Inactivated Mouse Embryonic Fibroblasts Research Articles

The human amniotic epithelium confers a bias to differentiate toward the neuroectoderm lineage in human embryonic stem cells.

Human embryonic stem cells (hESCs) derive from the epiblast and have pluripotent potential. To maintain the conventional conditions of the pluripotent potential in an undifferentiated state, inactivated mouse embryonic fibroblast (iMEF) is used as a feeder layer. However, it has been suggested that hESC under this conventional condition (hESC-iMEF) is an artifact that does not correspond to the in vitro counterpart of the human epiblast. Our previous studies demonstrated the use of an alternative feeder layer of human amniotic epithelial cells (hAECs) to derive and maintain hESC. We wondered if the hESC-hAEC culture could represent a different pluripotent stage than that of naïve or primed conventional conditions, simulating the stage in which the amniotic epithelium derives from the epiblast during peri-implantation. Like the conventional primed hESC-iMEF, hESC-hAEC has the same levels of expression as the 'pluripotency core' and does not express markers of naïve pluripotency. However, it presents a downregulation of HOX genes and genes associated with the endoderm and mesoderm, and it exhibits an increase in the expression of ectoderm lineage genes, specifically in the anterior neuroectoderm. Transcriptome analysis showed in hESC-hAEC an upregulated signature of genes coding for transcription factors involved in neural induction and forebrain development, and the ability to differentiate into a neural lineage was superior in comparison with conventional hESC-iMEF. We propose that the interaction of hESC with hAEC confers hESC a biased potential that resembles the anteriorized epiblast, which is predisposed to form the neural ectoderm.

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.

Evaluation of ATG5 Gene Expression in Human Induced Pluripotent Stem Cells During Endoderm Induction

Background: Autophagy is a vital cell survival mechanism that authorizes cells to assort to metabolic stress and is essential for the development and maintenance of cellular and tissue homeostasis, as well as the prevention of human disease. It has also been shown that autophagy plays a significant role in the development and differentiation of stem cells, as well as induced pluripotent stem cells (iPSCs). Objectives: The present study aimed to examine the mRNA expression of the ATG5 gene, one of the key markers of autophagy in human iPSCs (hiPSCs) during endoderm induction. Methods: In this study, we cultured the human iPSC line (R1-hiPSC1) on mitomycin-C, inactivated mouse embryonic fibroblasts (MEF) layer, and used hanging drop protocol to generate embryoid body (EB) and expose differentiation. The Real-time PCR method was used to examine the mRNA expression level of ATG5 in hiPSC during endoderm induction. Results: Our results demonstrated the high mRNA expression of ATG5 in the mesendoderm induction (MEI) stage, which shows the high rate of autophagy in MEI days rather than the other stages of differentiation. Conclusions: The modification of ATG5 gene expression within hiPSC during endoderm induction shows the importance of autophagy assessments in hiPSC differentiation. Therefore, subsequent studies are needed to clarify the details of autophagy effects on hiPSC differentiation.

Silk fibroin sponge combined with cell-derived ECM for tissue-engineered 3D functional neural tissues

The construction of engineered neural tissue using cell-derived extracellular matrix (ECM)-modified scaffolds is a promising approach. Silk fibroin (SF) sponge is an advantageous scaffold for the construction of engineered neural tissues, but it can still be modified to enhance its bioactivity. Inactivated mouse embryonic fibroblasts (MEFs) are proven that they can secrete amounts of ECM and soluble factors including neurotrophic factors. MEFs-derived ECM is expected to improve the activity of the scaffold. Here we aimed to construct 3D functional neural tissues based on MEFs-derived ECM modified SF sponge. MEFs were cultured on porous SF sponges and decellularized with TritonX-100 and NH4OH. The decellularized ECM deposited scaffolds were characterized through scanning electron microscopy and confocal microscopy. The efficiency of decellularization was evaluated by quantifying remaining DNA. Besides, we investigated the primary cortical neuronal growth and 3D neural network formation effect on MEFs-derived ECM modified SF sponges. Compared to bare SF sponges, ECM-SF sponges showed improved neuronal growth and axon extension indicated by immunofluorescence staining and RT-PCR. Specifically, ECM-SF sponges showed increased 3D neural network formation with functional connectivity. Hence, this study demonstrated that functionalization of SF scaffolds using cell-derived ECM could improve the bioactivity of SF materials and provide an ideal microenvironment for functional neural tissue formation.

