Mouse Embryonic Fibroblast Feeder Research Articles

WNT/\u03b2-Catenin signaling pathway regulates non-tumorigenesis of human embryonic stem cells co-cultured with human umbilical cord mesenchymal stem cells

Human pluripotent stem cells harbor hope in regenerative medicine, but have limited application in treating clinical diseases due to teratoma formation. Our previous study has indicated that human umbilical cord mesenchymal stem cells (HUCMSC) can be adopted as non-teratogenenic feeders for human embryonic stem cells (hESC). This work describes the mechanism of non-tumorigenesis of that feeder system. In contrast with the mouse embryonic fibroblast (MEF) feeder, HUCMSC down-regulates the WNT/β-catenin/c-myc signaling in hESC. Thus, adding β-catenin antagonist (FH535 or DKK1) down-regulates β-catenin and c-myc expressions, and suppresses tumorigenesis (3/14 vs. 4/4, p = 0.01) in hESC fed with MEF, while adding the β-catenin enhancer (LiCl or 6-bromoindirubin-3′-oxime) up-regulates the expressions, and has a trend (p = 0.056) to promote tumorigenesis (2/7 vs. 0/21) in hESC fed with HUCMSC. Furthermore, FH535 supplement does not alter the pluripotency of hESC when fed with MEF, as indicated by the differentiation capabilities of the three germ layers. Taken together, this investigation concludes that WNT/β-catenin/c-myc pathway causes the tumorigenesis of hESC on MEF feeder, and β-catenin antagonist may be adopted as a tumor suppressor.

Culture Methods and Choice of Cells Makes a Difference in Outcome of Tissue Engineering- a Perspective

This perspective reviews the effects of different tissue engineering techniques, for induced differentiation of human embryonic stem cells into pulmonary lineage specific cells, and validating cell based regenerative therapy to ameliorate pulmonary degeneration. The etiology of degeneration differs from disease to disease. Likewise, strategies to regenerate lost tissues in its heterogeneity, should also cater to the regeneration process. To customize the same, researchers ought to device strategies to repair, replace or regenerate, in the spatio- temporal format, healthy tissues in their heterogeneous multi-functional existence, such that when transplanted in vitro, they may find their niche and integrate seamlessly into the tissue system and manifest full-fledged functional competence in order to reverse the erstwhile effects of degeneration. Cell based lung repair or regeneration is unquestionably the most promising agenda of regenerative medicine. In one of our studies, human embryonic stem cell (hESC) line H7 was successfully differentiated into three non-ciliated alveolar epithelial cells. In another study, BJNhem19 and BJNhem20 stem cell lines were used to induce similar guided endodermal differentiation into pulmonary lineage cells. Both studies were done using similar protocols, where the hESCs were first plated on gamma irradiated mouse embryonic fibroblast (MEF) feeder cells and undifferentiated hESCs were taken through embroid body formation. They were then subjected to induction into lung specific cells, by defined growth factors in Small Airways Growth Medium (SAGM) and Bronchiolar Endothelial Growth Media (BEGM). The H7 cells were found to express, both intracellularly and on their surfaces, characteristic marker proteins. However, the BJNhem19 and BJNhem20 cells showed poor differential potential, and could not be successfully differentiated into lung- specific cells. This difference in differentiation potential may be due to several intrinsic limitations, like genetic heterogeneity, anomalous morphogen receptivity or dormant/ unresponsive cytoplasmic determinants. These observations indicate that the choice of cells may play an important factor in tissue engineering. This review addresses the different culture methods and conditions needed for guided differentiation of stem cells. This perspective also discusses culture conditions of mesenchymal stem cells from different origins and dendritic stem cells from bone marrow.

Genome-Wide Transcriptome and Binding Sites Analyses Identify Early FOX Expressions for Enhancing Cardiomyogenesis Efficiency of hESC Cultures.

