Design of dual peptide-conjugated hydrogels for proliferation and differentiation of human pluripotent stem cells.

Completely synthetic cell cultivation materials for human pluripotent stem cells (hPSCs) are important for the future clinical use of hPSC-derived cells. Currently, cell culture materials conjugated with extracellular matrix (ECM)-derived peptides are being prepared using only one specific integrin-targeting peptide. We designed dual peptide-conjugated hydrogels, for which each peptide was selected from different ECM sites: the laminin ß4 chain and fibronectin or vitronectin, which can target α6ß1 and α2ß1 or αVß5. hPSCs cultured on dual peptide-conjugated hydrogels, especially on hydrogels conjugated with peptides obtained from the laminin ß4 chain and vitronectin with a low peptide concentration of 200 µg/mL, showed high proliferation ability over the long term and differentiated into cells originating from 3 germ layers in vivo as well as a specific lineage of cardiac cells. The design of grafting peptides was also important, for which a joint segment and positive amino acids were added into the designed peptide. Because of the designed peptides on the hydrogels, only 200 µg/mL peptide solution was sufficient for grafting on the hydrogels, and the hydrogels supported hPSC cultures long-term; in contrast, in previous studies, greater than 1000 µg/mL peptide solution was needed for the grafting of peptides on cell culture materials.

Comparison of retinal degeneration treatment with four types of different mesenchymal stem cells, human induced pluripotent stem cells and RPE cells in a rat retinal degeneration model.

BACKGROUND: Retinal degeneration (RD) is a group of disorders on irreversible vision loss. Multiple types of stem cells were used in clinical trials for RD treatment. However, it remains unknown what kinds of stem cells are most effective for the treatment. Therefore, we investigated the subretinal transplantation of several types of stem cells, human adipose-derived stem cells (hADSCs), amniotic fluid stem cells (hAFSCs), bone marrow stem cells (hBMSCs), dental pulp stem cells (hDPSCs), induced pluripotent stem cell (hiPSC), and hiPSC-derived retinal pigment epithelium (RPE) cells for protection effects, paracrine effects and treatment efficiency in an RD disease model rats. METHODS: The generation and characterization of these stem cells and hiPSC-derived RPE cells were performed before transplantation. The stem cells or hiPSC-derived RPE cell suspension labelled with CellTracker Green to detect transplanted cells were delivered into the subretinal space of 3-week-old RCS rats. The control group received subretinal PBS injection or non-injection. A series of detections including fundus photography, optomotor response (OMR) evaluations, light-dark box testing, electroretinography (ERG), and hematoxylin and eosin (HE) staining of retinal sections were conducted after subretinal injection of the cells. RESULTS: Each stem cell, hiPSC-derived RPE cell or PBS (blank experiment) was successfully transplanted into at least six RCS rats subretinally. Compared with the control rats, RCS rats subjected to subretinal transplantation of any stem cells except hiPSCs showed higher ERG waves (p < 0.05) and quantitative OMR (qOMR) index values (hADSCs: 1.166, hAFSCs: 1.249, hBMSCs: 1.098, hDPSCs: 1.238, hiPSCs: 1.208, hiPSC-RPE cells: 1.294, non-injection: 1.03, PBS: 1.06), which indicated better visual function, at 4 weeks post-injection. However, only rats that received hiPSC-derived RPE cells maintained their visual function at 8 weeks post-injection (p < 0.05). The outer nuclear layer thickness observed in histological sections after HE staining showed the same pattern as the ERG and qOMR results. CONCLUSIONS: Compared to hiPSC-derived RPE cells, adult and fetal stem cells yielded improvements in visual function for up to 4 weeks post-injection; this outcome was mainly based on the paracrine effects of several types of growth factors secreted by the stem cells. Patients with RD will benefit from the stem cell therapy.

Universal and hypoimmunogenic pluripotent stem cells for clinical usage.

