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Companion animals, such as felines and canines, could provide an excellent platform for translational research from veterinary to human medicine. However, the use of feline induced pluripotent stems (fiPSCs) of quality in basic or clinical research has not been reported. Here, we generated footprint-free fiPSCs derived from embryonic cells, as well as juvenile feline uterus-derived cells using Sendai virus vector harboring six feline-specific pluripotency-associated genes. The fiPSCs were confirmed to be of high quality with the potential to form teratomas including all three germ layers. Furthermore, our fiPSCs were maintained under feeder-free and chemically-defined conditions using StemFit® AK02N and recombinant laminin 511, iMatrix-511. Further research on fiPSCs could result in their widespread application in veterinary regenerative medicine, which could pave the way for their use in advanced regenerative medicine research for humans.

Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs. [ABSTRACT FROM AUTHOR]

We generated a human induced pluripotent stem cell (hiPSC) line from erythroid progenitor cells (EPCs) of a 20-year-old female healthy donor using Sendai virus vector encoding Yamanaka factors OCT3/4, SOX2, c-MYC, and KLF4. The established hiPSCs showed a standard morphology and expression of typical undifferentiated stem cell markers, a normal karyotype (46, XX), and demonstrated potential for differentiation in vitro. Furthermore, they were successfully differentiated into cardiomyocytes that expressed cardiomyocyte-specific markers. The iPSC line and iPSC-derived cardiomyocytes will provide new avenues for future drug testing/development and personalized cell therapy for cardiovascular diseases (CVDs).

Introduction: Lung transplantation is the only effective treatment option for many patients with irreversible pulmonary injury, and the demand for lung transplantation is increasing worldwide and expected to continue to outstrip the number of available donors. Regenerative therapy with alveolar epithelial cells (AECs) holds promise as an alternative option to organ transplantation. AECs are usually co-cultured with mouse-derived 3T3 feeder cells, but the use of xenogeneic tissues for regenerative therapy raises safety concerns. Fabrication of AEC sheets under feeder-free conditions would avoid these safety issues. We describe a novel feeder-free method of fabricating AEC sheets that may be suitable for pulmonary regenerative therapy. Methods: Lung tissues excised from male outbred rats or transgenic rats expressing green fluorescent protein (GFP) were finely minced and dissociated with elastase. The isolated AECs were cultured under four different feeder-free conditions according to whether a rho kinase (ROCK) inhibitor was included in the low-calcium medium (LCM) and whether the tissue culture dish was coated with recombinant laminin-511 E8 fragment (rLN511E8). The expanded cells were cultured on temperature-responsive dishes and subsequently harvested as AEC sheets. Engraftment of GFP-AEC sheets after their transplantation onto a partially resected region of the left lung was assessed in athymic rats. Results: AECs proliferated and reached confluence when cultured in LCM containing a ROCK inhibitor on tissue culture dishes coated with rLN511E8. When both the ROCK inhibitor and rLN511E8-coated culture dish were used, the number of AECs obtained after 7 days of culture was significantly higher than that in the other three groups. Immunohistochemical analyses revealed that aquaporin-5, surfactant protein (SP)-A, SP-C, SP-D and Axin-2 were expressed by the cultured AECs. AEC sheets were harvested successfully from temperature-responsive culture dishes (by lowering the temperature) when the expanded AECs were cultured for 7 days in LCM + ROCK inhibitor and then for 3 days in LCM + ROCK inhibitor supplemented with 200 mg/L calcium chloride. The AEC sheets were firmly engrafted 7 days after transplantation onto the lung defect and expressed AEC marker proteins. Conclusions: AEC sheets fabricated under feeder-free conditions retained the features of AECs after transplantation onto the lung in vivo. Further improvement of this technique may allow the bioengineering of alveolar-like tissue for use in pulmonary regenerative therapy.

Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications.

Natural killer (NK) cells are emerging as a candidate for next-generation allogeneic cell immunotherapy. Genetically engineered feeder cells enabling an effective expansion of NK cells have become the market's mainstream. However, there are safety concerns regarding contaminating cells in the final therapeutic product, and current feeder-free methods result in much lower yields. Herein, we demonstrate a novel bead-based platform for NK cell manufacturing. iKNOBeads is the world's first magnetic microbeads with controllable uniform sizes and bumpy morphology. We have screened numerous stimulatory candidates to be modified and proved that iKNOBeads are efficient for peripheral blood and cord blood NK cell expansion with comparable quality and quantity to feeder cells reported. A triple-negative breast cancer patient study demonstrates the NK cell cytotoxic potential against breast cancer cell lines and patient-derived organoids. We also develop a scale-up process to meet the needs of clinical applications. These results show that iKNOBeads offers a more effective and economical feeder-free platform for generating NK cells for therapy. iKNOBeads will be commercially available in 2025. [ABSTRACT FROM AUTHOR]

We have established the footprint-free Vietnamese human induced pluripotent stem cell (hiPSC) line, VRISGi002-A, from CD71 + CD235a + erythroid progenitor cells (EPCs) of a 27-year-old healthy donor. The EPCs were enriched from isolated peripheral blood and reprogrammed using Sendai viruses which carried the reprogramming factors c-MYC, SOX2, KLF4, and OCT4 under a feeder-free culture system. The established VRISGi002-A cell line expressed typical pluripotency markers, displayed a normal karyotype, and demonstrated the potential to differentiate into the three germ layers. This hiPSC line could serve as a Vietnamese healthy control model for physiological processes and drug screening.

