Mouse Embryonic Fibroblast Feeder Research Articles

A rapid and stable spontaneous reprogramming system of Spermatogonial stem cells to Pluripotent State

BackgroundThe scarcity of pluripotent stem cells poses a major challenge to the clinical application, given ethical and biosafety considerations. While germline stem cells commit to gamete differentiation throughout life, studies demonstrated the spontaneous acquisition of pluripotency by spermatogonial stem cells (SSCs) from neonatal testes at a low frequency (1 in 1.5 × 107). Notably, this process occurs without exogenous oncogenes or chemical supplementation. However, while knockout of the p53 gene accelerates the transformation of SSCs, it also increases risk and hampers their clinical use.ResultsWe report a transformation system that efficiently and stably convert SSCs into pluripotent stem cells around 10 passages with the morphology similar to that of epiblast stem cells, which convert to embryonic stem (ES) cell-like colonies after change with ES medium. Epidermal growth factor (EGF), leukemia inhibitory factor (LIF) and fresh mouse embryonic fibroblast feeder (MEF) are essential for transformation, and addition of 2i (CHIR99021 and PD0325901) further enhanced the pluripotency. Transcriptome analysis revealed that EGF activated the RAS signaling pathway and inhibited p38 to initiate transformation, and synergically cooperated with LIF to promote the transformation.ConclusionThis system established an efficient and safe resource of pluripotent cells from autologous germline, and provide new avenues for regenerative medicine and animal cloning.

Transplantation of Induced Pluripotent Stem Cells (iPSCs) into Cuprizone Model of Demyelination in Rat

Introduction: Multiple Sclerosis (MS) is a neurodegenerative inflammatory disease with a wide range of axonal demyelination and sensory-motor disorders. Today, stem cell therapy is an appropriate choice for MS management. Induced Pluripotent Stem Cells (iPSC) are pluripotent stem cells that have neuroprotective effects with neuroglial differentiation potential. In this study, we used iPSCs to improve remyelination and behavioral function in model of cuprizone multiple sclerosis disease in rats. Method: Human iPSCs were cultured and extended on a mouse embryonic fibroblast feeder cell layer inactivated with mitomycin-C in DMEM/F12 supplemented with 20% knockout serum replacement, 0.1 mmol/L nonessential amino acids, and 10 ng/mL of recombinant human basic fibroblast growth factor in a 5% CO2 and 95% humidity. The demyelination model was induced using a 0.2 oral Cuprizone regime. Thereafter, iPSCs were transplanted after six weeks. Remyelination was investigated via histological assessments and immunocytochemistry following six-week post-transplantation. The functionality was evaluated using behavioral tests, BBB, and Footprint following six-week post-transplantation. Results: The results of in vivo studies showed that after specific myelin tissue staining, the cuprizone diet led to the induction of demyelinated areas in the brain tissue six weeks after the cuprizone diet. Differentiation of transplanted cells was confirmed via the immunocytochemistry technique of PLP. The results of the Footprint test showed that the motor function of the paws of the animals improved compared to the control group. Moreover, BBB behavioral tests showed improved symptoms in the experimental groups that received the cells. Conclusion: In total, iPSCs can improve remyelination and functional recovery following transplantation into the Cuprizone demyelination model in rats. Therefore, iPSC therapy could improve behavioral function in multiple sclerosis disease

Generation of Mouse Primitive Endoderm Stem Cells.