It is urgent to prepare and store large numbers of clinical trial grade human pluripotent stem (hPS) cells for off-the-shelf use in stem cell therapies. However, stem cell banks, which store off-the-shelf stem cells, need financial support and large amounts of technicians for daily cell maintenance. Therefore, it is valuable to create "universal" or "hypoimmunogenic" hPS cells with genome editing engineering by knocking in or out immune-related genes. Only a small number of universal or hypoimmunogenic hPS cell lines should be needed to store for off-the-shelf usage and reduce the large amounts of instruments, consumables and technicians. In this article, we consider how to create hypoimmunogenic or universal hPS cells as well as the demerits of the technology. ß2-Microglobulin-knockout hPS cells did not harbor human leukocyte antigen (HLA)-expressing class I cells but led to the activation of natural killer cells. To escape the activities of macrophages and natural killer cells, homozygous hPS cells having a single allele of an HLA class I gene, such as HLA-C, were proposed. Major HLA class Ia molecules were knocked out, and CD47, HLA-G and PD-L1 were knocked in hPS cells utilizing CRISPR/Cas9 genome editing. Finally, some researchers are trying to generate universal hPS cells without genome editing. The cells evaded the activation of not only T cells but also macrophages and natural killer cells. These universal hPS cells have high potential for application in cell therapy.

Next-generation materials for RNA-lipid nanoparticles: lyophilization and targeted transfection.

RNA, including mRNA, siRNA and miRNA, is part of a new class of patient treatments that prevent and treat several diseases. As an alternative to DNA therapy using plasmid DNA, RNA functions in the cellular cytosol, avoiding the potential risks of insertion into patient genomes. RNA drugs, including mRNA vaccines, need carrier materials for delivery into the patient's body. Several delivery carriers of mRNA, such as cationic polymers, lipoplexes, lipid-polymer nanoparticles and lipid nanoparticles (LNPs), have been investigated. For clinical applications, one of the most commonly selected types of RNA delivery carrier is LNPs, which are typically formed with (a) ionizable lipids, which bind to RNA; (b) cholesterol for stabilization; (c) phospholipids to form the LNPs; and (d) polyethylene glycol-conjugated lipids to prevent aggregation and provide stealth characteristics. Most RNA-LNP research has been devoted to achieving highly efficient RNA expression in vitro and in vivo. It is also necessary to study the extended storage of RNA-LNPs under mild conditions. One of the most efficient methods to store RNA-LNPs for a long time is to prepare freeze-dried (lyophilized) RNA-LNPs. Future research should include investigating LNP materials for the development of freeze-dried RNA-LNPs using optimal lipid components and compositions with optimal cryoprotectants. Furthermore, the development of sophisticated RNA-LNP materials for targeted transfection into specific tissues, organs or cells will be a future direction in the development RNA therapeutics. We will discuss the prospects for the development of next-generation RNA-LNP materials.

Cell-binding peptides on the material surface guide stem cell fate of adhesion, proliferation and differentiation.

Human cells, especially stem cells, need to communicate and interact with extracellular matrix (ECM) proteins, which not only serve as structural components but also guide and support cell fate and properties such as cell adhesion, proliferation, survival and differentiation. The binding of the cells with ECM proteins or ECM-derived peptides via cell adhesion receptors such as integrins activates several signaling pathways that determine the cell fate, morphological change, proliferation and differentiation. The development of synthetic ECM protein-derived peptides that mimic the biological and biochemical functions of natural ECM proteins will benefit academic and clinical application. Peptides derived from or inspired by specific ECM proteins can act as agonists of each ECM protein receptor. Given that most ECM proteins function in cell adhesion via integrin receptors, many peptides have been developed that bind to specific integrin receptors. In this review, we discuss the peptide sequence, immobilization design, reaction method, and functions of several ECM protein-derived peptides. Various peptide sequences derived from mainly ECM proteins, which are used for coating or grafting on dishes, scaffolds, hydrogels, implants or nanofibers, have been developed to improve the adhesion, proliferation or differentiation of stem cells and to culture differentiated cells. This review article will help to inform the optimal choice of ECM protein-derived peptides for the development of scaffolds, implants, hydrogels, nanofibers and 2D cell culture dishes to regulate the proliferation and direct the differentiation of stem cells into specific lineages.