Abstract Background Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. Methods We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. Results We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. Conclusion This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases.

Background/Aims: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. Methods: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. Results: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. Conclusion: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications.

Abstract Background While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming. Methods Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13–18 days). Results The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation. Conclusion In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs.

Noonan syndrome is an autosomal dominant developmental disorder. Although it is relatively common, and its phenotypical variability is well documented, its pathophysiology is not fully understood. Previously, with the aim of revealing the pathogenesis of genetic disorders, we reported the induction of cleidocranial dysplasia-specific human-induced pluripotent stem cells (hiPSCs) from patient's dental pulp cells (DPCs) under serum-free, feeder-free, and integration-free conditions. Notably, these cells showed potential for application to genetic disorder disease models. Furthermore, using similar procedures, we reported the induction of hiPSCs derived from peripheral blood mononuclear cells (PBMCs) of healthy volunteers. These methods are beneficial, because they are carried out without invasive and painful biopsies. Using those procedures, we reprogrammed DPCs and PBMCs that were derived from a patient with Noonan syndrome (NS) to establish NS-specific hiPSCs (NS-DPC-hiPSCs and NS-PBMC-hiPSCs, respectively). The induction efficiency of NS-hiPSCs was higher than that of WT-hiPSCs. We hypothesize that this was caused by high NANOG expression. Here, we describe the experimental results and findings related to NS-hiPSCs. This is the first report on the establishment of NS-hiPSCs and their disease modeling. [ABSTRACT FROM AUTHOR]

We generated a human induced pluripotent stem cell (hiPSC) line from erythroid progenitor cells (EPCs) of a 20-year-old female healthy donor using Sendai virus vector encoding Yamanaka factors OCT3/4, SOX2, c-MYC, and KLF4. The established hiPSCs showed a standard morphology and expression of typical undifferentiated stem cell markers, a normal karyotype (46, XX), and demonstrated potential for differentiation in vitro. Furthermore, they were successfully differentiated into cardiomyocytes that expressed cardiomyocyte-specific markers. The iPSC line and iPSC-derived cardiomyocytes will provide new avenues for future drug testing/development and personalized cell therapy for cardiovascular diseases (CVDs). [ABSTRACT FROM AUTHOR]

Abstract Background Human pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs). Methods Multiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation. Results The defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing. Conclusions We propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basal method, followed by adherent culture, passaging, and cryobanking.

Muscular dystrophy is a disease characterized by progressive muscle weakness and degeneration. There are currently no available treatments for most muscular diseases, such as muscular dystrophy. Moreover, current therapeutics are focused on improving the quality of life of patients by relieving the symptoms or stress caused by the disease. Although the causative genes for many muscular diseases have been identified, the mechanisms underlying their pathogenesis remain unclear. Patient-derived induced pluripotent stem cells (iPSCs) have become a powerful tool for understanding the pathogenesis of intractable diseases, as well as for phenotype screening, which can serve as the basis for developing new drugs. However, it is necessary to develop an efficient and reproducible myogenic differentiation system. Previously, we reported a tetracycline-inducible MyoD overexpression model of myogenic differentiation using human iPSCs (hiPSCs). However, this model has certain disadvantages that limit its use in various applications, such as a drug screening. In this study, we developed an efficient and reproducible myogenic differentiation system by further modifying our previous protocol. The new protocol achieves efficient differentiation of feeder-free hiPSCs to myogenic cells via small-scale culture in six-well microplates to large-scale culture in 384-well microplates for high-throughput applications.

Development and improvement of in vitro culture system supporting self‐renewal and unlimited proliferation of porcine pluripotent stem cells (pPSCs) is an indispensable process for the naïve pPSCs establishment. In this study, we modified the previous culture system and attempted to develop a novel chemically defined medium (KOFL) for the establishment of pPSCs. It has been cultured >45 passages with flat colony morphology and normal karyotypes in in vitro environment. These cells exhibited alkaline phosphatase activity and expressed pluripotency markers such as OCT4, SOX2, and NANOG, and also possessed differentiation abilities both in vitro and in vivo, proving by the formation of embryonic bodies and teratomas into three germ layers. Then the cells transfected with a green fluorescent protein (GFP) and the GFP positive cells contribute to the porcine preimplantation embryo development. In addition, these cells maintained long duration under feeder‐free condition. In conclusion, our results demonstrated that the pPSCs could be derived from preimplantation porcine embryos in serum‐free medium and cultured under the feeder‐free condition, providing an effective reference for further optimization of the pPSCs culture system. [ABSTRACT FROM AUTHOR]