Inducing Pluripotency in the Domestic Cat (Felis catus).

Domestic cats suffer from a range of inherited genetic diseases, many of which display similarities with equivalent human conditions. Developing cellular models for these inherited diseases would enable drug discovery, benefiting feline health and welfare as well as enhancing the potential of cats as relevant animal models for translation to human medicine. Advances in our understanding of these diseases at the cellular level have come from the use of induced pluripotent stem cells (iPSCs). iPSCs can differentiate into virtually any cell type and can be derived from adult somatic cells, therefore overcoming the ethical implications of destroying embryos to obtain embryonic stem cells. No studies, however, report the generation of iPSCs from domestic cats [feline iPSCs (fiPSCs)]. Feline adipose-derived fibroblasts were infected with amphotropic retrovirus containing the coding sequences for human Oct4, Sox2, Klf4, cMyc, and Nanog. Isolated iPSC clones were expanded on inactivated mouse embryonic fibroblasts in the presence of feline leukemia inhibitory factor (fLIF). Retroviral delivery of human pluripotent genes gave rise to putative fiPSC colonies within 5-7 days. These iPS-like cells required fetal bovine serum and fLIF for maintenance. Colonies were domed with refractile edges, similar to mouse iPSCs. Immunocytochemistry demonstrated positive staining for stem cell markers: alkaline phosphatase, Oct4, Sox2, Nanog, and SSEA1. Cells were negative for SSEA4. Expression of endogenous feline Nanog was confirmed by quantitative polymerase chain reaction. The cells were able to differentiate in vitro into cells representative of the three germ layers. These results confirm the first generation of induced pluripotent stem cells from domestic cats. These cells will provide valuable models to study genetic diseases and explore novel therapeutic strategies.

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.

Biophysical regulation of mouse embryonic stem cell fate and genomic integrity by feeder derived matrices.

For maintaining pluripotency, mouse embryonic stem cells (mESCs) are typically grown on mitotically inactivated mouse embryonic fibroblasts (MEFs). While the role of MEF conditioned media (MEFCM) and leukemia inhibitory factor (LIF) in regulating mESC pluripotency has led to culturing of mESCs on LIF/MEFCM supplemented gelatin-coated substrates, the role of physical interactions between MEFs and mESCs in regulating mESC pluripotency remains to be fully understood. Here, we address this question by characterizing the physicochemical properties of MEF derived matrices (MEFDMs), and probing their role in regulating mESC fate. We show that MEFDM composition and stiffness-dictated by MEF contractility-regulates mESC pluripotency by modulating mESC contractility through integrin-mediated mechanoadaptation. While baseline mESC pluripotency is maintained at early time points, activation of mESC contractility by LPA leads to drop in pluripotency levels. In contrast, addition of blebbistatin and LIF independently increases pluripotency by suppressing mechanoadaptation, highlighting the role of mechanoadaptation in regulating pluripotency and illustrating the role of LIF as a mechano-inhibitor in mESCs. Long-term culture of mESCs on MEFDMs under LIF-free conditions triggers loss of pluripotency, and induces ligand-dependent expression of the osteogenic transcription factor Runx2. Maintenance of genomic integrity (euploidy) on MEFDMs but not on gelatin-coated substrates, combined with the ability of MEFDMs in supporting LIF-free expansion and differentiation of mESCs, illustrates the suitability of MEFDMs for clinical and regenerative medicine applications.