Designed peptide-grafted hydrogels for human pluripotent stem cell culture and differentiation.

Human pluripotent stem cells (hPSCs) have the ability to differentiate into cells derived from three germ layers and are an attractive cell source for cell therapy in regenerative medicine. However, hPSCs cannot be cultured on conventional tissue culture flasks but can be cultured on biomaterials with specific hPSC integrin interaction sites. We designed hydrogels conjugated with several designed peptides that had laminin-ß4 active sites, optimal elasticities and different zeta potentials. A higher expansion fold of hPSCs cultured on the hydrogels was found with the increasing zeta potential of the hydrogels conjugated with designed peptides, where positive amino acid (lysine) insertion into the peptides promoted higher zeta potentials of the hydrogels and higher expansion folds of hPSCs when cultured on the hydrogels using xeno-free protocols. The hPSCs cultured on hydrogels conjugated with the optimal peptides showed a higher expansion fold than those on recombinant vitronectin-coated plates, which are the gold standard of hPSC cultivation dishes. The hPSCs could differentiate into specific cell lineages, such as mesenchymal stem cells (MSCs) and MSC-derived osteoblasts, even after being cultivated on hydrogels conjugated with optimal peptides for long periods of time, such as 10 passages.

Neuronal Cell Differentiation of Human Dental Pulp Stem Cells on Synthetic Polymeric Surfaces Coated With ECM Proteins.

Stem cells serve as an ideal source of tissue regeneration therapy because of their high stemness properties and regenerative activities. Mesenchymal stem cells (MSCs) are considered an excellent source of stem cell therapy because MSCs can be easily obtained without ethical concern and can differentiate into most types of cells in the human body. We prepared cell culture materials combined with synthetic polymeric materials of poly-N-isopropylacrylamide-co-butyl acrylate (PN) and extracellular matrix proteins to investigate the effect of cell culture biomaterials on the differentiation of dental pulp stem cells (DPSCs) into neuronal cells. The DPSCs cultured on poly-L-ornithine (PLO)-coated (TPS-PLO) plates and PLO and PN-coated (TPS-PLO-PN) plates showed excellent neuronal marker (ßIII-tubulin and nestin) expression and the highest expansion rate among the culture plates investigated in this study. This result suggests that the TPS-PLO and TPS-PN-PLO plates maintained stable DPSCs proliferation and had good capabilities of differentiating into neuronal cells. TPS-PLO and TPS-PN-PLO plates may have high potentials as cell culture biomaterials for the differentiation of MSCs into several neural cells, such as cells in the central nervous system, retinal cells, retinal organoids and oligodendrocytes, which will expand the sources of cells for stem cell therapies in the future.

Effect of extracellular matrix proteins on the differentiation of human pluripotent stem cells into mesenchymal stem cells.

The transplantation of human mesenchymal stem cells (hMSCs), such as bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs), has shown beneficial effects in protecting transplanted tissues and cells against graft-versus-host disease (GVHD). Human pluripotent stem cell (hPSC)-derived mesenchymal stem cells (MSCs) can also be used to generate hMSCs with stable characteristics without limitations. Therefore, we differentiated human induced pluripotent stem cells (hiPSCs, H-M5) and human embryonic stem cells (hESCs, H9) into hMSCs on dishes coated with different extracellular matrix (ECM) proteins to study the effect of cell culture biomaterials on hPSC differentiation into hMSCs. hPSC-derived MSCs cultured on Matrigel (MAT)-coated, collagen (COL)-coated and laminin-521 (LN-521)-coated tissue culture polystyrene (TCP) dishes showed excellent proliferation speed and reduced aging over 10 passages. High MSC surface marker (CD44, CD73, CD90 and CD105) expression was also observed on hPSC-derived MSCs cultured on MAT-coated, COL-coated and LN-521-coated TCP dishes as well as uncoated TCP dishes. Analysis of late osteogenic differentiation by evaluation of mineral deposition revealed that hPSC-derived MSCs cultured on fibronectin (FN)-coated and LN-521-coated TCP dishes showed high osteogenic differentiation. ECM proteins are effective as coating materials on cell culture biomaterials to regulate the proliferation and differentiation fate of hPSC-derived MSCs.