The blastocysts consist of dozens of cells of three distinct lineages: epiblast (Epi), trophoblast (TB), and primitive endoderm (PrE). All embryonic and extraembryonic tissues are derived from Epi, TB, and PrE. Stem cell lines representing preimplantation Epi and TB have been established and are known as embryonic stem cells (ESCs) and trophoblast stem cells (TSCs). Extraembryonic endoderm cells (XENCs) constitute a cell line that has been established from PrE. Although in vivo, PrE gives rise to visceral endoderm (VE), parietal endoderm (PE), and marginal zone endoderm (MZE); XENCs, on blastocyst injection into chimeras, primarily contribute to the distal region of PE. Here, we provide a comprehensive protocol for the establishment of fully potent primitive endoderm stem cell (PrESC) lines. PrESCs are established and maintained on mouse embryonic fibroblast (MEF) feeder cells in a serum-free medium supplemented with fibroblast growth factor 4 (FGF4), heparin, CHIR99021, and platelet-derived growth factor-AA (PDGF-AA). PrESCs co-express markers indicative of pluripotency and endoderm lineage commitment, exhibiting characteristics akin to those of PrE. On transplantation of PrESCs into blastocysts, they demonstrate a high efficiency in contributing to VE, PE, and MZE. PrESCs serve as a valuable model for studying PrE, sharing similarities in gene expression profiles and differentiation potential. PrESCs constitute a pivotal cornerstone for in vitro analysis of early developmental mechanisms and for studies of embryo reconstitution in vitro, particularly in conjunction with ESCs and TSCs. Key features • Establishment and maintenance of primitive endoderm stem cell (PrESCs) capable of recapitulating the developmental prowess inherent to PrE. • Offering a source of PrE lineage for embryo-like organoid reconstitution studies.

Naïve-like conversion of bovine induced pluripotent stem cells from Sertoli cells

Feeder cells are essential to derive pluripotent stem cells (PSCs). Mouse embryonic fibroblasts (MEF) are widely used as feeder to generate and culture embryonic stem cells (ESCs) and induced PSCs (iPSCs) in many species. However it may not be suitable for livestock ESCs/iPSCs due to interspecies difference. Previously we derived bovine iPSCs from bovine Sertoli cells using MEF feeder. Here we compared the effects of MEF feeder and bovine embryonic fibroblasts (BEF) feeder on the maintenance of bovine iPSC pluripotency and morphology as well their contributions to the naïve-like conversion, based on a naïve medium (NM). The results showed successful conversion of the primed bovine iPSCs to naïve-like state within 3–4 days both on MEF feeder and BEF feeder in NM (termed as MNM and BNM respectively). These naïve-like iPSCs showed normal karyotype. There were more iPSC colonies under BNM condition than MNM condition. Epigenetically, histone modification H3K4 was upregulated, while H3K27 was downregulated in the naïve-like iPSCs. We further analyzed the naïve markers and differentiation potential both in vitro and in vivo of these cells, which were all reserved throughout the maintenance. Together, bovine naïve-like iPSCs can be generated both on MEF and BEF feeder in NM condition. The BNM condition is able to sustain the pluripotency and differentiation potential of the naïve-like bovine iPSCs, and improve the conversion efficiency.

TGFβ superfamily signaling regulates the state of human stem cell pluripotency and capacity to create well-structured telencephalic organoids.

SummaryTelencephalic organoids generated from human pluripotent stem cells (hPSCs) are a promising system for studying the distinct features of the developing human brain and the underlying causes of many neurological disorders. While organoid technology is steadily advancing, many challenges remain, including potential batch-to-batch and cell-line-to-cell-line variability, and structural inconsistency. Here, we demonstrate that a major contributor to cortical organoid quality is the way hPSCs are maintained prior to differentiation. Optimal results were achieved using particular fibroblast-feeder-supported hPSCs rather than feeder-independent cells, differences that were reflected in their transcriptomic states at the outset. Feeder-supported hPSCs displayed activation of diverse transforming growth factor β (TGFβ) superfamily signaling pathways and increased expression of genes connected to naive pluripotency. We further identified combinations of TGFβ-related growth factors that are necessary and together sufficient to impart broad telencephalic organoid competency to feeder-free hPSCs and enhance the formation of well-structured brain tissues suitable for disease modeling.

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves­tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 μm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments.