Molecular markers of putative spermatogonial stem cells in the domestic cat

Spermatogonial stem cells (SSCs) are an important tool for fertility preservation and species conservation. The ability to expand SSCs by in vitro culture is a crucial premise for their use in assisted reproduction. Because SSCs represent a small proportion of the germ cells in the adult testis, culture success is aided by pre-enrichment through sorting techniques based on cell surface-specific markers. Given the importance of the domestic cat as a model for conservation of endangered wild felids, herein we sought to examine culture conditions as well as molecular markers for cat SSCs. Using a cell culture medium for mouse SSCs supplemented with glial cell-derived neurotrophic factor (GDNF), germ cells from prepuberal cat testes remained viable in culture for up to 43days. Immunohistochemistry for promyelocytic leukaemia zinc finger (PLZF) protein on foetal, prepuberal and adult testis sections revealed a pattern of expression consistent with the labelling of undifferentiated spermatogonia. Fluorescence-activated cell sorting (FACS) with an antibody against epithelial cell adhesion molecule (EPCAM) was used to sort live cells. Then, the gene expression profile of EPCAM-sorted cells was investigated through RT-qPCR. Notably, EPCAM (+) cells expressed relatively high levels of CKIT (CD117), a surface protein typically expressed in differentiating germ cells but not SSCs. Conversely, EPCAM (-) cells expressed relatively high levels of POU domain class 5 transcription factor 1 (POU1F5 or OCT4), clearly a germ line stem cell marker. These results suggest that cat SSCs would probably be found within the population of EPCAM (-) cells. Future studies should identify additional surface markers that alone or in combination can be used to further enrich SSCs from cat germ cells.

Improved Oocyte Isolation and Embryonic Development of Outbred Deer Mice.

In this study, we improved the protocol for isolating cumulus-oocyte complexes (COCs) from the outbred deer mice by using only one hormone (instead of the widely used combination of two hormones) with reduced dose. Moreover, we identified that significantly more metaphase II (MII) oocytes could be obtained by supplementing epidermal growth factor (EGF) and leukemia inhibition factor (LIF) into the previously established medium for in vitro maturation (IVM) of the COCs. Furthermore, we overcame the major challenge of two-cell block during embryonic development of deer mice after either in vitro fertilization (IVF) or parthenogenetic activation (PA) of the MII oocytes, by culturing the two-cell stage embryos on the feeder layer of inactivated mouse embryonic fibroblasts (MEFs) in the medium of mouse embryonic stem cells. Collectively, this work represents a major step forward in using deer mice as an outbred animal model for biomedical research on reproduction and early embryonic development.

Ethanol Inactivated Mouse Embryonic Fibroblasts Maintain the Self-Renew and Proliferation of Human Embryonic Stem Cells.

Conventionally, mouse embryonic fibroblasts (MEFs) inactivated by mitomycin C or irradiation were applied to support the self-renew and proliferation of human embryonic stem cells (hESCs). To avoid the disadvangtages of mitomycin C and irradiation, here MEFs were treated by ethanol (ET). Our data showed that 10% ET-inactivated MEFs (eiMEFs) could well maintain the self-renew and proliferation of hESCs. hESCs grown on eiMEFs expressed stem cell markers of NANOG, octamer-binding protein 4 (OCT4), stage-specific embryonic antigen-4 (SSEA4) and tumour related antigen-1-81 (TRA-1-81), meanwhile maintained normal karyotype after long time culture. Also, hESCs cocultured with eiMEFs were able to form embryoid body (EB) in vitro and develop teratoma in vivo. Moreover, eiMEFs could keep their nutrient functions after long time cryopreservation. Our results indicate that the application of eiMEF in hESCs culture is safe, economical and convenient, thus is a better choice.