Background: Disease modeling with patient-derived induced pluripotent stem cells (iPSCs) is a powerful tool for elucidating the mechanisms underlying disease pathogenesis and developing safe and effective treatments. Patient peripheral blood (PB) cells are used for iPSC generation in many cases since they can be collected with minimum invasiveness. To derive iPSCs that lack immunoreceptor gene rearrangements, hematopoietic stem and progenitor cells (HSPCs) are often targeted as the reprogramming source. However, the current protocols generally require HSPC mobilization and/or ex vivo expansion owing to their sparsity at the steady state and low reprogramming efficiencies, making the overall procedure costly, laborious, and time-consuming. Methods: We have established a highly efficient method for generating iPSCs from non-mobilized PB-derived CD34+ HSPCs. The source PB mononuclear cells were obtained from 1 healthy donor and 15 patients and were kept frozen until the scheduled iPSC generation. CD34+ HSPC enrichment was done using immunomagnetic beads, with no ex vivo expansion culture. To reprogram the CD34+-rich cells to pluripotency, the Sendai virus vector SeVdp-302L was used to transfer four transcription factors: KLF4, OCT4, SOX2, and c-MYC. In this iPSC generation series, the reprogramming efficiencies, success rates of iPSC line establishment, and progression time were recorded. After generating the iPSC frozen stocks, the cell recovery and their residual transgenes, karyotypes, T cell receptor gene rearrangement, pluripotency markers, and differentiation capability were examined. Results: We succeeded in establishing 223 iPSC lines with high reprogramming efficiencies from 15 patients with 8 different disease types. Our method allowed the rapid appearance of primary colonies (~ 8 days), all of which were expandable under feeder-free conditions, enabling robust establishment steps with less workload. After thawing, the established iPSC lines were verified to be pluripotency marker-positive and of non-T cell origin. A majority of the iPSC lines were confirmed to be transgene-free, with normal karyotypes. Their trilineage differentiation capability was also verified in a defined in vitro assay. Conclusion: This robust and highly efficient method enables the rapid and cost-effective establishment of transgene-free iPSC lines from a small volume of PB, thus facilitating the biobanking of patient-derived iPSCs and their use for the modeling of various diseases. [ABSTRACT FROM AUTHOR]

Although it is in its early stages, canine induced pluripotent stem cells (ciPSCs) hold great potential for innovative translational research in regenerative medicine, developmental biology, drug screening, and disease modeling. However, almost all ciPSCs were generated from fibroblasts, and available canine cell sources for reprogramming are still limited. Furthermore, no report is available to generate ciPSCs under feeder-free conditions because of their low reprogramming efficiency. Here, we reanalyzed canine pluripotency-associated genes and designed canine LIN28A, NANOG, OCT3/4, SOX2, KLF4, and C-MYC encoding Sendai virus vector, called 159cf. and 162cf. We demonstrated that not only canine fibroblasts but also canine urine-derived cells, which can be isolated using a noninvasive and straightforward method, were successfully reprogrammed with or without feeder cells. ciPSCs existed in undifferentiated states, differentiating into the three germ layers in vitro and in vivo. We successfully generated ciPSCs under feeder-free conditions, which can promote studies in veterinary and consequently human regenerative medicines., Competing Interests: Declaration of interests This study was also funded by Anicom Specialty Medical Institute. T.A. and K.W. are employees of Anicom Specialty Medical Institute. M.I. and M.N. are the inventors of the patent application (JP 6770224 B2, JP 7174958 B2, US 10544431 B1, EP 3246406 B1, TW I 696700 B, CN 107109400 B, SG 11201705820U, KR 6102459458 B1)., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background/Aims: Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine, disease modeling, and drug development. Thus, generation of non-integration and feeder-free iPSCs is highly desirable for clinical applications. Peripheral blood mononuclear cells (PBMCs) are an attractive resource for cell reprogramming because of their properties of easy accessibility and the limited invasiveness of blood collection. However, derivation of iPSCs is technically demanding due to the low reprogramming efficiency and nonadherent features of PBMCs. Methods: iPSCs were generated from PBMCs using non-integrative Sendai viruses carrying the reprogramming factors Oct4, Sox2, Klf4, and cMyc. The derived iPSCs were fully characterized at the levels of gene and protein, and then they were transplanted into immunocompromised mice for evaluation of in vivo differentiation potential. Three types of extracellular substrates (Geltrex, vitronectin, and rhLaminn-521) were tested for their influences on cell reprogramming under feeder-free conditions. We also sought to establish approaches to efficient cell recovery post-thaw and single cell passaging of iPSCs employing Rock inhibitors. Results: iPSCs were efficiently generated from PBMCs under feeder-free conditions. The derived iPSCs proved to be pluripotent and transgene-free. Furthermore, they demonstrated multi-lineage differentiation potentials when transplanted into immunocompromised mice. Among the three substrates, Geltrex and rhLaminin-521 could effectively support the initial cell reprogramming process, but vitronectin failed. However, the vitronectin, similar to Geltrex and rhLaminin-521, could effectively maintain cell growth and expansion of passaged iPSCs. In addition, RevitaCell supplement (RVC) was more potent on cell recovery post-thaw than Y-27632. And RVC and Y-27632 could significantly increase the cell survival when the cells were passaged in single cells, and they showed comparable effectiveness on cell recovery. Conclusion: We have successfully derived non-integration and feeder-free human iPSCs from peripheral blood cells, and established effective strategies for efficient cell recovery and single cell passaging. This study will pave the way to the derivation of clinical-grade human iPSCs for future clinical applications. [ABSTRACT FROM AUTHOR]

