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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.

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.

The purification of human adipose-derived stem cells (hADSCs) from human adipose tissue cells (stromal vascular fraction) was investigated using membrane filtration through poly(lactide-co-glycolic acid)/silk screen hybrid membranes. Membrane filtration methods are attractive in regenerative medicine because they reduce the time required to purify hADSCs (i.e., less than 30 min) compared with conventional culture methods, which require 5-12 days. hADSCs expressing the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the permeation solution from the hybrid membranes. Expression of the surface markers CD44, CD73, and CD99 on the cells in the permeation solution from the hybrid membranes, which were obtained using 18 mL of feed solution containing 50 × 10⁴ cells, was statistically significantly higher than that of the primary adipose tissue cells, indicating that the hADSCs can be purified in the permeation solution by the membrane filtration method. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the permeation solution, whereas CD34⁺ cells could not be purified by the conventional culture method. The hADSCs in the permeation solution demonstrated a superior capacity for osteogenic differentiation based on their alkali phosphatase activity, their osterix gene expression, and the results of mineralization analysis by Alizarin Red S and von Kossa staining compared with the cells from the suspension of human adipose tissue. These results suggest that the hADSCs capable of osteogenic differentiation preferentially permeate through the hybrid membranes.

Umbilical cord blood (UCB) is an attractive source of hematopoietic stem and progenitor cells (HSPCs) for transplantation. However, the low number of HSPCs from a single UCB donor limits the direct transplantation of UCB to patients. Because little is known about the effects of the physical microenvironment on HSPC expansion, we investigated the ex vivo expansion of HSPCs cultured on biomaterials with different elasticities and grafted with different nanosegments. Polyvinylalcohol-co-itaconic acid (PVA-IA)-coated dishes with different stiffnesses ranging from a 3.7 kPa to 30.4 kPa storage modulus were used. Fibronectin or an oligopeptide (CS1, EILDVPST) was grafted onto the PVA-IA substrates. High ex vivo fold expansion of HSPCs was observed in the PVA-IA dishes grafted with fibronectin or CS1, which displayed an intermediate stiffness ranging from 12.2 kPa to 30.4 kPa. The fold expansion was more than 1.4 times higher than that cultured in tissue culture polystyrene dishes (TCPS, 12 GPa). Furthermore, HSPCs cultured in fibronectin or CS1-grafted PVA-IA-coated dishes with a stiffness of 12.2-30.4 kPa generated more pluripotent colony-forming units (CFU-GM and CFU-GEMM) than those in TCPS dishes. This result indicates that both the physical and biological properties of biomaterials affect the ex vivo expansion of HSPCs.

Human adipose-derived stem cells (hADSCs) were purified from a suspension of human adipose tissue cells (stromal vascular fraction) by the conventional culture method and by membrane filtration through polyurethane (PU) foam membranes. hADSCs can be obtained from a suspension of human adipose tissue cells using the membrane filtration method in less than 30 min, whereas the conventional culture method requires 5-12 days. hADSCs that express the mesenchymal stem cell markers CD44, CD73, and CD90 were concentrated in the recovery solution from the PU membranes; no hADSCs were isolated in the permeate. After filtration, the cells expressing the mesenchymal stem cell markers were 3-4.5 times more concentrated than in the initial suspension of human adipose tissue cells, with the level of concentration depending on the surface modification of the PU membrane. Cells expressing the stem cell-associated marker CD34 could be successfully isolated in the recovery solutions, whereas CD34(+) cells could not be purified by the conventional culture method. The hADSCs in the recovery solution demonstrated a superior capacity for osteogenic differentiation than did the cells in the suspension of human adipose tissue cells. These results suggested that the hADSCs with the capability for osteogenic differentiation adhered to the PU membranes.