An Efficient Culture Method of CD3-Positive T Cells from Human Cryopreserved Buffy Coat Specimens.

Context: In the National Cerebral and Cardiovascular Center (NCVC) Biobank, buffy coats have been collected from patients and stored with cryoprotective agents as a possible source for viable blood cells, using cost-efficient methods for storage. However, whether viable cells for in vitro studies can be recovered from these biospecimens has not been verified. Objective: To investigate whether T cells can be collected and expanded as viable cells from cryopreserved human buffy coats. Design: After thawing of cryopreserved buffy coat specimens, CD3-positive cells were isolated from the cell suspension using a leukocyte separation filter coated with an anti-CD3 antibody, and the filter-attached cells were cultured in T cell culture medium. To analyze the characteristics of these cultured cells, histocytological analyses of Giemsa staining, immunocytochemical (ICC) staining for CD3, and flow cytometry for CD3 in live cells were conducted. Results: A few days after starting cell culture, cell clusters were observed, and they gradually grew in size. Using Giemsa staining, the expanded cells were found to be ∼15 µm in diameter, having round nuclei, a high nucleus/cytoplasm ratio, and cytoplasm stained light blue, which is characteristic of lymphocytes. From ICC staining, these cells were CD3 positive, a pan-T cell marker among lymphocytes. Furthermore, CD3 immunoreactivity in live cells was detected in a flow cytometry assay, though that for CD19 was not detected, which is a marker of pan-B cells. Conclusions: These results suggest that T cells can be expanded from buffy coats cryopreserved at -180°C as an adequate method of NCVC Biobank, highlighting these biospecimens as a possible useful source for future in vitro studies.

Effect of stem cell niche elasticity/ECM protein on the self-beating cardiomyocyte differentiation of induced pluripotent stem (iPS) cells at different stages.

Stem cell-based myocardial regeneration therapies have emerged as alternative strategies to heart transplantation for serious heart diseases, but autologous beating mature cardiomyocytes are not available. Here we investigated the effect of culture substrates on the cardiomyocyte differentiation of induced pluripotent stem cells (iPSs) in vitro by separately evaluating the following continuous three steps: (1) cardiac marker gene expression, (2) contractile gene expression and self-beating, and (3) beating duration. To this end, we used iPS cells to study the cardiac differentiation, and neonatal rat cardiomyocytes (NCMs) to study beating behavior. These cells were cultured on substrates with different natures, i.e., an elastic substrate (Es) with the modulus of 9, 20, or 180 kPa, and hard tissue culture polystyrene dishes (TCPS) coated with collagen type I (Col), gelatin (Gel), or fibronectin (FN). The results revealed that the effective niches in each step were very different. The cardiac marker gene (GATA4, Tbx5, MEF2C) expression of iPSs at the 1st step was very high on the TCPS coated with FN or Gel, whereas on the FN-coated Es (especially with the 9 kPa modulus), the undifferentiated marker gene (Nanog) expression of iPSs was maintained. The expression of the contractile genes α-MHC, TnC1, and TnT2 and the self-beating (the 2nd step) of the NCMs were high on FN-coated TCPS and Col-coated Es. The 3rd step (beating duration) of the NCMs was effective on the Es, and at 21 days both the iPSs and NCMs stopped beating on the TCPS but were still beating on the Es. Overall, cardiac differentiation 'preferred' ECM-rigid culture substrates, and beating-behavior 'preferred' Col-soft culture substrates. These results are important for understanding and designing cardiac differentiation niches for regenerative medicine, and they suggest that a single culture substrate is not suitable for preparing self-beating cardiomyocytes. STATEMENT OF SIGNIFICANCE: The transplantation of beating cardiomyocytes (BCMs) is expected to be made more effective for serious heart diseases. The identification of the appropriate engineering processes and suitable culture substrates for inducing stem cell differentiation into BCMs is thus indispensable. The differentiation can be divided into three major processes, the cardiac differentiation step, the beating-induction step and the beating-duration step. A protocol with the higher efficiency in all of the steps must be useful. In this study, we separately evaluated the effect of culture substrates at each three step. We clarified that the biological and the physical properties of the culture substrates required at these steps were different. We found useful criteria for effective cardiac cell niche systems design.

Hepatocytic differentiation of iPS cells on decellularized liver tissue.

Decellularized tissues (DETs) have been attracting great attention as scaffolds for tissue-engineering approaches. Recently, some studies have reported that decellularized liver tissues (DLT) can provide an excellent environment for the hepatocytic differentiation of hepatic stem/progenitor cells that were already committed to the hepatocyte lineage. However, the effects of DLT on the hepatocytic differentiation of induced pluripotent stem cells (iPSs) have not yet been established. Here we studied the hepatocytic differentiation of iPSs on DLT and decellularized heart tissues (DHT) in order to determine the tissue-specific effects of DETs on iPSs differentiation. Our results showed that DLTs led to higher gene expression levels of forkhead box A2 (a marker of endoderm) and CCAAT/enhancer binding protein-α (master transcription factor to hepatocyte differentiation), alpha-fetoprotein (a marker of fetal hepatocyte,), and albumin (a marker of fetal and mature hepatocyte) of iPSs than on DHTs. Furthermore, gene expression levels of tyrosine aminotransferase (a marker of mature hepatocyte) were higher on DLT than that on DHT, and immunocytochemical analysis and ELISA assay showed that albumin secretion level of iPSs on DLT was higher than that on DHT. Our study demonstrated that the use of DLTs led to mature hepatocytic differentiation levels of iPSs compared to DHTs, which provides a better niche for iPSs cell engineering and enables the preparation of useful mature cells for regenerative therapy.

Isolation and expansion of human pluripotent stem cell-derived hepatic progenitor cells by growth factor defined serum-free culture conditions.

