrity was well-preserved, regardless of the vitrification method. Vitrification using the needle immersion method resulted in greater follicular survival after 12 day of culture than the SSV method. Gene expression patterns during culture did not seem to explain the reduced survival rate observed in the solid surface group.

Safety and efficacy of granulocyte-colony-stimulating factor administration following autologous intramuscular implantation of bone marrow mononuclear cells: a randomized controlled trial in patients with advanced lower limb ischemia.

BACKGROUND AIMS The aim was to investigate the therapeutic effect of granulocyte-colony-stimulating factor (G-CSF) administration following implantation of autologous bone marrow mononuclear cells (BM MNC) for patients with lower limb ischemia. METHODS The design was a randomized controlled trial. Fifteen patients with severe chronic limb ischemia were treated with autologous BM MNC [without G-CSF (MNC-G-CSF) or combined with G-CSF administration for 5 days following transplantation (MNC+G-CSF)]. RESULTS All clinical parameters, including ankle brachial index, visual analog scale and pain-free walking distance, showed a mean improvement from baseline, which was measured at 4 and 24 weeks after transplantation in both groups. However, in three (20%) patients, the clinical course did not improve and limb salvage was not achieved. No significant difference was observed among the patients treated in the MNC-G-CSF and MNC+G-CSF groups. No severe adverse reactions were reported during the study period. No relationship was observed between both the numbers of viable MNC or CD34+ cells and the clinical outcome. CONCLUSIONS Autologous transplantation of BM MNC into ischemic lower limbs is safe, feasible and efficient for patients with severe peripheral artery disease. However, the administration of G-CSF following cell transplantation does not improve the effect of BM MNC implantation and therefore would not have any beneficial value in clinical applications of such cases.

Inhibition of glycogen synthase kinase-3 promotes efficient derivation of pluripotent stem cells from neonatal mouse testis

BACKGROUND Several studies have demonstrated the derivation of multi- or pluripotent stem cells from testicular cells of both newborn and adult mice by a spontaneous conversion process, when these cells are cultured in vitro for an extended time. To obtain a better and robust derivation, we have attempted to identify small molecules (SMs) that induce reprogramming of testicular cells in culture into germline-derived pluripotent stem cells (gPSCs). METHODS We tested several SMs based on previous reports that have shown enhancement of establishment of induced pluripotent stem cells or embryonic stem cells (ESCs) on mouse NMRI (outbred strain) and C57BL/6 (inbred strain) testicular cells. After appearance of ESC-like colonies at Day 6, they were passaged on mitotically arrested mouse embryonic fibroblasts in mouse ESC medium in the absence or presence of SMs up to Day 14. The generated cells were characterized using a variety of experimental approaches. RESULTS The application of several SMs involved in pluripotent reprogramming led to the discovery that CHIR99021 (CHIR), a glycogen synthase kinase-3 (GSK-3) inhibitor, promotes efficient derivation of gPSCs from neonatal mouse NMRI and C57BL/6 testes. The pluripotency of the generated cell lines has been confirmed by in vitro spontaneous and direct differentiation toward cardiac and neural lineages, and formation of chimeras after injection of gPSCs into blastocysts. We have shown that the generated gPSCs could be maintained and expanded under chemically defined serum and feeder-free conditions by inhibition of both the extracellular signal-regulated kinases (Erk1/2) and GSK-3. CONCLUSIONS To our knowledge, this is the first report of a simple and efficient protocol to reprogram gPSCs from testicular cells solely by inhibition of GSK-3 in two strains of mice with different genetic backgrounds. Additionally, this brings us closer to eliminating the need for genetic modification in pluripotent reprogramming. Future studies will determine whether the inhibition of GSK-3 could affect the generation of naïve gPSCs lines in other mammals.