The differentiation efficiency of human embryonic stem cells (hESCs) into heart muscle cells (cardiomyocytes) is highly sensitive to culture conditions. To elucidate the regulatory mechanisms involved, we investigated hESCs grown on three distinct culture platforms: feeder-free Matrigel, mouse embryonic fibroblast feeders, and Matrigel replated on feeders. At the outset, we profiled and quantified their differentiation efficiency, transcriptome, transcription factor binding sites and DNA-methylation. Subsequent genome-wide analyses allowed us to reconstruct the relevant interactome, thereby forming the regulatory basis for implicating the contrasting differentiation efficiency of the culture conditions. We hypothesized that the parental expressions of FOXC1, FOXD1 and FOXQ1 transcription factors (TFs) are correlative with eventual cardiomyogenic outcome. Through WNT induction of the FOX TFs, we observed the co-activation of WNT3 and EOMES which are potent inducers of mesoderm differentiation. The result strengthened our hypothesis on the regulatory role of the FOX TFs in enhancing mesoderm differentiation capacity of hESCs. Importantly, the final proportions of cells expressing cardiac markers were directly correlated to the strength of FOX inductions within 72 hours after initiation of differentiation across different cell lines and protocols. Thus, we affirmed the relationship between early FOX TF expressions and cardiomyogenesis efficiency.

Abstract 2502: Bioengineered-induced cancer-like stem cells: characterization and applications

Abstract Cancer stem cells (CSCs) are a small subset of tumor cells capable of self-renewal, tumor initiation, and growth. Isolation, expansion, and differentiation of CSCs in the proper stem niches remain a topic of great debate. To address some of the challenges, we developed bioengineered-induced cancer like stem cells (iCLSCs) through in vitro oncogenic reprograming of C57BL/6 mouse embryonic stem cells (mESCs). We constructed retroviral plasmids through integration of well-defined oncogenic elements such as SV40LTg and HrasV12 into a mouse stem virus long terminal repeat (MSCV-LTR) plasmid fused with either a GFP or RFP gene. We successfully acquired these bioengineered-iCLSCs through retroviral introduction alongside with FACS sorting of positively infected mESCs. We then cultured the bioengineered-iCLSCs on mouse embryonic fibroblast (mEF) feeder cells. Bioengineered-iCLSCs expressed the introduced oncogenic genes and showed enhanced proliferation similar to cancer cells. In addition, we ascertained the unhampered stem properties of the bioengineered-iCLSCs by examining the maintenance of alkaline phosphatase activity as well as the expression of mouse stem cell marker SSEA-1. Furthermore, the orthotopic inoculation of these bioengineered-iCLSCs into either the mammalian fat pad or the ovarian bursa of female C57BL/6 mice lead to the formation of teratomas in supportive niches. Interestingly, the teratomas formed in the ovarian bursa exhibited malignant and immature features, whereas the iCLSCs introduced to the mammary fat pad were not able to generate teratomas with similar features. These observations call for additional research to identify the dependency on a supportive niche and their role in metastasis based on a tumor-favorable microenvironment. Our study helped to establish the use of in vitro oncogenic reprograming to generate bioengineered-iCLSCs and suggests the potential application for the initiation of organ-specific, malignant tumor formation in an orthotopic small animal cancer model. Citation Format: Sungpil Cho, Hongsuk Park, Elke A. Jarboe, C Matthew Peterson, You Han Bae, Margit Matthew Janat-Amsbury. Bioengineered-induced cancer-like stem cells: characterization and applications. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2502.