Derivation and characterization of human embryonic stem cells on human amnion epithelial cells

Culture conditions that support the growth of undifferentiated human embryonic stem cells (hESCs) have already been established using primary human amnion epithelial cells (hAECs) as an alternative to traditional mitotically inactivated mouse embryonic fibroblasts (MEFs). In the present work, inner cell masses (ICM) were isolated from frozen embryos obtained as donations from couples undergoing in vitro fertilization (IVF) treatment and four new hESC lines were derived using hAECs as feeder cells. This feeder system was able to support continuous growth of what were, according to their domed shape and markers, undifferentiated naïve-like hESCs. Their pluripotent potential were also demonstrated by embryoid bodies developing to the expected three germ layers in vitro and the productions of teratoma in vivo. The cell lines retained their karyotypic integrity for over 35 passages. Transmission electron microscopy (TEM) indicated that these newly derived hESCs consisted mostly of undifferentiated cells with large nuclei and scanty cytoplasm. The new hESCs cultured on hAECs showed distinct undifferentiated characteristics in comparison to hESCs of the same passage maintained on MEFs. This type of optimized culture system may provide a useful platform for establishing clinical-grade hESCs and assessing the undifferentiated potential of hESCs.

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.

Locust bean gum as an alternative polymeric coating for embryonic stem cell culture

Pluripotent embryonic stem cells (ESCs) have self-renewal capacity and the potential to differentiate into any cellular type depending on specific cues (pluripotency) and, therefore, have become a vibrant research area in the biomedical field. ESCs are usually cultured in gelatin or on top of a monolayer of feeder cells such as mitotically inactivated mouse embryonic fibroblasts (MEFsi). The latter is the gold standard support to maintain the ESCs in the pluripotent state. Examples of versatile, non-animal derived and inexpensive materials that are able to support pluripotent ESCs are limited. Therefore, our aim was to find a biomaterial able to support ESC growth in a pluripotent state avoiding laborious and time consuming parallel culture of MEFsi and as simple to handle as gelatin. Many of the new biomaterials used to develop stem cell microenvironments are using natural polymers adsorbed or covalently attached to the surface to improve the biocompatibility of synthetic polymers. Locust beam gum (LBG) is a natural, edible polymer, which has a wide range of potential applications in different fields, such as food and pharmaceutical industry, due to its biocompatibility, adhesiveness and thickening properties. The present work brings a natural system based on the use of LBG as a coating for ESC culture. Undifferentiated mouse ESCs were cultured on commercially available LBG to evaluate its potential in maintaining pluripotent ESCs. In terms of morphology, ESC colonies in LBG presented the regular dome shape with bright borders, similar to the colonies obtained in co-cultures with MEFsi and characteristic of pluripotent ESC colonies. In short-term cultures, ESC proliferation in LBG coating was similar to ESC cultured in gelatin and the cells maintained their viability. The activity of alkaline phosphatase and Nanog, Sox2 and Oct4 expression of mouse ESCs cultured in LBG were comparable or in some cases higher than in ESCs cultured in gelatin. An in vitro differentiation assay revealed that mouse ESCs cultured in LBG preserve their tri-lineage differentiation capacity. In conclusion, our data indicate that LBG coating promotes mouse ESC growth in an undifferentiated state demonstrating to be a viable, non-animal derived alternative to gelatin to support pluripotent mouse ESCs in culture.

Generation of Human Induced Pluripotent Stem (iPS) Cells in Serum- and Feeder-Free Defined Culture and TGF-β1 Regulation of Pluripotency

Human Embryonic Stem cells (hESCs) and human induced Pluripotent Stem cells (hiPSCs) are commonly maintained on inactivated mouse embryonic fibroblast as feeder cells in medium supplemented with FBS or proprietary replacements. Use of culture medium containing undefined or unknown components has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. In addition, there is no consensus as to the optimal formulation, or the nature of the cytokine requirements of the cells to promote their self-renewal and inhibit their differentiation. In this study, we successfully generated hiPSCs from human dental pulp cells (DPCs) using Yamanaka's factors (Oct3/4, Sox2, Klf4, and c-Myc) with retroviral vectors in serum- and feeder-free defined culture conditions. These hiPSCs retained the property of self-renewal as evaluated by the expression of self-renewal marker genes and proteins, morphology, cell growth rates, and pluripotency evaluated by differentiation into derivatives of all three primary germ layers in vitro and in vivo. In this study, we found that TGF-β1 increased the expression levels of pluripotency markers in a dose-dependent manner. However, increasing doses of TGF-β1 suppressed the growth rate of hiPSCs cultured under the defined conditions. Furthermore, over short time periods the hiPSCs cultured in hESF9 or hESF9T exhibited similar morphology, but hiPSCs maintained in hESF9 could not survive beyond 30 passages. This result clearly confirmed that hiPSCs cultured in hESF9 medium absolutely required TGF-β1 to maintain pluripotency. This simple serum-free adherent monoculture system will allow us to elucidate the cell responses to growth factors under defined conditions and can eliminate the risk might be brought by undefined pathogens.

