Feeder-free Conditions Research Articles

Induction of integration-free human-induced pluripotent stem cells under serum- and feeder-free conditions

Human-induced pluripotent stem cells (hiPSCs) have shown great potential toward practical and scientific applications. We previously reported the generation of human dental pulp stem cells using non-integrating replication-defective Sendai virus (SeVdp) vector in feeder-free culture with serum-free medium hESF9. This study describes the generation of hiPSCs from peripheral blood mononuclear cells to increase the donor population, while reducing biopsy invasiveness. From 6-d-old primary culture of peripheral blood mononuclear cells (PBMCs) with IL-2, hiPSCs were established using SeVdp(KOSM)302L with recombinant Laminin-511 E8 fragments under serum-free condition. The established PBMC-derived hiPSCs showed pluripotency and differentiation ability both in vivo and in vitro. In addition, we evaluated microarray data from PBMC- and dental pulp–derived hiPSCs. These hiPSCs will be beneficial for characterizing the molecular mechanisms of cellular differentiation and may provide useful substrates for developing cellular therapeutics.

Effect of cell culture biomaterials for completely xeno-free generation of human induced pluripotent stem cells

Human induced pluripotent stem cells (hiPSCs) were generated on several biomaterials from human amniotic fluid in completely xeno-free and feeder-free conditions via the transfection of pluripotent genes using a nonintegrating RNA Sendai virus vector. The effect of xeno-free culture medium on the efficiency of the establishment of human amniotic fluid stem cells from amniotic fluid was evaluated. Subsequently, the effect of cell culture biomaterials on the reprogramming efficiency was investigated during the reprogramming of human amniotic fluid stem cells into hiPSCs. Cells cultured in laminin-511, laminin-521, and Synthemax II-coated dishes and hydrogels having optimal elasticity that were engrafted with specific oligopeptides derived from vitronectin could be reprogrammed into hiPSCs with high efficiency. The reprogrammed cells expressed pluripotency proteins and had the capability to differentiate into cells derived from all three germ layers in vitro and in vivo. Human iPSCs could be generated successfully and at high efficiency (0.15–0.25%) in completely xeno-free conditions from the selection of optimal cell culture biomaterials.

Identification of active sequences in human laminin α5 G domain.

Human laminin-511 (α5β1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6β1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder-free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin-511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1-hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide-coated plate and peptide-chitosan matrix (peptide-ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26-coated plates and hA5G74-ChtMs promoted integrin β1-mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin-511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.

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

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

Highly sulfated hyaluronic acid maintains human induced pluripotent stem cells under feeder-free and bFGF-free conditions

Human induced pluripotent stem (hiPS) cells are attracting attention as a tool for regenerative medicine. However, several problems need to be overcome for their widespread and safe use, for example, the high cost of maintaining hiPS cells and the possibility of xenogeneic cell contamination in hiPS cell cultures. One of the main contributors to the high cost of maintaining hiPS cells is basic fibroblast growth factor (bFGF), which is essential for such cultures. Xenogeneic contamination can occur because of the use of mouse-derived feeder cells to culture hiPS cells. To overcome the problems of cell culture cost and xenogeneic contamination, we have developed a novel culture method in which the undifferentiated state and pluripotency of hiPS cells can be maintained under feeder-free and bFGF-free conditions. Our new approach involves the addition to the culture medium of highly sulfated hyaluronic acid (HA-HS), in which the hydroxyl groups of d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) are chemically sulfated. HA-HS promotes bFGF signaling and maintains the undifferentiated state and pluripotency of hiPS cells under feeder-free and bFGF-free conditions. By contrast, non-sulfated hyaluronic acid and low sulfated hyaluronic acid do not maintain the undifferentiated state and pluripotency of hiPS cells. These results indicate that the maintenance of hiPS cells under feeder-free and bFGF-free conditions is an HA-HS specific effect. This study is the first to demonstrate the effects of sulfated hyaluronic acid on mammalian pluripotent stem cells, and provides a novel method for maintaining hiPS cells using HA-HS.

Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds.

SummaryA simple induction protocol to differentiate chondrocytes from pluripotent stem cells (PSCs) using small-molecule compounds is beneficial for cartilage regenerative medicine and mechanistic studies of chondrogenesis. Here, we demonstrate that chondrocytes are robustly induced from human PSCs by simple combination of two compounds, CHIR99021, a glycogen synthase kinase 3 inhibitor, and TTNPB, a retinoic acid receptor (RAR) agonist, under serum- and feeder-free conditions within 5–9 days. An excellent differentiation efficiency and potential to form hyaline cartilaginous tissues in vivo were demonstrated. Comprehensive gene expression and open chromatin analyses at each protocol stage revealed step-by-step differentiation toward chondrocytes. Genome-wide analysis of RAR and β-catenin association with DNA showed that retinoic acid and Wnt/β-catenin signaling collaboratively regulated the key marker genes at each differentiation stage. This method provides a promising cell source for regenerative medicine and, as an in vitro model, may facilitate elucidation of the molecular mechanisms underlying chondrocyte differentiation.

Abstract 543: The Metabolites of Human Cardiac Precursors Enhanced Cardiomyogenic Differentiation of hiPSC

Background: Wnt signaling pathways are involved in cardiomyogenic differentiation. Besides that, nutrients and metabolism may also influence cell differentiation. However, it is still unclear whether the cell lineage commitment could be controlled by the metabolites of its precursor cells. The aim of this study is to examine whether the metabolites of cardiac precursors could determine cardiomyogenic differentiation fate of hiPSC. Methods: hiPSC was maintained in serum-free and feeder-free monolayer condition. After CHIR99021-induced mesendodermal differentiation for two days, IWP4 was added to inhibit Wnt signaling and to induce myogenic differentiation for another two days. After wash, the culture medium was collected during cell differentiation period of day 5-10 (d5-10-diff-medium). ExoQuick-TC kit was used to extract the exosomes in the medium. Cardiomyocytes were identified by immunocytochemistry staining with mouse monoclonal antibody against cardiac sarcomeric actinin alpha, and were counted the percentage by flow cytometry. Results: The exosome-free d5-10-diff-medium was found to be able to dramatically induce cardiomyogenic differentiation only in CHIR99021-induced mesendodermal cells, rather than directly in hiPSC, in the absence of Wnt inhibitor. The metabolic profiling of the exosome-free d5-10-diff-medium was further characterized by LC-MS. After functional validation, the effective metabolites for the induction of cardiomyogenic differentiation were identified and the possible signaling pathways were analyzed. Conclusion: This study demonstrated that the metabolites of cardiac precursors could control cardiac cell commitment by driving the development of cardiac metabolic system.

Baboon induced pluripotent stem cell generation by piggyBac transposition of reprogramming factors.

Clinical application of regenerative therapies using embryonic or induced pluripotent stem cells is within reach. Progress made during recent years has encouraged researchers to address remaining open questions in order to finally translate experimental cell replacement therapies into application in patients. To achieve this, studies in translationally relevant animal models are required to make the final step to the clinic. In this context, the baboon (Papio anubis) may represent a valuable nonhuman primate (NHP) model to test cell replacement therapies because of its close evolutionary relationship to humans and its large body size. In this study, we describe the reprogramming of adult baboon skin fibroblasts using the piggyBac transposon system. Via transposon-mediated overexpression of six reprogramming factors, we generated five baboon induced pluripotent stem cell (iPSC) lines. The iPSC lines were characterized with respect to alkaline phosphatase activity, pluripotency factor expression analysis, teratoma formation potential, and karyotype. Furthermore, after initial cocultivation with mouse embryonic fibroblasts, we were able to adapt iPSC lines to feeder-free conditions. In conclusion, we established a robust and efficient protocol for iPSC generation from adult baboon fibroblasts.