Simplification of culture conditions and feeder-free expansion of bovine embryonic stem cells

Bovine embryonic stem cells (bESCs) extend the lifespan of the transient pluripotent bovine inner cell mass in vitro. After years of research, derivation of stable bESCs was only recently reported. Although successful, bESC culture relies on complex culture conditions that require a custom-made base medium and mouse embryonic fibroblasts (MEF) feeders, limiting the widespread use of bESCs. We report here simplified bESC culture conditions based on replacing custom base medium with a commercially available alternative and eliminating the need for MEF feeders by using a chemically-defined substrate. bESC lines were cultured and derived using a base medium consisting of N2B27 supplements and 1% BSA (NBFR-bESCs). Newly derived bESC lines were easy to establish, simple to propagate and stable after long-term culture. These cells expressed pluripotency markers and actively proliferated for more than 35 passages while maintaining normal karyotype and the ability to differentiate into derivatives of all three germ lineages in embryoid bodies and teratomas. In addition, NBFR-bESCs grew for multiple passages in a feeder-free culture system based on vitronectin and Activin A medium supplementation while maintaining pluripotency. Simplified conditions will facilitate the use of bESCs for gene editing applications and pluripotency and lineage commitment studies.

Differentiation of chemically induced liver progenitor cells to cholangiocytes: Investigation of the optimal conditions

Chemically induced liver progenitor (CLiP) cells, converted invitro from mature hepatocytes, possess the bipotentiality to differentiate into both hepatocytes and cholangiocytes. Here, we aimed to investigate the optimal conditions for bile duct (BD) induction from rat CLiPs. A two-step induction protocol was used for the differentiation of cholangiocytes. We investigated the effects of passage number, preincubation times, Matrigel, and mouse embryonic fibroblast (MEF) feeder cells on the induction of cholangiocytes. Earlier passages of CLiPs were better for BD induction compared with stable CLiPs. Extending the preincubation time of CLiPs before induction delayed the formation of the BD. Matrigel provided cells with space to form three-dimensional (3D) structures, but the long-term use of Matrigel from the induction step did not benefit the differentiation of CLiPs to cholangiocytes. MEF feeder cells, through the Jag/Notch pathway, affected BD formation and function, as well as gene and protein expression. CLiPs were a good cell source for cholangiocyte differentiation under appropriate conditions and may offer a key vehicle for the study of cholangiopathies invitro.