WNT3 Is a Biomarker Capable of Predicting the Definitive Endoderm Differentiation Potential of hESCs

Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However, the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here, we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition, manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally, analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively, our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs.

Comparative Proteomic Analysis of Supportive and Unsupportive Extracellular Matrix Substrates for Human Embryonic Stem Cell Maintenance

Human embryonic stem cells (hESCs) are pluripotent cells that have indefinite replicative potential and the ability to differentiate into derivatives of all three germ layers. hESCs are conventionally grown on mitotically inactivated mouse embryonic fibroblasts (MEFs) or feeder cells of human origin. In addition, feeder-free culture systems can be used to support hESCs, in which the adhesive substrate plays a key role in the regulation of stem cell self-renewal or differentiation. Extracellular matrix (ECM) components define the microenvironment of the niche for many types of stem cells, but their role in the maintenance of hESCs remains poorly understood. We used a proteomic approach to characterize in detail the composition and interaction networks of ECMs that support the growth of self-renewing hESCs. Whereas many ECM components were produced by supportive and unsupportive MEF and human placental stromal fibroblast feeder cells, some proteins were only expressed in supportive ECM, suggestive of a role in the maintenance of pluripotency. We show that identified candidate molecules can support attachment and self-renewal of hESCs alone (fibrillin-1) or in combination with fibronectin (perlecan, fibulin-2), in the absence of feeder cells. Together, these data highlight the importance of specific ECM interactions in the regulation of hESC phenotype and provide a resource for future studies of hESC self-renewal.

SPARC is involved in the maintenance of mitotically inactivated mouse embryonic fibroblast cells

Mitotically inactivated feeder cells such as mouse embryonic fibroblast (MEFs) cells have been widely applied for physical and physiological support in the pluripotency maintenance of human pluripotent stem cells (hPSCs). However, accurate supporting mechanism or factors of feeder cells are poorly understood. Here, we isolated differentially expressed genes between wild-type MEFs and mitotically inactivated MEFs (miMEFs) by employing annealing control primer-based GeneFishing polymerase chain reaction. We identified a secreted protein acidic cysteine-rich glycoprotein (SPARC) gene that is upregulated in miMEFs. Suppression of SPARC expression in miMEFs using small interference RNA (siRNA) displayed gradual detachment of miMEFs. Furthermore, we found a significant reduction of OCT4- and SSEA3-positive hPS cell population maintained on SPARC siRNA-miMEFs compared to on miMEFs by flow cytometrical analysis. These findings suggest that SPARC plays a critical role in the maintenance of miMEFs without loss of cell number and might be a key component for supporting the culture of hPSCs.

Long-term serial cultivation of mouse induced pluripotent stem cells in serum-free and feeder-free defined medium

Mouse embryonic stem (mES) cells and mouse induced pluripotent stem (miPS) cells are commonly maintained on inactivated mouse embryonic fibroblast feeder cells in medium supplemented with fetal bovine serum or proprietary replacements. An undefined medium containing unknown quantities of reagents has limited the development of applications for pluripotent cells because of the relative lack of knowledge regarding cell responses to differentiating growth factors. Therefore we developed a serum-free medium, designated ESF7, in which mES cells can be maintained in an undifferentiated state without feeder cells. The medium was tested for culturing miPS cells. The miPS cells have been maintained in ESF7 medium for more than 3 years with an undifferentiated phenotype manifested by the expression of pluripotency marker genes and alkaline phosphatase, and these cells exhibited largely normal karyotypes. Furthermore, we found that fibroblast growth factor-2 (FGF-2) with heparin induced miPS cell differentiation into neuronal cells, both in an adherent monolayer and in embryoid body suspension culture. Moreover, we found that FGF-2 with bone morphogenetic protein 2 induced miPS cell differentiation into cardiomyocytes in embryoid body suspension culture. Furthermore, we transplanted subcutaneously miPS cells maintained in ESF7 into the dorsal flanks of SCID mice; all of the transplants produced tumors with tissues derived from all three embryonic germ layers. As this simple serum-free adherent monoculture system supports the long-term propagation of pluripotent iPS cells in vitro, it will allow us to elucidate cell responses to growth factors under defined conditions, and it should provide useful information for differentiation protocols for human iPS cells.