Robust induction of retinal pigment epithelium cells from human induced pluripotent stem cells by inhibiting FGF/MAPK signaling

Functional decline and loss of the retinal pigment epithelium (RPE) cause retinal diseases. Clinical studies using human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived RPE cells have shown the safety and potential efficacy of hESC/iPSC-RPE cell transplantation. However, the production of RPE cells remains somewhat problematic. hESCs/iPSCs co-cultured with mouse feeder cells carry the risk of xeno-transmitted infections and immune reactions. Moreover, increasing the rate of cell division to ensure the quantity and purity of cells with low differentiation efficiency elevates the risk of gene mutations and chromosomal abnormalities. Here, we show that the transient inhibition of the FGF/MAPK signaling pathway during the hiPSC maintenance period markedly promotes RPE differentiation efficiency under feeder-free culture conditions. Blockage of FGF/MAPK signal induces neural differentiation and generates RPE cells without subsequent inhibition of Wnt and Nodal signals, which is known to be effective for retinal specification. We also found that additional inhibition of the PKC or BMP signaling pathway together with FGF/MAPK signal inhibition further elevates RPE differentiation efficiency. Our study will be helpful for producing clinical-grade RPE cells and will facilitate the development of therapies using hESC/hiPSC-RPE cells.

Robust and highly efficient hiPSC generation from patient non-mobilized peripheral blood-derived CD34+ cells using the auto-erasable Sendai virus vector

BackgroundDisease 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.MethodsWe 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.ResultsWe 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.ConclusionThis 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.

Synergistic effect of basic fibroblast growth factor (bFGF) and epidermal growth factor on derivation of camel (Camelus dromedarius) trophoblast stem cells.

This study aimed to optimize the derivation of trophectoderm from in vitro-produced camel embryos under feeder-free culture conditions using the basement membrane matrix Matrigel. Trophoblastic vesicles were obtained through mechanical microdissection of in vitro-produced camel (Camelus dromedarius) embryos. Supplementing the culture medium with 10 ng/ml of epidermal growth factor and 10 ng/ml fibroblast growth factor improved the attachment and subsequent outgrowths of cultured trophoblastic vesicles when compared with the control group and the groups supplemented individually with each growth factor. The expression levels of pluripotency genes octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), myelocytomatosis proto-oncogene (c-Myc) and anti-apoptotic gene B-cell lymphoma 2 (Bcl2) were increased in trophoblastic vesicles supplemented with both growth factors when compared with the control group. Conversely, both growth factors decreased the expression of apoptotic genes tumour protein p53 (p53) and Bcl-2-associated X protein (Bax). To the best of our knowledge, this may be the first report describing the derivation of trophoblast stem cells from in vitro-produced camel embryos.

Applying hydrodynamic pressure to efficiently generate induced pluripotent stem cells via reprogramming of centenarian skin fibroblasts.

Induced pluripotent stem cell (iPSC)-technology is an important platform in medicine and disease modeling. Physiological degeneration and disease onset are common occurrences in the aging population. iPSCs could offer regenerative medical options for age-related degeneration and disease in the elderly. However, reprogramming somatic cells from the elderly is inefficient when successful at all. Perhaps due to their low rates of replication in culture, traditional transduction and reprogramming approaches with centenarian fibroblasts met with little success. A simple and reproducible reprogramming process is reported here which enhances interactions of the cells with the viral vectors that leads to improved iPSC generation. The improved methods efficiently generates fully reprogrammed iPSC lines from 105–107 years old subjects in feeder-free conditions using an episomal, Sendai-Virus (SeV) reprogramming vector expressing four reprogramming factors.In conclusion, dermal fibroblasts from human subjects older than 100 years can be efficiently and reproducibly reprogrammed to fully pluripotent cells with minor modifications to the standard reprogramming procedures. Efficient generation of iPSCs from the elderly may provide a source of cells for the regeneration of tissues and organs with autologous cells as well as cellular models for the study of aging, longevity and age-related diseases.