231 Novel protocol for the invitro maintenance and expansion of adult epidermal LGR5+ stem cells

Currently, the standard for treatment of full-thickness skin wounds is skin grafts or bioengineered skin substitutes; however, this method is limited by the amount of intact donor skin and lack of follicles and architecture. Thus, a protocol is needed for the expansion and differentiation of adult epidermal and hair follicle stem cells for use in scaffold mediated tissue engineering. Recently, we developed a transgenic porcine model in which H2B-GFP is under the control of the LGR5 promoter. LGR5 is an established marker of stem cells, meaning this model can be used to track the development and behaviour of these cells. The focus of this project was to create a novel culture method for the maintenance and expansion of LGR5+ epidermal adult stem cells utilising the green fluorescent protein (GFP) tag. Single cell epidermal stem cells were isolated from porcine skin using dispase II (10mgmL−1; Sigma) and trypsin (0.05%; Corning). Porcine fetal fibroblasts (PFF) or mouse embryonic fibroblasts (MEF) were grown to 95% confluence in a 6-well plate. Feeder layer cells were mitotically inactivated by incubation with mitomycin C (Sigma Aldrich, 10μgmL−1). Three different media were tested: basal medium [Dulbecco's modified Eagle's medium (DMEM), penicillin/streptomycin, Corning; Ham's F12, ThermoFisher; fetal bovine serum, Gemini Bio-Products], basal media + 5-azacytidine (Sigma Aldrich) and CHIR99021 (Tocris), or basal media + keratinocyte growth supplements (transferrin, hydrocortisone, T3, adenine, insulin, cholera toxin; Sigma Aldrich, epidermal growth factor; R&D Systems). Epidermal cells were plated in each medium for both PFF and MEF feeder layers. Experiments were performed in technical duplicates and replicated 3 times. On Day 9, total numbers of colonies in each well were counted and number of GFP-positive cells were quantified using ImageJ (National Institutes of Health). Results in Table 1 show that overall, the MEF feeder layer was able to support a higher rate of growth (P<0.05) and maintain the LGR5+ lineage at a higher proportion under all of the experimental conditions (P<0.05). In the growth-supplemented media, MEFs had fewer colonies than PFFs, but MEF colonies were, on average, 2.5 times larger (P<0.05). Conditions containing 5-aza and CHIR were the only conditions to maintain the LGR5+ lineage on the feeder layer. Statistically significant differences (P<0.05) were determined using two-way ANOVA, followed by Tukey's HSD test. Next, LGR5+ cells will be plated in media containing additional growth factors to stimulate expansion, while using CHIR and 5-aza to maintain the LGR5+ lineage. This protocol could be used in scaffolds to create three-dimensional growth of skin invitro and lead to better grafts for burn victims. Table 1.Growth of LGR5+ cells in different media including 5-azacytidine (5-aza), CHIR 99021 (CHIR), and keratinocyte growth supplements Group1 Basal medium (BM) BM + 5-aza+ CHIR BM + growth supplements No. of colonies/well MEF 127.7±40.8AB 189.3±16.9A 87.3±14.6B PFF 65.0±14.1A 83.3±17.0AB 148±33.7B Average no. of GFP+ cells per frame MEF 0.5±0.8B 65.7±18.4A 1.8±1.7B PFF 0.9±1.0B 22.6±4.5A 0.3±0.6B A,BValues within rows with different superscripts differ (P ≤ 0.05). 1MEF=mouse embryonic fibroblasts; PFF=porcine fetal fibroblasts.

Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

Abstract 209: iPSC Derived Cardiomyocytes Reproduce Divergent Phenotypes Caused by a LQTS Type-1 Likely Pathogenic Mutation

The purpose of this work was to investigate if iPSC derived cardiomyocytes would reproduce the divergent phenotypes observed in a proband and mother that carried the same likely-pathogenic variant in KCNQ1 gene. A 15 years old proband had syncope and cardiac arrest while swimming being diagnosed with LQTS based on a 12 lead ECG. The mother’s ECG showed no abnormalities. Genomic DNA from blood of daughter and mother was extracted using standard procedures. All coding exons and flanking intronic sequences of KCNQ1 (NM_000218) genes were amplified by polymerase chain reaction. Direct sequencing was performed on a 3500XL Genetic Analyzer. Data was analyzed with Geneious software for identification of mutations. For iPSC generation CD71+ CD36+ erythroblasts were reprogrammed using CytoTune™-iPS 2.0 Sendai Reprogramming Kit on irradiated mouse embryonic fibroblast feeder layers. Differentiation into cardiomyocytes was accomplished using the protocol described by Lian et al. (2012). Electrophysiological recordings were obtained from cardiomyocytes after 30–45 days of differentiation. Cells were transferred to acrylic plates pretreated with Matrigel and kept in culture for 3 days before microelectrode action potential (AP) were registered. The ECG recordings showed a corrected QT interval (QTc) of 516 ms for the daughter and 436 ms for the mother. Sequencing showed that both daughter and mother had a c.1763C>T mutation resulting in p.Ile588Thr substitution. iPSC generated from daughter, mother and an unrelated control displayed normal karyotype and expressed pluripotent genes, besides spontaneously differentiating into cells of the three germ layers. Differentiation into cardiomyocytes was ascertained by immunofluorescence and flow cytometry for troponin T. In all differentiations more than 70% of cells were cardiomyocytes. Action potentials recordings showed that ventricular cardiomyocytes from the daughter exhibited significantly longer action potential durations at 90% repolarization than the mother’s or the unrelated control’s cardiomyocytes. Cardiomyocytes derived from iPS cells reproduced the clinical phenotypes exhibited by daughter and mother.

