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.

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.

Retrospectively investigating the 12-year experience of prenatal diagnosis of small supernumerary marker chromosomes through array comparative genomic hybridization.

OBJECTIVE: This study retrospectively evaluated the incidences of small supernumerary marker chromosomes (sSMCs) in prenatal diagnoses and detected with gain of pathogenic copy number variation through array comparative genomic hybridization (CGH) in a laboratory in Taiwan. MATERIALS AND METHODS: We retrospectively searched and reviewed the sSMC cases detected during prenatal diagnoses in the Youthgene medical laboratory, between 2004 and 2015 and used array CGH to successfully analyze 45 of 47,XN,+mar or 47,XN + mar/46,XN. RESULTS: A total of 68,087 cases of amniocentesis were analyzed, of which 59 were identified as sSMCs. The overall frequency of sSMCs was 0.087%, and 7 of 45 sSMCs were identified with gain of pathogenic copy number variation (CNV). CONCLUSION: Array CGH offers useful tools that can be used to detect small fragments of chromosomal abnormalities and sSMC origins in prenatal diagnosis. In this study, we successfully used array CGH to detect 7 out of 45 sSMCs, which were identified with gain in pathogenic CNV.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods.

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 µm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not.

Proliferation and osteogenic differentiation of amniotic fluid-derived stem cells.

Human amniotic fluid-derived stem cells (hAFCs) are pluripotent fetal cells capable of differentiating into multiple lineages, including cell types of each of the three embryonic germ layers. Proper differentiation and maintenance of pluripotency, the defining characteristics of stem cells, are regulated not only by the cells themselves but also by their microenvironment. Furthermore, the physical characteristics of the cell culture materials, such as material elasticity, influence the results of stem cell differentiation. We investigated the osteogenic differentiation efficiency of hAFCs cultured on cell culture materials with different elasticities that were grafted with extracellular matrix-derived oligopeptides. Polyvinyl alcohol-co-itaconic acid (PV) hydrogels with different elasticities were prepared by controlling the crosslinking intensity, and the resulting PV hydrogels were grafted with and without extracellular matrix (ECM)-derived oligopeptides. Specific ECM-derived oligopeptides were used to maintain the pluripotency of AFCs and were determined by evaluation of pluripotent gene expression (Sox2 and Oct4). The osteogenic differentiation efficiency of the hAFCs, cultured on PV hydrogels grafted with and without ECM-derived oligopeptides, was analyzed by alkaline phosphatase activity, Alizarin Red S staining, and von Kossa staining. Unmodified PV hydrogels induced osteoblast differentiation of hAFCs with high efficiency. We conclude that the hAFCs interacting with ECM-derived oligopeptides tended to maintain an undifferentiated state.

Generation of pluripotent stem cells without the use of genetic material.

Induced pluripotent stem cells (iPSCs) provide a platform to obtain patient-specific cells for use as a cell source in regenerative medicine. Although iPSCs do not have the ethical concerns of embryonic stem cells, iPSCs have not been widely used in clinical applications, as they are generated by gene transduction. Recently, iPSCs have been generated without the use of genetic material. For example, protein-induced PSCs and chemically induced PSCs have been generated by the use of small and large (protein) molecules. Several epigenetic characteristics are important for cell differentiation; therefore, several small-molecule inhibitors of epigenetic-modifying enzymes, such as DNA methyltransferases, histone deacetylases, histone methyltransferases, and histone demethylases, are potential candidates for the reprogramming of somatic cells into iPSCs. In this review, we discuss what types of small chemical or large (protein) molecules could be used to replace the viral transduction of genes and/or genetic reprogramming to obtain human iPSCs.

Pluripotency maintenance of amniotic fluid-derived stem cells cultured on biomaterials.

The stem cell fates of pluripotency and differentiation are regulated by not only soluble biological cues but also insoluble biochemical cues (i.e., extracellular matrix (ECM)) and the physical cues of cell culture biomaterials (i.e., elasticity). We investigated the maintenance of pluripotency and the differentiation lineages of human amniotic fluid-derived stem cells (hAFSCs) cultured on poly(vinyl alcohol-co-itaconic acid) (PVA) hydrogels grafted with several types of ECM and corresponding oligopeptides in expansion medium. hAFSCs cultured on soft PVA hydrogels (12.2 kPa) that were grafted with oligopeptides derived from fibronectin and vitronectin showed high pluripotency, which was evaluated by Oct4, Sox2 and Nanog expression. The hAFSCs grown on soft PVA hydrogels (12.2 kPa) grafted with each oligopeptide showed higher pluripotency, as assessed by Oct4 and Nanog expression, than hAFSCs grown on stiff PVA hydrogels (25.3 kPa) grafted with the same oligopeptides and a much higher pluripotency than those grown on rigid tissue-culture polystyrene dishes. Soft biomaterials appeared to be adequate to maintain the pluripotency of hAFSCs. Surprisingly, hAFSCs that showed higher pluripotency on PVA hydrogels grafted with oligopeptides derived from fibronectin and vitronectin also expressed higher levels of early differentiation markers for three germ layers in expansion medium. This result suggests that hAFSCs are heterogeneous and that this population contains highly pluripotent stem cells and stem cells that can be easily differentiated.

Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes.

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.

The isolation and differentiation of human adipose-derived stem cells using membrane filtration.

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.

Osteoblast differentiation of amniotic fluid-derived stem cells irradiated with visible light.

The effect of visible light irradiation on the expression of pluripotent genes (Oct-4, Sox2, and Nanog) in amniotic fluid-derived stem cells (AFSCs) and on the osteogenic differentiation ability of AFSCs was investigated using light-emitting diodes (LEDs) at 0-2 mW/cm(2) in various wavelengths: [blue (470 nm), green (525 nm), yellow (600 nm), and red (630 nm)]. Pluripotent gene expression in AFSCs was up-regulated by visible light irradiation from a LED for more than 6 h. Green light irradiation of AFSCs up-regulated the expression of pluripotent genes more significantly than irradiation with other light. The osteogenic differentiation of AFSCs was facilitated by green and blue light irradiation. Facilitated differentiation into osteogenic cells by visible light irradiation was not mediated by reactive oxygen species (ROS); alkaline phosphatase activity (a marker of early osteogenic differentiation) and gene expression of osteopontin (a marker of late osteogenic differentiation) did not change significantly between AFSCs in differentiation medium with or without a ROS scavenger (vitamin C). The mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway, as well as other unknown signaling pathways, may be responsible for the activation of signaling pathways that facilitate the differentiation of AFSCs into osteogenic cells on light irradiation.