Cooperation of FGF/MEK/ERK and Wnt/\u03b2-catenin pathway regulators to promote the proliferation and pluripotency of mouse embryonic stem cells in serum- and feeder-free conditions

The FGF/MEK/ERK and Wnt/β-catenin signaling pathways have previously been proved to regulate mouse embryonic stem cell (mESCs) function. However, the relationships between these two pathways, especially their different functions on proliferation and pluripotency of mESCs, were rarely mentioned. Here, we investigated the effects of FGF/MEK/ERK and Wnt/β-catenin pathway regulators and their combinations on the proliferation and pluripotency of mESCs under serum- and feeder-free conditions. We found that MEK inhibitor PD0325901 and FGFR inhibitor SU5402 has paradoxical function on mESCs; one could promote proliferation along with differentiation and the other one could improve pluripotency while impairing cell proliferation. The combination of these two kinds of inhibitors could better regulate FGF/MEK/ERK pathway. Wnt/β-catenin pathway regulators SB216763 led to differentiation while promoting proliferation of mESCs. When we used FGF/MEK/ERK and Wnt/β-catenin pathway regulators in combination, the total expansion fold of mESCs reached 318.78 ± 47.95 and the proportion of SSEA-1-positive cells reached 82.40 ± 2.74% which were significantly higher than using the regulators alone. This finding indicates that regulators of FGF/MEK/ERK and Wnt/β-catenin pathways play different roles in the regulatory networks of mESCs. Their combination can better maintain the undifferentiated state and promote the proliferation of mESCs under serum- and feeder-free conditions.

Clinical Application of Cultured Stratified Epithelial Sheets Grown Under Feeder or Feeder-Free Conditions for Stable Vitiligo.

Autologous cultured therapy has emerged as an effective treatment for stable vitiligo. However, culture methods may include harmful agents and be unsuitable for therapeutic use in humans. To investigate the safety and efficacy of autologous cultured epithelial sheets propagated under serum-free and feeder-free conditions for the treatment of stable vitiligo. Twenty-eight patients with stable vitiligo were included in this study. Keratinocytes and melanocytes from 14 patients were cultured under serum-free, feeder-free conditions (Group A). Epithelial cells from the remaining 14 patients were cultured according to Rheinward and Green's technique (Group B). Patients were followed up at 1, 3, 6, and 12 months after transplantation. The epithelial sheets cultured in Group A were thinner and more fragile than Group B, but there were no significant differences in repigmentation between the 2 groups. At 12-month follow-up, in Group A, repigmentation at graft sites was classified as excellent in 9 patients and good in 2 patients. In Group B, repigmentation was excellent in 8 patients and good in 4 patients. Scars at the donor sites were the most frequent adverse events associated with the procedure. Autologous epithelial sheet cultured in serum-free, feeder-free conditions is a safe and efficacious approach to cure stable vitiligo.

Commentary on Clinical Application of Cultured Stratified Epithelial Sheets Grown Under Feeder or Feeder-Free Conditions for Stable Vitiligo.

Commentary on Clinical Application of Cultured Stratified Epithelial Sheets Grown Under Feeder or Feeder-Free Conditions for Stable Vitiligo.

Expansion and maintenance of primary corneal epithelial stem/progenitor cells by inhibition of TGFβ receptor I-mediated signaling