Long‐term culture of human pluripotent stem cells on coverslips coated with peptide‐poly(styrene‐co‐vinyl benzoic acid) copolymer

Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have the potential to grow indefinitely (self‐renewal) in culture and differentiate into any cell type in adult human body. The hESCs and hiPSCs represent important resources for regenerative medicine and for disease modeling. Therefore, growing stem cells in undifferentiated state is important. The human pluripotent stem cells are expanded traditionally using either mitotically inactivated mouse embryonic fibroblasts (feeder cells) or Matrigel (MG). Both feeder cells and MG are prepared using mouse tissues that have several disadvantages. These manufactured expensive biological materials that have limited scalability also have high batch‐to‐batch variability. In addition, the animal‐derived (xenogenic) materials have potential to spread inter‐species pathogens. Thus, in order to replace these animal based biological materials, great efforts have been invested to develop synthetic materials that could be used for long‐term pluripotent stem cell growth and proliferation. As a consequence, materials such as poly [2‐(methacryloyloxy) ethyl dimethyl‐(3‐sulfopropyl) ammonium hydroxide] (PMEDSAH) and UV treated peptide‐acrylate materials have been used to culture hESCs. Since these synthetic materials are polymerized directly on the surface of cell culture dish, its application in broad sector and large‐scale expansion of stem cells for downstream applications has limitation. Thus, in the present study, we used a copolymer system containing poly (vinyl benzoic acid‐co‐styrene) (PVBA‐St), which could be coated onto various cell culture surfaces, such as glass, polystyrene cell culture plate etc. We synthesized PVBA‐St via nitroxide‐mediated polymerization to obtain polymer with narrow polydispersity index (PDI) and controlled molecular weight. In this study, we tested the stem cell growth on glass coverslips coated with peptide (extracellular matrix related) conjugated‐PVBA‐St. Up on initial screening, three active peptides were identified suitable for pluripotent stem cells expansion and self‐renewal. The long‐term culture of three pluripotent stem cell lines (H7, HUES‐7 and DF699) further confirmed the applicability and robustness of the copolymer system.Support or Funding InformationFunding was provided by the Institute of Bioengineering and Nanotechnology (Biomedical Research Council, Agency for Science, Technology and Research, Singapore). VMR was supported by funding from National Institutes of Health grant (NIH/NIDDK RO1DK104791)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Wnt3a Activates the WNT-YAP/TAZ Pathway to Sustain CDX2 Expression in Bovine Trophoblast Stem Cells.

Trophoblast stem cells (TSCs), the precursors of placental cells, are effective for studying placental formation in vitro. Using a dual inhibition (2i) medium and mixed L-Wnt3a/mouse embryonic fibroblast feeder cells, we previously established the bovine trophoblast cell line BTS-1. In this study, we used bovine fetal fibroblasts and added Wnt3a to the 2i medium to establish another bovine TSC line (BTSW). BTSW cells expressed pluripotency markers, including NANOG, SOX2, OCT4, TRA-1-60, TRA-1-81, SSEA4, CDH1, and KRT18, and TSC markers CDX2, TEAD4, and ESRRB. Methylation sequencing of the promoter regions of NANOG, OCT4, and CDX2 revealed no significant differences between BTS-1 and BTSW cells. Removal of Wnt3a from the culture medium resulted in downregulation (p < 0.05) of NANOG, OCT4, CDX2, and TSC marker genes, and upregulation of TSC differentiation markers, including MASH2, GCM1, and PAG. Western blotting indicated activation of the WNT-YAP/TAZ signaling pathway in BTS-1 and BTSW cells, consequently activating TEAD4 transcription. However, this pathway was not activated in BCFF cells, an established bovine embryonic stem-like cell line that expresses OCT4, SOX2, and NANOG, but not CDX2. Thus, Wnt3a may play a critical role in bovine TSC maintenance by activating and regulating CDX2 expression through the WNT-YAP/TAZ signaling pathway.