MP62-18 CONVERSION OF ADENOCARCINOMA TO SMALL CELL CARCINOMA-LIKE BY REPROGRAMMING

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology I1 Apr 2016MP62-18 CONVERSION OF ADENOCARCINOMA TO SMALL CELL CARCINOMA-LIKE BY REPROGRAMMING Gisely Borges, Eneida Vencio, Sue-Ing Quek, Adeline Chen, Diego Salvanha, Ricardo Vencio, Holly Nguyen, Robert Vessella, Christopher Cavanaugh, Carol Ware, Pamela Troisch, and Alvin Liu Gisely BorgesGisely Borges More articles by this author , Eneida VencioEneida Vencio More articles by this author , Sue-Ing QuekSue-Ing Quek More articles by this author , Adeline ChenAdeline Chen More articles by this author , Diego SalvanhaDiego Salvanha More articles by this author , Ricardo VencioRicardo Vencio More articles by this author , Holly NguyenHolly Nguyen More articles by this author , Robert VessellaRobert Vessella More articles by this author , Christopher CavanaughChristopher Cavanaugh More articles by this author , Carol WareCarol Ware More articles by this author , Pamela TroischPamela Troisch More articles by this author , and Alvin LiuAlvin Liu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.926AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Most prostate tumors are adenocarcinoma with glandular differentiation, and a small percentage, found most frequently in advanced diseases, are small cell neuroendocrine carcinoma without glandular differentiation. The LuCaP series is a family of prostate cancer xenografts propagated in male mice. LuCaP CR variants were obtained from passages in castrated hosts. Expression of stem cell transcription factor (TF) genes LIN28A, NANOG, POU5F1, SOX2 was examined in the LuCaP lines because some LuCaP lines were found to express stem cell markers. These four genes when forced expressed in somatic cells could induce reprogramming. METHODS The LuCaP cells were harvested and frozen by using a special protocol that preserved their viability. Gene expression was studied by RT-PCR. Adenocarcinoma LuCaP cells were plated on mouse embryonic fibroblast (MEF) feeder, which allowed them to grow in vitro. The cancer cells were transfected with lentiviral vectors containing the TF genes. Transfected cells on MEF were monitored over a period of 1 month. DNA microarrays were used to obtain cell type transcriptomes. RESULTS Most LuCaP lines expressed POU5F1, and LIN28A was upregulated in LuCaP 73CR vs. LuCaP 73. The small cell carcinoma LuCaP 145.1 expressed all four. Its expression of B2M (β-2 microglobulin) was also lower than that of other LuCaP lines. Low B2M expression is characteristic of stem cells. Many other LuCaP 145.1 genes (COTL1, ENO2, IGFBP2, ROBO1, chromogranin A) were also found in embryonic stem (ES) cells. TF transfection was carried out on five (AR+/PSA+) adenocarcinoma LuCaP lines: 70CR, 73CR, 86.2, 92, 105CR. All produced small dark cells as compared to large bright parental cells. The colony morphology was unlike that of ES cells, and more like that of PC3 cancer cell line, which was established from a bone metastasis of high-grade undifferentiated prostate carcinoma. The small cells started to appear around day 10 post-transfection and multiplied until individual colonies were visible at day 21. No such cells were seen in mock transfected. The transcriptome of LuCaP 70CR* (* to indicate transfected progeny) was very different from that of parental LuCaP 70CR as measured by Δ=91.72 in a prostate principal components analysis space. Gene expression of LuCaP 70CR* was closest to that of LuCaP 145.1 by Δ=45.85 (vs. Δ=78.04 with ES cells). CONCLUSIONS The adenocarcinoma phenotype could be converted to that of small cell carcinoma-like by stem cell TF genes (as in reprogramming). Cancer progression can be characterized molecularly as cellular de-differentiation from differentiated adenocarcinoma to undifferentiated stem-like small cell carcinoma. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e819 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Gisely Borges More articles by this author Eneida Vencio More articles by this author Sue-Ing Quek More articles by this author Adeline Chen More articles by this author Diego Salvanha More articles by this author Ricardo Vencio More articles by this author Holly Nguyen More articles by this author Robert Vessella More articles by this author Christopher Cavanaugh More articles by this author Carol Ware More articles by this author Pamela Troisch More articles by this author Alvin Liu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Propagation of Human Embryonic Stem Cells on Human Amniotic Fluid Cells as Feeder Cells in Xeno-Free Culture Conditions.