Poly(vinyl alcohol-co-itaconic acid) hydrogels grafted with several designed peptides for human pluripotent stem cell culture and differentiation into cardiomyocytes.

We developed poly(vinyl alcohol-co-itaconic acid) (PV) hydrogels grafted with laminin-derived peptides that had different joint segments and several specific designs, including dual chain motifs. PV hydrogels grafted with a peptide derived from laminin-ß4 (PMQKMRGDVFSP) containing a joint segment, dual chain motif and cationic amino acid insertion could attach human pluripotent stem (hPS) cells and promoted high expansion folds in long-term culture (over 10 passages) with low differentiation rates, whereas hPS cells attached poorly on PV hydrogels grafted with laminin-α5 peptides that had joint segments with and without a cationic amino acid or on PV hydrogels grafted with laminin-ß4 peptides containing the joint segment only. The inclusion of a cationic amino acid in the laminin-ß4 peptide was critical for hPS cell attachment on PV hydrogels, which contributed to the zeta potential shifting to higher values (3-4 mV enhancement). The novel peptide segment-grafted PV hydrogels developed in this study supported hPS cell proliferation, which induced better hPS cell expansion than recombinant vitronectin-coated dishes (gold standard of hPS cell culture dishes) in xeno-free culture conditions. After long-term culture on peptide-grafted hydrogels, hPS cells could be induced to differentiate into specific lineages of cells, such as cardiomyocytes, with high efficiency.

Transient characteristics of universal cells on human-induced pluripotent stem cells and their differentiated cells derived from foetal stem cells with mixed donor sources.

INTRODUCTION: It is important to prepare 'hypoimmunogenic' or 'universal' human pluripotent stem cells (hPSCs) with gene-editing technology by knocking out or in immune-related genes, because only a few hypoimmunogenic or universal hPSC lines would be sufficient to store for their off-the-shelf use. However, these hypoimmunogenic or universal hPSCs prepared previously were all genetically edited, which makes laborious processes to check and evaluate no abnormal gene editing of hPSCs. METHODS: Universal human-induced pluripotent stem cells (hiPSCs) were generated without gene editing, which were reprogrammed from foetal stem cells (human amniotic fluid stem cells) with mixing 2-5 allogenic donors but not with single donor. We evaluated human leucocyte antigen (HLA)-expressing class Ia and class II of our hiPSCs and their differentiated cells into embryoid bodies, cardiomyocytes and mesenchymal stem cells. We further evaluated immunogenic response of transient universal hiPSCs with allogenic mononuclear cells from survival rate and cytokine production, which were generated by the cells due to immunogenic reactions. RESULTS: Our universal hiPSCs during passages 10-25 did not have immunogenic reaction from allogenic mononuclear cells even after differentiation into cardiomyocytes, embryoid bodies and mesenchymal stem cells. Furthermore, the cells including the differentiated cells did not express HLA class Ia and class II. Cardiomyocytes differentiated from transient universal hiPSCs at passage 21-22 survived and continued beating even after treatment with allogenic mononuclear cells.

Generation of universal and hypoimmunogenic human pluripotent stem cells.

There is a need to store very large numbers of conventional human pluripotent stem cell (hPSC) lines for their off-the-shelf usage in stem cell therapy. Therefore, it is valuable to generate "universal" or "hypoimmunogenic" hPSCs with gene-editing technology by knocking out or in immune-related genes. A few universal or hypoimmunogenic hPSC lines should be enough to store for their off-the-shelf usage. Here, we overview and discuss how to prepare universal or hypoimmunogenic hPSCs and their disadvantages. ß2-Microglobulin-knockout hPSCs did not harbour human leukocyte antigen (HLA)-expressing class I cells but rather activated natural killer (NK) cells. To avoid NK cell and macrophage activities, homozygous hPSCs expressing a single allele of an HLA class I molecule, such as HLA-C, were developed. Major HLA class I molecules were knocked out, and PD-L1, HLA-G and CD47 were knocked in hPSCs using CRISPR/Cas9 gene editing. These cells escaped activation of not only T cells but also NK cells and macrophages, generating universal hPSCs.