183 Generation of porcine embryonic stem cell lines derived from nuclear transfer embryos reconstructed with induced pluripotent stem cells

To establish a porcine embryonic stem (ES) cell line that not only maintains self-renewing capacity but also exhibits pluripotency [Haraguchi et al. 2012 J. Reprod. Dev. 58, 707-716], 6 synthetic porcine RNAs (Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28) were chemically transfected into outgrowth cultured cells derived from the inner cell mass of in vitro-produced porcine embryos. Subsequently, cells grew as compact, dome-shaped colonies displaying alkaline phosphatase activity and were cultured for more than 20 passages. Although 13 candidate cell lines were generated (13/43, 30%), none formed teratomas after injection of the cells into SCID (sever combined immunodeficiency) mice. We also observed that when transfection of the exogeneous RNAs was discontinued, the cells no longer maintained a stem cell morphology and began to differentiate (13/13, 100%). This suggests that continuous expression of exogenous reprogramming factors is necessary to maintain induced pluripotency in the pig. Next, we used cloned embryos reconstructed with porcine induced pluripotent stem cells (piPSC), which were created using a recombinant lentivirus expression vector carrying 6 mouse reprogramming factor genes (the same as above) and green fluorescent protein (GFP) (Fukuda et al. 2017 J. Cell Biochem. 118, 537-553]. The piPSC were dispersed to a single cell suspension and electrically fused to cytoplasts prepared following enucleation of in vitro-matured zona-free metaphase II-arrested oocytes. A second cytoplast was then fused to the first reconstruct (double cytoplast nuclear transfer). Reconstructs were electrically activated and cultured in microwells with porcine zygote medium-3 (PZM3). After 5 days, reconstructed embryos developed to GFP-positive blastocysts (10/93, 11%) and 4- to 8-cell stages (25/93, 27%). The blastocysts (10) and 4- to 8-cell-stage embryos (25) were transferred onto mouse embryonic fibroblast feeder cells for outgrowth culture in FCS-based ES cell medium supplemented with 2% polyvinylpyrrolidone. After 24h, the medium was changed to piPSC medium containing CHIR99021, PD0325901, thiazovivin, and GF-109203x. Embryos attached to the feeder cells began to outgrow (8/10 of blastocysts and 6/25 of 4- to 8-cell-stage embryos). To date, 3 ES-like cell lines have been established from blastomeres of embryos (3/25, 12%) but not from blastocysts (0/10, 0%). They show GFP fluorescence and have been maintained continuously in culture for more than 20 passages without any overt changes in morphology. These results suggest that the constant expression of reprogramming factors and the use of combinations of specific small molecule inhibitors largely contribute to the establishment of pluripotent cells in the pig. Further characterisation of the cells is ongoing, including methylation status of the X chromosome and the capacity for in vivo differentiation.

Feeders facilitate telomere maintenance and chromosomal stability of embryonic stem cells

Feeder cells like mouse embryonic fibroblasts (MEFs) have been widely applied for culture of pluripotent stem cells, but their roles remain elusive. Noticeably, ESCs cultured on the feeders display transcriptional heterogeneity. We investigated roles of feeder cells by examining the telomere maintenance. Here we show that telomere is longer in mESCs cultured with than without the feeders. mESC cultures without MEF feeders exhibit telomere loss, chromosomal fusion, and aneuploidy with increasing passages. Notably, feeders facilitate heterogeneous transcription of 2-cell genes including Zscan4 and telomere elongation. Moreover, feeders produce Fstl1 that together with BMP4 periodically activate Zscan4. Interestingly, Zscan4 is repressed in mESCs cultured in 2i (inhibitors of Mek and Gsk3β signaling) media, associated with shorter telomeres and increased chromosome instability. These data suggest the important role of feeders in maintaining telomeres for long-term stable self-renewal and developmental pluripotency of mESCs.

Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

Preparation of mouse embryonic fibroblast (MEF) feeder cells to maintain pluripotent stem cells (PSCs) is time consuming and involved in animal issues. Here, we demonstrated a novel method to prepare feeder cells with high efficiency, timesaving, and low costs. MEFs in 3 × 104 cell/cm2 were fixed by methanol for 5 min and air drying for 5 min. Thereafter, the methanol fixed MEF cells (MT-MEF) were able to be used directly to culture PSCs or stored at room temperature for the future usage. PSCs cultured on MT-MEF could be continuously expanded for over 40 passages with the naïve pluripotency. MT-MEFs could also be used to maintain human and pig iPSCs. Moreover, methanol fixed MEFs’ culture dish was able to be reused for at least 4 times, and to be applied for antibiotic resistant screening assay to establishing stable transfected PSC lines. Alternatively, the immortalized cell lines, for instance NIH3T3 cells, could also be fixed by methanol and used as feeder cells to maintain PSCs. Thus, this novel means of methanol fixed feeder cells can completely replace the mitomycin C and gamma radiation treated MEF feeder cells, and be used to maintain PSCs derived from mouse as well as other animal species.

Temporal Gene Expression and DNA Methylation during Embryonic Stem Cell Derivation.

Objective Dual inhibition of mitogen-activated protein kinase (MAPK) kinase (also known as MEK) and transforming growth factor β (TGFβ) type I receptors by PD0325901 and SB431542, known as R2i has been introduced as a highly efficient approach to the generation of mouse embryonic stem cells (ESC). In the present study, we investigated the molecular mechanisms underlying ESC derivation in the R2i condition. Materials and Methods In this experimental study, zona-free whole E3.5 blastocysts were seeded on mouse embryonic fibroblast (MEF) feeder cells in both R2i and serum conventional media. The isolated inner cell mass (ICM), ESCs and the ICM-outgrowths were collected on days 3, 5 and 7 post-blastocyst culture for quantitative real time- polymerase chain reaction (qRT-PCR) analysis as well as to assess the DNA methylation status at the time points during the transition from ICM to ESC. Results qRT-PCR revealed a significantly higher expression of the pluripotency-related genes (Oct4, Nanog, Sox2, Rex1, Dppa3, Tcf3, Utf1, Nodal, Dax1, Sall4 and β-Catenin) and lower expression of early differentiation genes (Gata6, Lefty2 and Cdx2) in R2i condition compared to the serum condition. Moreover, the upstream region of Oct4 and Nanog showed a progressive increase in methylation levels in the upstream regions of the genes following in R2i or serum conditions, followed by a decrease of DNA methylation in ESCs obtained under R2i. However, the methylation level of ICM outgrowths in the serum condition was much higher than R2i, at levels that could have a repressive effect and therefore explain the absence of expression of these two genes in the serum condition. ConclusionOur investigation revealed that generation of ESCs in the ground-state of pluripotency could be achieved by inhibiting the MEK and TGF-β signaling pathways in the first 5 days of ESC derivation.