Transforming growth factor β (TGFβ) signaling is one of the most important signaling pathways regulating cell behavior in ocular tissues. Its functions are mainly linked to tissue fibrosis and inflammatory responses in ophthalmology. In epithelial cells, however, the growth inhibitory activity of TGFβ was reported in both non-ocular and ocular tissues. Since TGFβ is a bifunctional regulator that either inhibits or stimulates cell proliferation according to the specific context, we examined the effect of inhibition of TGFβ receptor (TβR) I-mediated signaling on primary corneal epithelial cells (CECs) in serum- and feeder-free conditions. The mouse CECs were isolated from the eyeballs of 6–8 weeks old female C57BL/6 mice using dispase and trypsin separately, cultivated in defined Keratinocyte serum-free medium (KSFM) with supplements (the complete medium) without feeder layer. Cells were divided into three groups, those cultured in complete medium additionally supplemented with 10 μM SB-431542, a specific inhibitor of TβR-I, were SB-CECs; those cultured in complete medium additionally supplemented with 10 ng/ml SRI-011381, a TGF-beta signaling agonist, were SRI-CECs; those cultured in complete medium without SB-431542 or SRI-011381 were control CECs. The growth rate and morphology were analyzed by light microscopy. The identity and stemness of cells was investigated through marker staining of p63, inhibitor of differentiation 1 (ID1), cytokeratin 12 (K12), cytokeratin 14 (K14), PAX6, pSmad3, alpha smooth muscle Actin (αSMA) and E-cadherin (E-cad); Real-time quantitative (RT-PCR) analysis of p63; Western blot analysis of ID1; as well as colony forming assay, sphere forming assay, healing wound in vitro assay and air-lifting interface assay. The results showed SB-CECs subcultured steadily, achieved sustained expansion, and expanded almost thrice faster than control CECs. Expanded SB-CECs exhibited smaller and more compact morphology, up-regulated p63 and ID1, as well as better performed colony-forming capacity, sphere-forming capacity, in vitro wound healing capacity, and the capacity to stratify and differentiate on air-lifting interface. Preliminary tests on human limbal epithelial cells (HLECs) showed the same results as mouse CECs. Interestingly, the ID1 expression pattern was almost identical to p63, the typical marker for corneal epithelial stem/progenitor cell (CESC/CEPC), in cultured CECs and normal corneal sections. Since ID1 has been proven to be regulated negatively by TGFβ signaling in epithelial cells and plays a role in blocking cell differentiation, its derepression by TβR-I inhibitor could be, at least in part, the underlying cause of CESC/CEPC expansion and the synchronously up-regulated expression of p63 in SB-CECs. In conclusion, inhibition of TβR-I-mediated signaling, CESCs/CEPCs achieved efficient long-term expansion in a feeder- and serum-free condition in vitro. And derepression of ID1 could be the underlying cause. Meanwhile, ID1 could serve as a marker for CESC/CEPC. These results may advance the basic and clinical CESC/CEPC research.

Commentary on Clinical Application of Cultured Stratified Epithelial Sheets Grown Under Feeder or Feeder-Free Conditions for Stable Vitiligo

Commentary on Clinical Application of Cultured Stratified Epithelial Sheets Grown Under Feeder or Feeder-Free Conditions for Stable Vitiligo

A novel chemically defined serum- and feeder-free medium for undifferentiated growth of porcine pluripotent stem cells.

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.

In Vitro Differentiation of T Cell: From Human iPS Cells in Feeder-Free Condition.

In order to differentiate T cells in vitro, co-culture systems with Notch ligand-expressing feeder cells have been in use for a long time. Here we describe a feeder-free culture condition for differentiating T cells from hematopoietic cells that are cultured on Fc-DLL4-coated plate with T-lineage cytokines. This condition is capable of efficiently differentiating hematopoietic progenitor cells (HPCs) to immature T cells expressing both CD4 and CD8. To mature those cells into functional T cells, further stimulation and culture is necessary.

Chemically Defined Neural Conversion of Human Pluripotent Stem Cells.

Human pluripotent stem cells (hPSCs) are characterized by their ability to self-renew and differentiate into any cell type of the human body. To fully utilize the potential of hPSCs for translational research and clinical applications, it is critical to develop rigorous cell differentiation protocols under feeder-free conditions that are efficient, reproducible, and scalable for high-throughput projects. Focusing on neural conversion of hPSCs, here we describe robust small molecule-based procedures that generate neural stem cells (NSCs) in less than a week under chemically defined conditions. These protocols can be used to dissect the mechanisms of neural lineage entry and to further develop systematic protocols that produce the cellular diversity of the central nervous system at industrial scale.