The 3D bioprinting and cell/tissue printing techniques open new possibilities for future applications. To facilitate the 3D bioprinting process, a large amount of living cells are required. Induced pluripotent stem cells (iPSCs) represent a promising cell source for bioprinting. However, the maintenance and expansion of undifferentiated iPSCs are expensive and time consuming. Therefore, in this study a culture method to obtain a sufficient amount of healthy and undifferentiated iPSCs in a short-term period was established. The iPSCs could be passaged for twice on tissue culture polystyrene (TCPS) dish with the conditional medium and could adapt to the feeder-free environment. Feeder-free dishes were further prepared from chitosan, chitosan-hyaluronan, silk fibroin, and polyurethane (PU1 and PU2) two-dimensional substrates. The iPSCs cultured on the chitosan substrates showed a higher proliferation rate without losing the stemness feature. Among the different materials, PU2 could be prepared as a thermoresponsive hydrogel, which was a potential ink for 3D bioprinting. The iPSCs cultured on PU2 substrates well survived when further embedded in PU2 hydrogel. Moreover, PU2 hydrogel printed with iPSCs remained structural integrity. The use of PU2 hydrogel to embed iPSCs reduced the injury to iPSCs by shear stress. These results indicate that iPSCs could be expanded on chitosan or PU2 membranes without the feeder layer and then printed in PU2 hydrogel. The combination of these steps could offer a new possibility for future applications of iPSC-based 3D bioprinting in tissue engineering. [ABSTRACT FROM AUTHOR]

Background: Human pluripotent stem cells (hPSCs) provide a promising cell source for ocular cell replacement therapy, but often lack standardized and xenogeneic-free culture and differentiation protocols. We aimed to develop a xeno- and feeder cell-free culture system for undifferentiated hPSCs along with efficient methods to derive ocular therapy target cells: retinal pigment epithelial (RPE) cells and corneal limbal epithelial stem cells (LESCs). Methods: Multiple genetically distinct hPSC lines were adapted to a defined, xeno-, and feeder-free culture system of Essential 8™ medium and laminin-521 matrix. Thereafter, two-stage differentiation methods toward ocular epithelial cells were established utilizing xeno-free media and a combination of extracellular matrix proteins. Both differentiation methods shared the same basal elements, using only minor inductive modifications during early differentiation towards desired cell lineages. The resulting RPE cells and LESCs were characterized after several independent differentiation experiments and recovery after xeno-free cryopreservation. Results: The defined, xeno-, and feeder-free culture system provided a robust means to generate high-quality hPSCs with chromosomal stability limited to early passages. Inductive cues introduced during the first week of differentiation had a substantial effect on lineage specification, cell survival, and even mature RPE properties. Derivative RPE formed functional epithelial monolayers with mature tight junctions and expression of RPE genes and proteins, as well as phagocytosis and key growth factor secretion capacity after 9 weeks of maturation on inserts. Efficient LESC differentiation led to cell populations expressing LESC markers such as p40/p63α by day 24. Finally, we established xeno-free cryobanking protocols for pluripotent hPSCs, hPSC-RPE cells, and hPSC-LESCs, and demonstrated successful recovery after thawing. Conclusions: We propose methods for efficient and scalable, directed differentiation of high-quality RPE cells and LESCs. The two clinically relevant cell types are generated with simple inductive modification of the same basalmethod, followed by adherent culture, passaging, and cryobanking. [ABSTRACT FROM AUTHOR]

Muscular dystrophy is a disease characterized by progressive muscle weakness and degeneration. There are currently no available treatments for most muscular diseases, such as muscular dystrophy. Moreover, current therapeutics are focused on improving the quality of life of patients by relieving the symptoms or stress caused by the disease. Although the causative genes for many muscular diseases have been identified, the mechanisms underlying their pathogenesis remain unclear. Patient-derived induced pluripotent stem cells (iPSCs) have become a powerful tool for understanding the pathogenesis of intractable diseases, as well as for phenotype screening, which can serve as the basis for developing new drugs. However, it is necessary to develop an efficient and reproducible myogenic differentiation system. Previously, we reported a tetracycline-inducible MyoD overexpression model of myogenic differentiation using human iPSCs (hiPSCs). However, this model has certain disadvantages that limit its use in various applications, such as a drug screening. In this study, we developed an efficient and reproducible myogenic differentiation system by further modifying our previous protocol. The new protocol achieves efficient differentiation of feeder-free hiPSCs to myogenic cells via small-scale culture in six-well microplates to large-scale culture in 384-well microplates for high-throughput applications. [ABSTRACT FROM AUTHOR]

Background: Embryonic stem (ES) cells are pluripotent cells with the ability to differentiate to any cell type of the resident organism. In recent years, significant advances have been made in using these cells to obtain large numbers of cardiomyocyte (CM) -like cells for scientific research and clinical application. A vast number of protocols have emerged describing differentiation methods without the use of animal serum or extracts restrictive for use in a human clinical setting. These techniques follow a complicated procedure, which although successful, show a relatively varied yield among cell batches. Results: We have designed a three-step differentiation protocol using defined reagents and a monolayer culture without feeder cells, avoiding embryoid body formation and multiple trypsin treatment, in which beating foci appeared as early as day 6 in in vitro differentiating conditions. Our results show a high yield of CM reaching approximately 60% of the differentiated cells after 13 days in vitro. Conclusions: We provide a fast, simple, reliable and reproducible protocol for inducing murine ES cells toward a CM-like phenotype comparable to available high-yield protocols, without the use of intermediate trypsinization/passage steps. Developmental Dynamics 245:157-165, 2016. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Human pluripotent stem cells hold great promise for their practical and scientific potentials. To improve understanding of self-renewal and differentiation, we previously reported a defined serum-free medium hESF9 could generate and maintain human induced pluripotent stem cells (iPSCs) in serum- and feeder-free culture conditions using retroviral vectors. To avoid the unpredictable side effects associated with retrovirus integration, we report here the successful generation of hiPSCs from dental pulp cells with a non-integrating replication-defective and persistent Sendai virus (SeVdp) vector expressing four key reprogramming genes. We found that hESF9 medium in combination with fibronectin are effective for generating and maintaining hiPSCs with SeVdp (KOSM). Using this system, pluripotent and self-renewing hiPSCs could be easily and stably generated and propagated. With this system, we successfully generated hiPSCs from cleidocranial dysplasia (CCD) caused by a heterozygous germ-line mutation of runt-related protein2 (RUNX2), which has an important role in the differentiation of osteoblasts and maturation of chondrocytes. This is the first report of the establishment of CCD-specific iPSCs. The cartilage in the teratomas of CCD-iPSCs showed abnormalities. These CCD-iPSCs would be beneficial to clarify the molecular mechanism and for development of medical applications. Moreover, it brings new pathophysiological role of RUNX2 in the differentiation of the human chondrocytes and osteocytes. [ABSTRACT FROM AUTHOR]