Human embryonic stem cells (hESCs) have been routinely cultured on mouse embryonic fibroblast feederlayers with a medium containing animal materials. For clinical application of hESCs, animal-derived products from the animal feeder cells, animal substrates such as gelatin or Matrigel and animal serum are strictly to be eliminated in the culture system. In this study, we performed that SNUhES32 and H1 were cultured on human amniotic fluid cells (hAFCs) with KOSR XenoFree and a humanized substrate. All of hESCs were relatively well propagated on hAFCs feeders with xeno-free conditions and they expressed pluripotent stem cell markers, alkaline phosphatase, SSEA-4, TRA1-60, TRA1-81, Oct-4, and Nanog like hESCs cultured on STO or human foreskin fibroblast feeders. In addition, we observed the expression of nonhuman N-glycolylneuraminic acid (Neu5GC) molecules by flow cytometry, which was xenotransplantation components of contamination in hESCs cultured on animal feeder conditions, was not detected in this xeno-free condition. In conclusion, SNUhES32 and H1 could be maintained on hAFCs for humanized culture conditions, therefore, we suggested that new xenofree conditions for clinical grade hESCs culture will be useful data in future clinical studies.

Generation of electrophysiologically functional cardiomyocytes from mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells can efficiently differentiate into the three germ layers similar to those formed by differentiated embryonic stem (ES) cells. This provides a new source of cells in which to establish preclinical allogeneic transplantation models. Our iPS cells were generated from mouse embryonic fibroblasts (MEFs) transfected with the Yamanaka factors, the four transcription factors (Oct4, Sox2, Klf4 and c-Myc), without antibiotic selection or MEF feeders. After the formation of embryoid bodies (EBs), iPS cells spontaneously differentiated into Flk1-positive cardiac progenitors and cardiomyocytes expressing cardiac-specific markers such as alpha sarcomeric actinin (α-actinin), cardiac alpha myosin heavy chain (α-MHC), cardiac troponin T (cTnT), and connexin 43 (CX43), as well as cardiac transcription factors Nk2 homebox 5 (Nkx2.5) and gata binding protein 4 (gata4). The electrophysiological activity of iPS cell-derived cardiomyocytes (iPS-CMs) was detected in beating cell clusters with optical mapping and RH237 a voltage-sensitive dye, and in single contracting cells with patch-clamp technology. Incompletely differentiated iPS cells formed teratomas when transplanted into a severe combined immunodeficiency (SCID) mouse model of myocardial infarction. Our results show that somatic cells can be reprogrammed into pluripotent stem cells, which in turn spontaneously differentiate into electrophysiologically functional mature cardiomyocytes expressing cardiac-specific makers, and that these cells can potentially be used to repair myocardial infarction (MI) in the future.

Abstract B45: Characterization of human fallopian tube epithelial stem-like cells.

Abstract Purpose: Recent studies have suggested a large part of ovarian high grade serous carcinomas arise from the fallopian tube epithelium (FTE). The purpose of this study was to derive the stem-like cells of fallopian tube epithelium (FTESC). Results: Immunohistochemistry of human fallopian tube revealed expression of Pax8 in secretory cells, Tubb4 in ciliated cells and Epcam in all the epithelial cells, whereas Lgr5 was expressed in a small subset of cells in the epithelial layer. The presumed FTESC were cultured from the fallopian tube fimbria epithelium exhibiting a cuboidal cell morphology and could be maintained at a constant proliferation rate up to P9. When cultured on matrigel, these cells formed spheroids with Pax8, Tubb4 and Lgr5 expression. Flowcytometry revealed decreasing expression of EpCAM, CD73, CD133 and CD105, and increasing of Pax 8 secretory cell marker (87% in P6, 78% in P3) with the advance of passage. Adding of estrogen to the culture resulted in an enlargement of cell body (4 nM) and differentiation toward ciliated cells (400 nM). When these FTESC were cultured on the mouse embryonic fibroblast feeder, or mixe-cultured with fallopian tube stromal cells and HUVECs, they formed tubal-like structures mimicking the fallopian tube. Conclusion: We successfully derived a presumed FTESC with self-renewal and differentiation capability. These cells may provide new insight to investigate how FTE regenerate, respond to hormone and ROS injury and subjected to cancer initiation. Citation Format: Dah-Ching Ding, Tang-Yuan Chu. Characterization of human fallopian tube epithelial stem-like cells. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B45.

Simplified three-dimensional culture system for long-term expansion of embryonic stem cells.