Limited growth potential, narrow ranges of sources, and difference in variability and functions from batch to batch of primary hepatocytes cause a problem for predicting drug-induced hepatotoxicity during drug development. Human pluripotent stem cell (hPSC)-derived hepatocyte-like cells in vitro are expected as a tool for predicting drug-induced hepatotoxicity. Several studies have already reported efficient methods for differentiating hPSCs into hepatocyte-like cells, however its differentiation process is time-consuming, labor-intensive, cost-intensive, and unstable. In order to solve this problem, expansion culture for hPSC-derived hepatic progenitor cells, including hepatic stem cells and hepatoblasts which can self-renewal and differentiate into hepatocytes should be valuable as a source of hepatocytes. However, the mechanisms of the expansion of hPSC-derived hepatic progenitor cells are not yet fully understood. In this study, to isolate hPSC-derived hepatic progenitor cells, we tried to develop serum-free growth factor defined culture conditions using defined components. Our culture conditions were able to isolate and grow hPSC-derived hepatic progenitor cells which could differentiate into hepatocyte-like cells through hepatoblast-like cells. We have confirmed that the hepatocyte-like cells prepared by our methods were able to increase gene expression of cytochrome P450 enzymes upon encountering rifampicin, phenobarbital, or omeprazole. The isolation and expansion of hPSC-derived hepatic progenitor cells in defined culture conditions should have advantages in terms of detecting accurate effects of exogenous factors on hepatic lineage differentiation, understanding mechanisms underlying self-renewal ability of hepatic progenitor cells, and stably supplying functional hepatic cells.

Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel.

When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the immobilized-antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state.

Individual evaluation of cardiac marker expression and self-beating during cardiac differentiation of P19CL6 cells on different culture substrates.

Cell-based therapies using self-beating cardiomyocytes have been attracting great attention for use in cardiac regeneration, although an effective procedure to improve cardiac differentiation and self-beating induction is required. The purpose of this study is to clarify the effect of the culture substrate on cardiac maturation by separately evaluating the cardiac differentiation step and the beating induction step in vitro. To this end, the well-studied cardiomyocyte-like progenitor cell line P19CL6 and neonatal cardiomyocytes (NCMs) were selected and cultured on substrates coated with collagen type I (Col-I), gelatin (Gel), fibronectin (FN), or poly-l-lysine (PLL). It was found that the cardiac differentiation step, which was assessed using cardiac marker gene expression (GATA-binding protein 4 (GATA4), myocyte-specific enhancer factor 2D (MEF2D), and hyperpolarization-activated cyclic nucleotide-gated potassium channel 4 (HCN4)) in the P19CL6 embryonal carcinoma cells, was greatly enhanced on Col-I, Gel, and PLL. In contrast, the spontaneous beating step, which was directly assessed by counting the beating colonies and measuring contractile protein gene expression (α-myosin heavy chain (α-MHC), troponin C type 1 (TnC1), and troponin T type 2 (TnT2)) in the rat NCMs, was enhanced on the FN and PLL surfaces. In the present study, for the first time, it was found that PLL enhances both the cardiac differentiation and the beating induction steps of cardiac maturation, which can aid in preparing beating cardiomyocytes for regenerative medicine. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1166-1174, 2017.

Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells.

Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However, the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study, we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line, H9, which is known to differentiate into hepatocytes, and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs, hPSC-derived hepatoblast-like differentiated cells, and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus, our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines.

Protein kinase C regulates human pluripotent stem cell self-renewal.

BACKGROUND: The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3ß (GSK-3ß), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3ß. Addition of activin A increased phosphorylation of GSK-3ß and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2 whereas activin A alone did not. GFX negated differentiation of hPS cells induced by the PKC activator, phorbol 12-myristate 13-acetate whereas Gö6976, a selective inhibitor of PKCα, ß, and γ isoforms could not counteract the effect of PMA. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3ß was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. CONCLUSIONS/SIGNIFICANCE: Our study suggested complicated cross-talk in hPS cells that FGF-2 induced the phosphorylation of phosphatidylinositol-3 kinase (PI3K)/AKT, mitogen-activated protein kinase/ERK-1/2 kinase (MEK), PKC/ERK-1/2 kinase, and PKC/GSK-3ß. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.

Growth factor-defined culture medium for human mesenchymal stem cells.

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.

Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture.

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

Establishment and characterization of hepatic stem-like cell lines from normal adult rat liver.

The liver is a unique organ with the potential to regenerate from injury. Hepatic stem cells contribute to liver regeneration when surviving hepatocytes in injured liver are unable to proliferate. To investigate the mechanism of liver regeneration in vitro, we established hepatic stem cell lines named HY1, HY2 and HY3, derived from a healthy liver of adult rat. HY cells showed an expression pattern similar to oval cells, and efficiently induced hepatic differentiation following sequential treatment with type I collagen, transforming growth factor-beta1 (TGF-beta1), and hepatocyte growth factor (HGF) or oncostatin M (OSM). These results suggested that HY cells are liver stem cells representing an excellent tool for in vitro studies on liver regeneration.

Transient expression of bone morphogenic protein-2 in acute liver injury by carbon tetrachloride.

Acute liver injury induced by administration of carbon tetrachloride (CCl4) was shown to be a model of wound-repair in rat liver. Albumin gene expression was significantly reduced at 24 h post injection with CCl4, but recovered at 48 h. We also observed significant and transient expression of bone morphogenic protein-2 (BMP-2) at 6-24 h post treatment. This expression was also shown with depletion of Kupffer cell by GdCl3, and immunostaining with anti-BMP-2 antibody showed BMP-2-producing cells interspersed in intralobular spaces of injured liver. These observations suggest that BMP-2 secreted from oval-like cells plays important roles in the wound healing response of injured liver.