Background: Heterogeneity of endothelial cells (ECs) is a hallmark of the vascular system which may impact the development and management of vascular disorders. Despite the tremendous progress in differentiation of human embryonic stem cells (hESCs) towards endothelial lineage, differentiation into arterial and venous endothelial phenotypes remains elusive. Additionally, current differentiation strategies are hampered by inefficiency, lack of reproducibility, and use of animal-derived products. Methods: To direct the differentiation of hESCs to endothelial subtypes, H1- and H9-hESCs were seeded on human plasma fibronectin and differentiated under chemically defined conditions by sequential modulation of glycogen synthase kinase-3 (GSK-3), basic fibroblast growth factor (bFGF), bone morphogenetic protein 4 (BMP4) and vascular endothelial growth factor (VEGF) signaling pathways for 5 days. Following the initial differentiation, the endothelial progenitor cells (CD34+CD31+ cells) were sorted and terminally differentiated under serum-free conditions to arterial and venous ECs. The transcriptome and secretome profiles of the two distinct populations of hESC-derived arterial and venous ECs were characterized. Furthermore, the safety and functionality of these cells upon in vivo transplantation were characterized. Results: Sequential modulation of hESCs with GSK-3 inhibitor, bFGF, BMP4 and VEGF resulted in stages reminiscent of primitive streak, early mesoderm/lateral plate mesoderm, and endothelial progenitors under feeder- and serumfree conditions. Furthermore, these endothelial progenitors demonstrated differentiation potential to almost pure populations of arterial and venous endothelial phenotypes under serum-free conditions. Specifically, the endothelial progenitors differentiated to venous ECs in the absence of VEGF, and to arterial phenotype under low concentrations of VEGF. Additionally, these hESC-derived arterial and venous ECs showed distinct molecular and functional profiles in vitro. Furthermore, these hESC-derived arterial and venous ECs were nontumorigenic and were functional in terms of forming perfused microvascular channels upon subcutaneous implantation in the mouse. Conclusions: We report a simple, rapid, and efficient protocol for directed differentiation of hESCs into endothelial progenitor cells capable of differentiation to arterial and venous ECs under feeder-free and serum-free conditions. This could offer a human platform to study arterial-venous specification for various applications related to drug discovery, disease modeling and regenerative medicine in the future. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem (iPS) cells are generated from adult cells and are potentially of great value in regenerative medicine. Recently, it was shown that iPS cells can differentiate into ameloblast-like cells in cultures using feeder cells. In the present study, we sought to induce differentiation of ameloblast-like cells from iPS cells under feeder-free conditions using medium conditioned by cultured epithelial cell rests of Malassez (ERM) cells and gelatin-coated dishes. Two culture conditions were compared: co-cultures of iPS cells and ERM cells; and, culture of iPS cells in ERM cell-conditioned medium. Differentiation of ameloblast-like cells in the cultures was assessed using real-time RT-PCR assays of expression of the marker genes keratin 14, amelogenin, and ameloblastin and by immunocytochemical staining for amelogenin. We found greater evidence of ameloblast-like cell differentiation in the cultures using the conditioned medium. In the latter, the level of amelogenin expression increased daily and was significantly higher than controls on the 7th, 10th, and 14th days. Expression of ameloblastin also increased daily and was significantly higher than controls on the 14th day. The present study demonstrates that mouse iPS cells can be induced to differentiate into ameloblast-like cells in feeder-free cell cultures using ERM cell-conditioned medium and gelatin-coated dishes. [ABSTRACT FROM AUTHOR]

Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum- and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells. [ABSTRACT FROM AUTHOR]

We have established the footprint-free Vietnamese human induced pluripotent stem cell (hiPSC) line, VRISGi002-A, from CD71 + CD235a + erythroid progenitor cells (EPCs) of a 27-year-old healthy donor. The EPCs were enriched from isolated peripheral blood and reprogrammed using Sendai viruses which carried the reprogramming factors c-MYC, SOX2, KLF4, and OCT4 under a feeder-free culture system. The established VRISGi002-A cell line expressed typical pluripotency markers, displayed a normal karyotype, and demonstrated the potential to differentiate into the three germ layers. This hiPSC line could serve as a Vietnamese healthy control model for physiological processes and drug screening. [ABSTRACT FROM AUTHOR]