To devise a simplified and efficient method for long-term culture and maintenance of embryonic stem cells requiring less frequent passaging. Mouse embryonic stem cells (ESCs) labeled with enhanced yellow fluorescent protein were cultured in three-dimensional (3-D) self-assembling scaffolds and compared with traditional two-dimentional (2-D) culture techniques requiring mouse embryonic fibroblast feeder layers or leukemia inhibitory factor. 3-D scaffolds encapsulating ESCs were prepared by mixing ESCs with polyethylene glycol tetra-acrylate (PEG-4-Acr) and thiol-functionalized dextran (Dex-SH). Distribution of ESCs in 3-D was monitored by confocal microscopy. Viability and proliferation of encapsulated cells during long-term culture were determined by propidium iodide as well as direct cell counts and PrestoBlue (PB) assays. Genetic expression of pluripotency markers (Oct4, Nanog, Klf4, and Sox2) in ESCs grown under 2-D and 3-D culture conditions was examined by quantitative real-time polymerase chain reaction. Protein expression of selected stemness markers was determined by two different methods, immunofluorescence staining (Oct4 and Nanog) and western blot analysis (Oct4, Nanog, and Klf4). Pluripotency of 3-D scaffold grown ESCs was analyzed by in vivo teratoma assay and in vitro differentiation via embryoid bodies into cells of all three germ layers. Self-assembling scaffolds encapsulating ESCs for 3-D culture without the loss of cell viability were prepared by mixing PEG-4-Acr and Dex-SH (1:1 v/v) to a final concentration of 5% (w/v). Scaffold integrity was dependent on the degree of thiol substitution of Dex-SH and cell concentration. Scaffolds prepared using Dex-SH with 7.5% and 33% thiol substitution and incubated in culture medium maintained their integrity for 11 and 13 d without cells and 22 ± 5 d and 37 ± 5 d with cells, respectively. ESCs formed compact colonies, which progressively increased in size over time due to cell proliferation as determined by confocal microscopy and PB staining. 3-D scaffold cultured ESCs expressed significantly higher levels (P < 0.01) of Oct4, Nanog, and Kl4, showing a 2.8, 3.0 and 1.8 fold increase, respectively, in comparison to 2-D grown cells. A similar increase in the protein expression levels of Oct4, Nanog, and Klf4 was observed in 3-D grown ESCs. However, when 3-D cultured ESCs were subsequently passaged in 2-D culture conditions, the level of these pluripotent markers was reduced to normal levels. 3-D grown ESCs produced teratomas and yielded cells of all three germ layers, expressing brachyury (mesoderm), NCAM (ectoderm), and GATA4 (endoderm) markers. Furthermore, these cells differentiated into osteogenic, chondrogenic, myogenic, and neural lineages expressing Col1, Col2, Myog, and Nestin, respectively. This novel 3-D culture system demonstrated long-term maintenance of mouse ESCs without the routine passaging and manipulation necessary for traditional 2-D cell propagation.

REST–miR-21–SOX2 axis maintains pluripotency in E14Tg2a.4 embryonic stem cells

Our previous studies have shown that the regulatory network that maintains pluripotency in mouse embryonic stem cells (mESCs) is regulated in a context-dependent manner and can be modulated, at least in part, by re-calibration of an intracellular network of pluripotency factors as well as cues arising from the extracellular matrix. The transcriptional repressor REST represses miR-21 and, thus, regulates self-renewal in E14Tg2a.4 mESCs cultured in the absence of mouse embryonic fibroblast feeder cell effects. However, how miR-21 connects to the nuclear regulatory network has not been clear. Here, we show that miR-21, a direct target of REST-mediated repression, directly targets Sox2. Exogenously added miR-21 to mESCs decreases the expression of Sox2, decreasing mESC self-renewal, and this effect of miR-21 on mESC self-renewal can be blocked by expression of exogenous Sox2. Conversely, destabilization of Sox2 by miR-21 can be blocked by anti-miR-21. Thus, the REST–miR-21–Sox2 axis connects REST to the core nuclear pluripotency regulators in E14Tg2a.4 mESCs cultured in the absence of feeder cells.

Comparison between the cultures of human induced pluripotent stem cells (hiPSCs) on feeder-and serum-free system (Matrigel matrix), MEF and HDF feeder cell lines.