Enrichment of cancer-initiating cells from colon cancer cells through porous polymeric membranes by a membrane filtration method.

Cancer-initiating cells (CICs) or cancer stem cells (CSCs) are primarily responsible for tumor initiation, growth, and metastasis and represent a few percent of the total tumor cell population. We designed a membrane filtration protocol to enrich CICs (CSCs) from the LoVo colon cancer cell line via nylon mesh filter membranes with 11 and 20 µm pore sizes and poly(lactide-co-glycolic acid)/silk screen (PLGA/silk screen) porous membranes (pore sizes of 20-30 µm). The colon cancer cell solution was filtered through the membranes to obtain a permeate solution. Subsequently, the cell culture medium was filtered through the membranes to collect the recovery solution where the cells attached to the membranes were rinsed off into the recovery solution. Then, the membranes were cultivated in the cultivation medium to collect the migrated cells from the membranes. The cells migrated from any membrane had higher expression of the CSC surface markers CD44 and CD133, had higher colony formation levels, and produced more carcinoembryonic antigen (CEA) than the colon cancer cells cultivated on conventional tissue culture plates (control). We established a method to enrich the CICs (CSCs) of colon cancer cells from migrated cells through porous polymeric membranes by the membrane filtration protocol developed in this study.

Culture and differentiation of purified human adipose-derived stem cells by membrane filtration via nylon mesh filters.

Human adipose-derived stem cells (hASCs) cultured for 5 passages were filtered through nylon (NY) mesh filter membranes coated with and without extracellular matrix proteins to obtain the permeation solution. Subsequently, the culture media were filtered via the membranes to obtain the recovery solution. Then, the membranes were cultured in cell culture medium to obtain the migrated cells from the membranes. The hASCs in the permeation solution, through any type of NY mesh filter membrane having 11 and 20 µm pore sizes, had lower osteogenic differentiation ability than conventional hASCs cultured on tissue culture polystyrene (TCP) dishes for passage 5, whereas the hASCs purified by the membrane migration method through NY mesh filter membranes coated with recombinant vitronectin, which have 11 and 20 µm pore sizes, showed a higher proliferation speed as well as higher osteogenic differentiation potential than the conventional hASCs cultured on TCP dishes for passage 5. The membrane filtration and migration methods would be useful for cell sorting for specific cells, such as hASCs with high proliferation and high osteogenic differentiation ability, which do not need antibody binding or genetic modification of the cells for the specific isolation of the cells.

Efficient differentiation of human pluripotent stem cells into cardiomyocytes on cell sorting thermoresponsive surface.

The current differentiation process of human pluripotent stem cells (hPSCs) into cardiomyocytes to enhance the purity of hPSC-derived cardiomyocytes requires some purification processes, which are laborious processes. We developed cell sorting plates, which are prepared from coating thermoresponsive poly(N-isopropylacrylamide) and extracellular matrix proteins. After hPSCs were induced into cardiomyocytes on the thermoresponsive surface coated with laminin-521 for 15 days, the temperature of the cell culture plates was decreased to 8-9 °C to detach the cells partially from the thermoresponsive surface. The detached cells exhibited a higher cardiomyocyte marker of cTnT than the remaining cells on the thermoresponsive surface as well as the cardiomyocytes after purification using conventional cell selection. The detached cells expressed several cardiomyocyte markers, such as α-actinin, MLC2a and NKX2.5. This study suggested that the purification of hPSC-derived cardiomyocytes using cell sorting plates with the thermoresponsive surface is a promising method for the purification of hPSC-derived cardiomyocytes without conventional laborious processes.

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.

Efficient differentiation of human ES and iPS cells into cardiomyocytes on biomaterials under xeno-free conditions.