Microfabricated Modular Scale-Down Device for Regenerative Medicine Process Development

The capacity of milli and micro litre bioreactors to accelerate process development has been successfully demonstrated in traditional biotechnology. However, for regenerative medicine present smaller scale culture methods cannot cope with the wide range of processing variables that need to be evaluated. Existing microfabricated culture devices, which could test different culture variables with a minimum amount of resources (e.g. expensive culture medium), are typically not designed with process development in mind. We present a novel, autoclavable, and microfabricated scale-down device designed for regenerative medicine process development. The microfabricated device contains a re-sealable culture chamber that facilitates use of standard culture protocols, creating a link with traditional small-scale culture devices for validation and scale-up studies. Further, the modular design can easily accommodate investigation of different culture substrate/extra-cellular matrix combinations. Inactivated mouse embryonic fibroblasts (iMEF) and human embryonic stem cell (hESC) colonies were successfully seeded on gelatine-coated tissue culture polystyrene (TC-PS) using standard static seeding protocols. The microfluidic chip included in the device offers precise and accurate control over the culture medium flow rate and resulting shear stresses in the device. Cells were cultured for two days with media perfused at 300 µl.h−1 resulting in a modelled shear stress of 1.1×10−4 Pa. Following perfusion, hESC colonies stained positively for different pluripotency markers and retained an undifferentiated morphology. An image processing algorithm was developed which permits quantification of co-cultured colony-forming cells from phase contrast microscope images. hESC colony sizes were quantified against the background of the feeder cells (iMEF) in less than 45 seconds for high-resolution images, which will permit real-time monitoring of culture progress in future experiments. The presented device is a first step to harness the advantages of microfluidics for regenerative medicine process development.

Hypoxic Culture of Human Pluripotent Stem Cell Lines is Permissible Using Mouse Embryonic Fibroblasts

Hypoxia is used within in vitro stem cell culture to recreate conditions similar to the in vivo environment surrounding the early blastocyst, from which embryonic stem cells can be isolated. Traditionally, basic research has used a coculture feeder system to culture pluripotent stem cells; however, it is possible that lowered oxygen may restrict cellular metabolic activity of the inactivated mouse embryonic fibroblasts (iMEFs) by disrupting oxygen-dependent pathways, such as ATP production through aerobic respiration. In this work, we examined the potential to continue using routine culture methods, such as iMEFs, to support human pluripotent cell expansion under hypoxia instead of feeder-free methods that can cause cell instability and offer a poor cell attachment rate. Metabolic activity and viability studies were carried out in normoxic and hypoxic conditions. Pluripotent stem cells were introduced into hypoxia on iMEFs and the rate of colony expansion was compared with normoxic conditions. In addition, pluripotent stem cells were grown in hypoxia for over 6 months to demonstrate maintenance of pluripotency. Immunocytochemistry and western blotting evaluated the activity of the hypoxic transcription factor, HIF1A. Hypoxia does not significantly affect viability or metabolic activity of feeder cells, and there is no detrimental effect on the rate of pluripotent stem cell colony expansion when cells are cultured in hypoxia. In addition, hypoxic pluripotent stem cells maintain their pluripotent nature and ability to differentiate into the three germ layers. The traditional iMEF coculture method is suitable for use in hypoxia and does not need to be replaced with feeder-free systems for hypoxic culture of human pluripotent stem cell lines in basic research.