Human induced pluripotent stem cells (hiPSCs) are a type of pluripotent stem cells artificially derived from an adult somatic cell (typically human fibroblast) by forced expression of specific genes. In recent years, different feeders like inactivated mouse embryonic fibroblasts (MEFs), human dermal fibroblasts (HDFs), and feeder free system have commonly been used for supporting the culture of stem cells in undifferentiated state. In the present work, the culture of hiPSCs and their characterizations on BD Matrigel (feeder-and serum-free system), MEF and HDF feeders using cell culture methods and molecular techniques were evaluated and compared. The isolated HDFs from foreskin samples were reprogrammed to hiPSCs using gene delivery system. Then, the pluripotency ability of hiPSCs cultured on each layer was determined by teratoma formation and immunohistochemical staining. After EBs generation the expression level of three germ layers genes were evaluated by Q-real-time PCR. Also, the cytogenetic stability of hiPSCs cultured on each condition was analyzed by karyotyping and comet assay. Then, the presence of pluripotency antigens were confirmed by Immunocytochemistry (ICC) test and alkaline phosphatase staining. This study were showed culturing of hiPSCs on BD Matrigel, MEF and HDF feeders had normal morphology and could maintain in undifferentiated state for prolonged expansion. The hiPSCs cultured in each system had normal karyotype without any chromosomal abnormalities and the DNA lesions were not observed by comet assay. Moreover, up-regulation in three germ layers genes in cultured hiPSCs on each layer (same to ESCs) compare to normal HDFs were observed (p < 0.05). The findings of the present work were showed in stem cells culturing especially hiPSCs both MEF and HDF feeders as well as feeder free system like Matrigel are proper despite benefits and disadvantages. Although, MEFs is suitable for supporting of stem cell culturing but it can animal pathogens transferring and inducing immune response. Furthermore, HDFs have homologous source with hiPSCs and can be used as feeder instead of MEF but in therapeutic approaches the cells contamination is a problem. So, this study were suggested feeder free culturing of hiPSCs on Matrigel in supplemented media (without using MEF conditioned medium) resolves these problems and could prepare easy applications of hiPSCs in therapeutic approaches of regenerative medicine such as stem-cell therapy and somatic cell nuclear in further researches.

Increased risk of genetic and epigenetic instability in human embryonic stem cells associated with specific culture conditions.

The self-renewal and differentiation capacities of human pluripotent stem cells (hPSCs) make them a promising source of material for cell transplantation therapy, drug development, and studies of cellular differentiation and development. However, the large numbers of cells necessary for many of these applications require extensive expansion of hPSC cultures, a process that has been associated with genetic and epigenetic alterations. We have performed a combinatorial study on both hESCs and hiPSCs to compare the effects of enzymatic vs. mechanical passaging, and feeder-free vs. mouse embryonic fibroblast feeder substrate, on the genetic and epigenetic stability and the phenotypic characteristics of hPSCs. In extensive experiments involving over 100 continuous passages, we observed that both enzymatic passaging and feeder-free culture were associated with genetic instability, higher rates of cell proliferation, and persistence of OCT4/POU5F1-positive cells in teratomas, with enzymatic passaging having the stronger effect. In all combinations of culture conditions except for mechanical passaging on feeder layers, we noted recurrent deletions in the genomic region containing the tumor suppressor gene TP53, which was associated with decreased mRNA expression of TP53, as well as alterations in the expression of several downstream genes consistent with a decrease in the activity of the TP53 pathway. Among the hESC cultures, we also observed culture-associated variations in global gene expression and DNA methylation. The effects of enzymatic passaging and feeder-free conditions were also observed in hiPSC cultures. Our results highlight the need for careful assessment of the effects of culture conditions on cells intended for clinical therapies.