Comparison of dissociation methods on pluripotency of stem cells

Induced pluripotent stem cells (iPSCs) can be generated from adult cells. Somatic cells can be reprogrammed to form iPSCs by overexpressing transcription factors such as Oct4, Sox2, cMyc, and Klf4. To maintain undifferentiated state of iPSCs in vitro, cells have traditionally been maintained on mouse embryonic fibroblast feeders and passaged by enzymatic or mechanical dissociation methods. In this study, we compared the morphology and pluripotency of porcine iPSCs (piPSCs) after subsequent passaging using enzymatic and mechanical dissociation methods. Enzymatically and mechanically passaged piPSCs showed embryonic stem cell-like morphologies with compact cell adhesion and clear colony borders. In addition, alkaline phosphatase staining was positive for both enzymatically and mechanically passaged piPSCs. However, visual observation revealed that some colonies of enzymatically passaged piPSCs were spontaneously differentiated more than those of piPSCs mechanically passaged from 5 passage. Quantitative real-time RT-PCR demonstrated that enzymatically and mechanically passaged piPSCs expressed pluripotent genes such as Oct4, Sox2 and Nanog well at early passage. Immunofluorescent staining also confirmed that pluripotent markers such as Oct4, Sox2, and Nanog were positively expressed at early passage. However, expression levels of pluripotent genes in mechanically passaged piPSCs were also higher than those in enzymatically passaged piPSCs at early passage. Collectively, we found that mechanical passage method was better than enzymatic passage in terms of morphology and pluripotency of piPSCs at early passage. Further studies are needed to compare these dissociation methods with those obtained after more passages of piPSCs.

Generation of embryonic stem-like cells from in vivo-derived porcine blastocysts at a low concentration of basic fibroblast growth factor.

Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self-renewal and pluripotency, its high cost has limited previous studies, and the development of a low-cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo-derived porcine blastocysts on 5.0×105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium-based medium for primary culture, on 2.5×105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5×105 MEF feeder cells in DMEM/Ham's F10-based medium for the post-2nd subpassage could support the establishment and maintenance of porcine ES-like cells at the low concentration of bFGF. The established porcine ES-like cells showed ES cell-specific characteristics such as self-renewal and pluripotency. We confirmed that porcine ES-like cells could be generated from in vivo-derived porcine blastocysts at a low concentration of bFGF.

Hair follicle-derived mesenchymal cells support undifferentiated growth of embryonic stem cells.

The aim of the present study was to investigate whether feeder layers composed of human hair follicle-derived mesenchymal stem cells (hHFDCs) are able to support human embryonic stem cells (hESCs). hHFDCs and mouse embryonic fibroblasts (MEFs) were isolated and cultured in Dulbecco's modified Eagle's medium (DMEM)/F-12 and low-glucose DMEM, respectively. hHFDCs were passaged three times and subsequently characterized. hHFDCs and MEFs were mitotically inactivated with mitomycin C for 3 h prior to co-culture with H9-hESCs. hESCs were initially established on a mouse feeder layer, subsequently transferred onto a human feeder layer and split every 5 days. Cell morphology, expression of specific ‘undifferentiation’ markers and growth factors, and the differentiation capacity of hESCs grown on the hHFDC feeder layer were analyzed. hHFDCs are adherent to plastic, possess the classic mesenchymal stem cell phenotype [they express cluster of differentiation (CD)90, CD73 and CD105] and are able to differentiate into adipocytes, chondroblasts and osteocytes, indicating that these cells are multipotent. Population-doubling time analysis revealed that hHFDCs rapidly proliferate over 34.5 h. As a feeder layer, hHFDC behaved similarly to MEF in maintaining the morphology of hESCs. The results of alkaline phosphatase activity, reverse transcription-quantitative polymerase chain reaction analysis of the expression of pluripotency transcription factors [octamer-binding transcription factor 4 (Oct4), Nanog and sex determining region Y-box 2], and immunofluorescence assays of markers (stage-specific embryonic antigen-4 and Oct4) in hESCs co-cultured over hHFDC, indicated that the undifferentiated state of hESCs was preserved. No change in the level of growth factor transcripts (bone morphogenetic protein 4, fibroblast growth factor-2, vascular endothelial growth factor, Pigment epithelium-derived factor and transforming growth factor-β1) was detected for either feeder layer prior to or following inactivation. Similar phenotypes of embryoid body formation, size and morphology were observed in the hHFDC and MEF feeders. In conclusion, hHFDC maintained hESCs in an undifferentiated state comparable to MEF in standard conditions, which may be an important finding regarding the establishment of stem cell-based translational applications.