. Purpose: Ocular surface reconstruction with cultivated oral mucosal epithelial transplantation technique is a viable treatment option for severe ocular surface injuries and diseases with limbal stem cell deficiency. Currently, this technique is based on utilization of xenogenic, allogenic or undefined components such as murine 3T3 feeders, serum and amniotic membrane. In this study, we aimed to find a more defined culture method to generate stratified human oral mucosal epithelium. Methods: In this study, we have examined the formation of stratified cell sheets from human oral mucosal epithelial cells under serum-free culture environment both in the absence and presence of fibroblast-conditioned culture medium and elevated epidermal growth factor (EGF) concentration. Results: In all examined culture conditions, the cultivated oral epithelial cells formed a stratified tissue, which was positive for keratins K3/12, K4 and K13. The tissue-engineered oral epithelia also expressed proliferation and progenitor markers Ki67 and p63 in the basal layer of the cell sheets, suggesting that the epithelia still had regenerative capacity. The cultures presented expression of tight junction proteins ZO-1 and occludin and high transepithelial electrical resistance values. Conclusion: In this culture method, we have been able to produce stratified cell sheets successfully without serum, conditioning of the medium or increased EGF concentration. We provide a novel protocol to produce tight multi-layered epithelium with proliferative potential, which can be easily adapted for cultivated oral mucosal epithelial transplantation. [ABSTRACT FROM AUTHOR]

Abstract: Induced pluripotent stem cells (iPSC) have successfully been derived from somatic fibroblasts through transfection of synthetic modified mRNA encoding transcription factors. This technique obviates the use of recombinant DNA and viral vectors in cellular reprogramming. The present study derived iPSC from adipose-derived mesenchymal stem cells (of a 50-year-old female patient) by utilizing a similar technique, but with defined culture medium without feeder cells, during both reprogramming and propagation. Clonal selection was performed to yield 12 putative iPSC lines from individual colonies of nascent reprogrammed cells, starting from 150,000 cells. However, only seven lines maintained their undifferentiated state after 10 continuous serial passages. These seven lines were then subjected to a rigorous battery of analyses to confirm their identity as iPSC. These tests included immunostaining, flow cytometry, qRT-PCR, in vitro differentiation assay, and teratoma formation assay within SCID mice. Positive results were consistently observed in all analyses, thus verifying the cells as fully reprogrammed iPSC. While all 7 iPSC lines displayed normal karyogram up to passage 13, chromosomal anomalies occurred in 4 of 7 lines with extended in vitro culture beyond 24 serial passages. Only three lines retained normal karyotype of 46,XX. The remaining four lines displayed mosaicism of normal and abnormal karyotypes. Hence, this study successfully derived iPSC from abundant and easily accessible adipose tissues of a middle-aged patient; utilizing a mRNA-based integration-free technique under feeder-free conditions. This is a step forward in translating iPSC into personalized regenerative medicine within the clinic. [Copyright &y& Elsevier]

Abstract: The components of extracellular matrix (ECM) may substitute for feeder layers that promote the self-renewal pathways in embryonic stem cells. Surface modification of electrospun nanofibrous scaffolds have been studied to closely resemble natural ECMs and support in vitro and in vivo proliferation, pluripotency and differentiation of stem cells. In this study, we analyzed the maintenance of stemness and pluripotency of the mouse embryonic stem cell (mESC) following feeder-free culture on collagen-grafted polyethersulfone (PES-COL) electrospun nanofibrous scaffold. Our results showed that, the mESCs cultured for seven passages on PES-COL scaffolds had a typical undifferentiated morphology, enhanced proliferation, stable diploid normal karyotype, and continued expression of stemness and pluripotency-associated markers, Oct-4, Nanog, SSEA-1, and Alkaline phosphatase (ALP) in comparison with PES scaffolds and gelatin-coated plate. Moreover, these cells retained their in vitro and in vivo pluripotency. Our results indicated the enhanced infiltration and teratoma formation of mESCs in PES-COL. Collagen-grafted polyethersulfone nanofibrous scaffold has potential for feeder-free culture of pluripotent stem cells because of its 3-dimensional structure and bioactivity which enhance pluripotency, proliferation, differentiation, and infiltration of embryonic stem cells. [Copyright &y& Elsevier]

Patient-specific therapeutic cells derived from induced pluripotent stem (iPS) cells may bypass the ethical issues associated with embryonic stem (ES) cells and avoid potential immunological reactions associated with allogenic transplantation. It is critical, for the ultimate clinical applicability of iPS cell-derived therapies, to establish feeder-free cultures that ensure efficient differentiation of iPS cells into therapeutic progenitors. It is also necessary to understand if iPS cell-derived progenitors differ from those derived from ES cells. In this study, we compared the efficiency of three different feeder-free cultures for differentiating mouse iPS cells into ckit+sca1+ hematopoietic progenitor cells (HPCs) and compared how differentiation and functionality varies between ES and iPS cells. Our results indicated that both iPS and ES cells can be efficiently differentiated into HPCs in suspension cultures supplemented with secretion factors from mouse bone marrow stromal cells (OP9-DL1 conditioned medium). The functionality of these cells was demonstrated by differentiation into CD11c+ dendritic cells (DCs). Both ES and iPS-derived DCs expressed activation molecules (CD86, CD80) in response to LPS stimulation and stimulated T cell proliferation in a mixed lymphocyte reaction (MLR). Extensive quantitative RT-PCR studies were used to study the differences in gene expression profiles of ckit+sca1+ cells generated from the various culture systems as well as differences between ES-derived and iPS-derived cells. We conclude that a feeder-free system using stromal conditioned medium can efficiently generate HPCs as well as functional DCs from iPS cells and the generated cells have similar gene expression profile as those from ES cells. [ABSTRACT FROM AUTHOR]