Human iPS cell-derived fibroblast-like cells as feeder layers for iPS cell derivation and expansion

Mouse embryonic fibroblasts (MEFs) are commonly used as feeder cells for the generation of human induced pluripotent stem cells (hiPSCs). However, medical applications of cell derivatives of hiPSCs generated with a MEF feeder system run the risk of having xeno-factor contamination due to long-term cell culturing under an animal factor-containing environment. We developed a new method for the derivation of human fibroblast-like cells (FLCs) from a previously established hiPSC line in an FLC differentiation medium. The method was based on direct differentiation of hiPSCs seeded on Matrigel followed by expansion of differentiating cells on gelatin. Using inactivated FLCs as feeder layers, primary human foreskin fibroblasts were successfully reprogrammed into a state of pluripotency by Oct4, Sox2 Klf4, and c-Myc (OSKM) transcription factor genes, with a reprogramming efficiency under an optimized condition superior to that obtained on MEF feeder layers. Furthermore, the FLCs were more effective in supporting the growth of human pluripotent stem cells. The pluripotency and differentiation capability of the cells cultured on FLC feeder layers were well retained. Our results suggest that FLCs are a safe alternative to MEFs for hiPSC generation and expansion, especially in the clinical settings wherein hiPSC derivatives will be used for medical treatment.

Dgcr8 is Indispensable for Cardiac Lineage Specification in Embryonic Stem Cells

Objective: microRNAs have been shown to play important roles in cellular behavior and lineage specification including cardiogenic differentiation. However, full understanding of their roles in cardiomyocyte differentiation has been impeded due to lack of proper cellular model. Here, we used an embryonic stem cell (ESC) that is lacking the important microprocessor Dgcr8 (or Pasha), which allows the introduction of individual miRNAs to study their role in cardiac differentiation and for more precise target selection. Methods: Dgcr8 KO ESC was cultured in LIF-supplemented ESC medium with mouse embryonic fibroblast feeders and cardiac differentiation was induced using an embryonic body-based differentiation protocol. Differentiation was monitored by measuring mRNA and protein levels of cardiogenic markers and heterochromatin changes using immunofluorescent staining and semi-quantitative PCR. Results and conclusion: We showed that Dgcr8 KO ESCs indeed are lacking a large population of small RNAs, including but not limited to mature microRNAs. The KO cells had a lower proliferation rate and were unable to differentiate into the cardiac lineage. To our surprise, in addition to a defect in microRNA processing, Dgcr8 KO embryonic stem cells are unable to form proper heterochromatin and to inactivate genotoxic centromeric repetitive elements. Our results argue that, in addition to controlling microRNA processing, Dgcr8 may serve a previously unrecognized role in heterochromatin silencing.

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro.

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.

Osteogenic induction from marmoset embryonic stem cells cultured in feeder-dependent and feeder-independent conditions

Embryonic stem cells (ESCs) have become increasingly attractive for cell replacement therapies of osteodegenerative diseases; however, pre-clinical studies in large animal models to repair diseased or injured bone are lacking. As a first step into this direction, we describe here the feeder-free cultivation and directed osteogenic differentiation of marmoset ESCs. Owing to their potential to self-renew and their enormous differentiation capability, ESCs are an adequate cell source for cell replacement therapies. To implement stem cell technology clinically, standardized cultivation and differentiation protocols and appropriate animal models are needed. Here, we describe the feeder-free cultivation of Callithrix jacchus ESCs (cESCs) in a chemically defined medium and their subsequent osteogenic differentiation. cESCs were maintained on mouse embryonic fibroblast feeder layers or in feeder-free conditions with activin A and basic fibroblast growth factor. Differentiation into mature osteoblasts was steered with ascorbic acid, β-glycerophosphate and 1α,25-(OH)2 vitamin D3 employing various induction strategies. In feeder-free conditions, cESCs maintained pluripotency as indicated by Oct-4 and Nanog expression, positive immunostaining for typical primate ESC markers and high telomerase activity. Cells also remained karyotypically normal after 40 passages without feeder cells. The hanging drop protocol as well as omitting the embryoid body step proved unsuccessful to initiate osteogenic differentiation. The highest degree of osteogenesis was achieved by formation of embryoid bodies employing the cell cluster technique as indicated by the amount of deposited calcium and bone marker gene expression. Early addition of retinoic acid further improved the yield of osteoblasts and led to an increase in calcium deposition. The osteogenic differentiation potential of feeder-free cESCs was equal if not higher compared to cells grown on feeders. These findings open the field for near clinical transplantation studies in primate models to evaluate the effectiveness of ESC-derived osteoblasts.