Current xeno-free and chemically defined methods for the differentiation of hPSCs (human pluripotent stem cells) into cardiomyocytes are not efficient and are sometimes not reproducible. Therefore, it is necessary to develop reliable and efficient methods for the differentiation of hPSCs into cardiomyocytes for future use in cardiovascular research related to drug discovery, cardiotoxicity screening, and disease modeling. We evaluated two representative differentiation methods that were reported previously, and we further developed original, more efficient methods for the differentiation of hPSCs into cardiomyocytes under xeno-free, chemically defined conditions. The developed protocol successively differentiated hPSCs into cardiomyocytes, approximately 90-97% of which expressed the cardiac marker cTnT, with beating speeds and sarcomere lengths that were similar to those of a healthy adult human heart. The optimal cell culture biomaterials for the cardiac differentiation of hPSCs were also evaluated using extracellular matrix-mimetic material-coated dishes. Synthemax II-coated and Laminin-521-coated dishes were found to be the most effective and efficient biomaterials for the cardiac differentiation of hPSCs according to the observation of hPSC-derived cardiomyocytes with high survival ratios, high beating colony numbers, a similar beating frequency to that of a healthy adult human heart, high purity levels (high cTnT expression) and longer sarcomere lengths similar to those of a healthy adult human heart.

The effect of human platelet lysate on the differentiation ability of human adipose-derived stem cells cultured on ECM-coated surfaces.

Human mesenchymal stem cells (hMSCs), such as human adipose-derived stem cells (hADSCs), present heterogeneous characteristics, including varying differentiation abilities and genotypes. hADSCs isolated under different conditions exhibit differences in stemness. We isolated hADSCs from human fat tissues via culture on different cell culture biomaterials including tissue culture polystyrene (TCPS) dishes and extracellular matrix protein (ECM)-coated dishes in medium supplemented with 5% or 10% serum-converted human platelet lysate (hPL) or 10% fetal bovine serum (FBS) as a control. Currently, it is not clear whether xeno-free hPL in the cell culture medium promotes the ability of hMSCs such as hADSCs to differentiate into several cell lineages compared to the xenomaterial FBS. We investigated whether a synchronized effect of ECM (Matrigel, fibronectin, and recombinant vitronectin) coatings on TCPS dishes for efficient hADSC differentiation could be observed when hADSCs were cultured in hPL medium. We found that Matrigel-coated dishes promoted hADSC differentiation into osteoblasts and suppressed differentiation into chondrocytes in 10% hPL medium. Recombinant vitronectin- and fibronectin-coated dishes greatly promoted hADSC differentiation into osteoblasts and chondrocytes in 5% and 10% hPL media. hPL promoted hADSC differentiation into osteoblasts and chondrocytes compared to FBS on the fibronectin-coated surface and recombinant vitronectin-coated surface.

Xeno-free and feeder-free culture and differentiation of human embryonic stem cells on recombinant vitronectin-grafted hydrogels.

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

The design of a thermoresponsive surface for the continuous culture of human pluripotent stem cells.

Commonly, stem cell culture is based on batch-type culture, which is laborious and expensive. We continuously cultured human pluripotent stem cells (hPSCs) on thermoresponsive dish surfaces, where hPSCs were partially detached on the same thermoresponsive dish by decreasing the temperature of the thermoresponsive dish to be below the lower critical solution temperature for only 30 min. Then, the remaining cells were continuously cultured in fresh culture medium, and the detached stem cells were harvested in the exchanged culture medium. hPSCs were continuously cultured for ten cycles on the thermoresponsive dish surface, which was prepared by coating the surface with poly(N-isopropylacrylamide-co-styrene) and oligovitronectin-grafted poly(acrylic acid-co-styrene) or recombinant vitronectin for hPSC binding sites to maintain hPSC pluripotency. After ten cycles of continuous culture on the thermoresponsive dish surface, the detached cells expressed pluripotency proteins and had the ability to differentiate into cells derived from the three germ layers in vitro and in vivo. Furthermore, the detached cells differentiated into specific cell lineages, such as cardiomyocytes, with high efficiency.

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.