The greatest therapeutic promise of human embryonic stem cells (hESC) is to generate specialized cells to replace damaged tissue in patients suffering from various degenerative diseases. However, the signaling mechanisms involved in lineage restriction of ESC to adopt various cellular phenotypes are still under investigation. Furthermore, for progression of hESC-based therapies towards clinical applications, appropriate culture conditions must be developed to generate genetically stable homogenous populations of cells, to hinder possible adverse effects following transplantation. Other critical challenges that must be addressed for successful cell implantation include problems related to survival and functional efficacy of the grafted cells. This review initially describes the derivation of hESC and focuses on recent advances in generation, characterization, and maintenance of these cells. We also give an overview of original and emerging differentiation strategies used to convert hESC to different cell types. Finally, we will discuss transplantation studies of hESC-derived cells with respect to safety and functional recovery. [ABSTRACT FROM AUTHOR]

This report summarises our efforts in deriving, characterising and banking of 20 different human embryonic stem cell lines. We have derived a large number of human embryonic stem cell lines between 2001 and 2005. One of these cell lines was established under totally xeno-free culture conditions. In addition, several subclones have been established, including a karyoptypical normal clone from a trisomic mother line. A master cell banking system has been utilised in concert with an extensive characterisation programme, ensuring a supply of high quality pluripotent stem cells for further research and development. In this report we also present the first data on a proprietary novel antibody, hES-Cellect, that exhibits high specificity for undifferentiated hES cells. In addition to the traditional manual dissection approach of propagating hES cells, we here also report on the successful approaches of feeder-free cultures as well as single cell cultures based on enzymatic digestion. All culture systems used as reported here have maintained the hES cells in a karyotypical normal and pluripotent state. These systems also have the advantage of being the principal springboards for further scale up of cultures for industrial or clinical applications that would require vastly more cells that can be produced by mechanical means. [ABSTRACT FROM AUTHOR]

Abstract: Human embryonic stem cells (hESCs) are routinely cultured on fibroblast feeder layers or in fibroblast-conditioned medium, which requires continued supply of feeder cells and poses the risks of xenogenic contamination and other complications such as feeder-dependent outcome. Here, we demonstrate a strategy that supports sustained self-renewal of hESCs in a three-dimensional porous natural polymer scaffold, comprised of chitosan and alginate, without the support of feeder cells or conditioned medium. We assessed the pluripotency of the renewed hESCs both in vitro by evaluation of cellular proliferation, functionality, and gene activities for 21 days, and in vivo by implantation of the stem cell populated scaffolds in an immunodeficient mouse model to induce teratoma formation. The self-renewed stem cells can be easily recovered for subculture by decomposing the scaffold under a mild condition. We further subcultured recovered hESCs for 14 days and verified their pluripotency. In addition to providing a clean environment for stem cell renewal, this strategy, with the demonstrated biocompatibility and biodegradability of chitosan and alginate, may potentially allow for the direct implantation of stem cell populated scaffolds for a broad spectrum of applications in tissue engineering and regenerative medicine. [Copyright &y& Elsevier]

Abstract: Pluripotent stem cells can be induced from somatic cells by the transcription factors Oct3/4, Sox2, c-Myc and Klf4 when co-cultured with mouse embryonic fibroblast (MEF) feeder cells. To date, the role of the feeder cells in the reprogramming process remains unclear. In this study, using a comparative analysis, we demonstrated that MEF feeder cells did not accelerate reprogramming or increase the frequency of induced pluripotent stem (iPS) cell colonies. However, feeder conditions did improve the growth of primary iPS colonies and were necessary for passaging the primary colonies after reprogramming was achieved. We further developed a feeder-free culture system for supporting iPS growth and sustaining pluripotency by adding bFGF and activin A (bFA) to the medium. These data will facilitate the generation of human iPS cells without animal feeders for regenerative medicine. [Copyright &y& Elsevier]

Matrigel is routinely used as a coating material in the feeder-free culture system of human embryonic stem cells (hESCs). However, matrigel is costive and inconvenient to use. In this study, the possibility of using gelatin as an alternative coating material was investigated. The results showed that, after trypsinization, hESCs were maintained undifferentiated on gelatin. These hESCs expressed pluripotent markers, formed teratoma and maintained a normal karyotype. As measured at passage 10, the hESCs expressed a high level of Oct4 on both gelatin and Matrigel. hESCs growing on gelatin formed AP-positive colonies in similar size and number to those growing on Matrigel (P > 0.05). Moreover, hESCs growing on gelatin contained a comparable percentage of SSEA-4-positive cells to those growing on Matrigel (95.1% vs.94.3%, P > 0.05). H-1 hESCs were maintained undifferentiated on gelatin for 20 passages and remained the stable normal karyotype. This gelatin-based culture protocol may allow us to propagate hESCs in large scale, with less cost. [ABSTRACT FROM AUTHOR]