Analysis of essential pathways for self-renewal in common marmoset embryonic stem cells

Common marmoset (CM) is widely recognized as a useful non-human primate for disease modeling and preclinical studies. Thus, embryonic stem cells (ESCs) derived from CM have potential as an appropriate cell source to test human regenerative medicine using human ESCs. CM ESCs have been established by us and other groups, and can be cultured in vitro. However, the growth factors and downstream pathways for self-renewal of CM ESCs are largely unknown. In this study, we found that basic fibroblast growth factor (bFGF) rather than leukemia inhibitory factor (LIF) promoted CM ESC self-renewal via the activation of phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT) pathway on mouse embryonic fibroblast (MEF) feeders. Moreover, bFGF and transforming growth factor β (TGFβ) signaling pathways cooperatively maintained the undifferentiated state of CM ESCs under feeder-free condition. Our findings may improve the culture techniques of CM ESCs and facilitate their use as a preclinical experimental resource for human regenerative medicine.

A Comparative Study of Protocols for Mouse Embryonic Stem Cell Culturing

Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.

Optimization of Culture Conditions for Maintaining Porcine Induced Pluripotent Stem Cells

Ground state porcine induced pluripotent stem cells (piPSCs), which retain the potential to generate chimeric animal and germline transmission, are difficult to produce. This study investigated morphological and biological progression at the early stage of porcine somatic cell reprogramming, and explored suitable conditions to increase the induction efficiency of piPSCs. A cocktail of defined transcription factors was used to generate piPSCs. The amphotropic retrovirus, which carried human OCT4 (O), SOX2 (S), KLF4 (K), C-MYC (M), TERT (T), and GFP, were used to infect porcine embryonic fibroblasts (PEFs). The number of clones derived from OSKM (4F) and OSKMT (4F+T) was significantly higher than that from SKM (3F) and SKMT (3F+T), suggesting that OCT4 played a critical role in regulating porcine cell reprogramming. The number of alkaline phosphatase-positive clones from a medium with leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) (M1 medium) was significantly higher than that with insulin and 2i PD0325901/CHIR99021 (M2 medium), indicating that insulin and 2i could not effectively maintain piPSC propagation. In the M1 medium, piPSC lines could not maintain the typical self-renewal morphology on gelatin-coated and Matrigel-coated plates. Without the mouse embryonic fibroblast (MEF) feeder, piPSCs started to simultaneously differentiate. Based on the potential for self-renewal and activation of pluripotent markers, we found that the culture condition of 4F+T plus LIF and bFGF plus MEF feeder promoted PEF reprogramming more efficiently than the other conditions tested here. Two piPSC lines (IB-1 and IB-2) were derived and maintained for up to 20 passages in vitro.

Single‐cell PCR analysis of murine embryonic stem cells cultured on different substrates highlights heterogeneous expression of stem cell markers

In the last few years, recent evidence has revealed that inside an apparently homogeneous cell population there indeed appears to be heterogeneity. This is particularly true for embryonic stem (ES) cells where markers of pluripotency are dynamically expressed within the single cells. In this work, we have designed and tested a new set of primers for multiplex PCR detection of pluripotency markers expression, and have applied it to perform a single-cell analysis in murine ES cells cultured on three different substrates that could play an important role in controlling cell behaviour and fate: (i) mouse embryonic fibroblast (MEF) feeder layer, as the standard method for ES cells culture; (ii) Matrigel coating; (iii) micropatterned hydrogel. Compared with population analysis, using a single-cell approach, we were able to evaluate not only the number of cells that maintained the expression of a specific gene but, most importantly, how many cells co-expressed different markers. We found that micropatterned hydrogel seems to represent a good alternative to MEF, as the expression of stemness markers is better preserved than in Matrigel culture. This single-cell assay allows for the assessment of the stemness maintenance at a single-cell level in terms of gene expression profile, and can be applied in stem cell research to characterise freshly isolated and cultured cells, or to standardise, for instance, the method of culture closely linked to the transcriptional activity and the differentiation potential.