A paracrine regulation was recently proposed in human embryonic stem cells (hESCs) grown in mouse embryonic fibroblast (MEF)-conditioned media (MEF-CM), where hESCs spontaneously differentiate into autologous fibroblast-like cells to maintain culture homeostasis by producing TGF-β and insulin-like growth factor-II (IGF-II) in response to basic fibroblast growth factor (bFGF). Although the importance of TGF-β family members in the maintenance of pluripotency of hESCs is widely established, very little is known about the role of IGF-II. In order to ease hESC culture conditions and to reduce xenogenic components, we sought (i) to determine whether hESCs can be maintained stable and pluripotent using CM from human foreskin fibroblasts (HFFs) and human mesenchymal stem cells (hMSCs) rather than MEF-CM, and (ii) to analyze whether the cooperation of bFGF with TGF-β and IGF-II to maintain hESCs in MEF-CM may be extrapolated to hESCs maintained in allogeneic mesenchymal stem cell (MSC)-CM and HFF-CM. We found that MSCs and HFFs express all FGF receptors (FGFR1-4) and specifically produce TGF-β in response to bFGF. However, HFFs but not MSCs secrete IGF-II. Despite the absence of IGF-II in MSC-CM, hESC pluripotency and culture homeostasis were successfully maintained in MSC-CM for over 37 passages. Human ESCs derived on MSCs and hESCs maintained in MSC-CM retained hESC morphology, euploidy, expression of surface markers and transcription factors linked to pluripotency and displayed in vitro and in vivo multilineage developmental potential, suggesting that IGF-II may be dispensable for hESC pluripotency. In fact, IGF-II blocking had no effect on the homeostasis of hESC cultures maintained either on HFF-CM or on MSC-CM. These data indicate that hESCs are successfully maintained feeder-free with IGF-II-lacking MSC-CM, and that the previously proposed paracrine mechanism by which bFGF cooperates with TGF-β and IGF-II in the maintenance of hESCs in MEF-CM may not be fully extrapolated to hESCs maintained in CM from human MSCs.Cell Research (2009) 19:698–709. doi: 10.1038/cr.2009.35; published online 24 March 2009 [ABSTRACT FROM AUTHOR]

Human embryonic stem cells (hESCs) are often cocultured on mitotically inactive fibroblast feeder cells to maintain their undifferentiated state. Under these growth conditions, hESCs form multilayered colonies of morphologically heterogeneous cells surrounded by flattened mesenchymal cells. In contrast, hESCs grown in feeder cell-conditioned medium on Matrigel instead tend to grow as monolayers with uniform morphology. Using mass spectrometry and immunofluorescence microscopy, we showed that hESCs under these conditions primarily express proteins belonging to epithelium-related cell-cell adhesion complexes, including adherens junctions, tight junctions, desmosomes, and gap junctions. This indicates that monolayers of hESCs cultured under feeder-free conditions retain a homogeneous epithelial phenotype similar to that of the upper central cell layer of colonies maintained on feeder cells. Notably, feeder-free hESCs also coexpressed vimentin, which is usually associated with mesenchyme, suggesting that these cells may have undergone epithelium-to-mesenchyme transitions, indicating differentiation. However, if grown on a "soft" substrate (Hydrogel), intracellular vimentin levels were substantially reduced. Moreover, when hESCs were transferred back to feeder cells, expression of vimentin was again absent from the epithelial cell population. These results imply that on tissue culture substrates, vimentin expression is most likely a stress-induced response, unrelated to differentiation. [ABSTRACT FROM AUTHOR]

A major limitation in developing applications for the use of human embryonic stem cells (HESCs) is our lack of knowledge of their responses to specific cues that control self-renewal, differentiation, and lineage selection. HESCs are most commonly maintained on inactivated mouse embryonic fibroblast feeders in medium supplemented with FCS, or proprietary replacements such as knockout serum-replacement together with FGF-2. These undefined culture conditions hamper analysis of the mechanisms that control HESC behavior. We have now developed a defined serumfree medium, hESF9, for the culture of HESC5 on a type I-collagen substrate without feeders. In contrast to other reported media for the culture of HESCs, this medium has a lower osmolarity (292 mosmol/liter), i.-ascorbic acid-2-phosphate (0.1 μg/ml), and hepanfl. Insulin, transferrin, albumin conjugated with oleic acid, and FGF-2 (10 ng/ml) were the only protein components. Further, we found that HESCs would proliferate in the absence of exogenous FGF-2 if heparin was also present. However, their growth was enhanced by the addition of FGF-2 up to 10 ng/mI although higher concentrations were deleterious in the presence of heparin. [ABSTRACT FROM AUTHOR]

Abstract: Previous studies have shown that cultivation of undifferentiated human embryonic stem (hES) cells requires human fibroblasts (hF) or mouse embryonic fibroblast (mEF) feeders or a coating matrix such as laminin, fibronectin or Matrigel™ in combination with mEF or hF conditioned medium. We here demonstrate a successful feeder-free and matrix-free culture system in which undifferentiated hES cells can be cultured directly on plastic surfaces without any supportive coating, in a hF conditioned medium. The hES cells cultured directly on plastic surfaces grow as colonies with morphology very similar to cells cultured on MatrigelTM. Two hES cell lines SA167 and AS034.1 were adapted to matrix-free growth (MFG) and have so far been cultured up to 43 passages and cryopreserved successfully. The lines maintained a normal karyotype and expressed the expected marker profile of undifferentiated hES cells for Oct-4, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and SSEA-1. The hES cells formed teratomas in SCID mice and differentiated in vitro into derivates of all three germ layers. Thus, the MFG-adapted hES cells appear to retain pluripotency and to remain undifferentiated. The present culture system has a clear potential to be scaleable up to a manufacturing level and become the preferred culture system for various applications such as cell therapy and toxicity testing. [Copyright &y& Elsevier]