human-pluripotent-stem-cell-culture-on-polyvinyl-alcohol-co-itaconic

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 ...

Cathay General Hospital

National Central University

The decrease of arterial blood pressure and body temperature after epidural or spinal anesthesia is thought to be the result of sympathetic block, which could cause pooling and redistribution of blood into the lower extremities. Studies have demonstrated that leg wrapping with elastic bandages may reduce the incidence of hypotension after spinal anesthesia. We tried to extend these previous observations to epidural anesthesia by testing the hypothesis that leg wrapping with elastic bandages should decrease the incidence of hypotension in patients receiving epidural anesthesia. Moreover, we evaluated the effect of this maneuver as regards hypothermia and shivering. Sixty parturients were randomly allocated to receive either leg wrapping with tight elastic bandages (leg-wrapped group) or not (control group) before anesthesia. Sublingual temperature was observed at five periods: baseline, immediately after epidural anesthesia, abdominal skin disinfection, skin incision, and delivery. Hypotension and shivering during the observation periods were also recorded. The incidence of hypotension was significantly less frequent (P = 0.03) in the leg-wrapped group (23%) compared with the control group (50%). Shivering incidences were similar in both groups (70% versus 70%). Sublingual temperature decreased significantly (P < 0.001) throughout the procedure in each group. However, no differences were found between the two groups at each designated observation, even if compared by the magnitude of temperature decrease. We conclude that although leg wrapping with elastic bandages prevents maternal hypotension after epidural anesthesia, it does not reduce the incidence or magnitude of hypothermia or prevent shivering.

Lower limb wrapping prevents hypotension, but not hypothermia or shivering, after the introduction of epidural anesthesia for cesarean delivery.

To define the prevalence rate of cervical human papilloma virus (HPV) using DNA oligonucleotide microarray and its correlation with risk factors in Taiwanese women in metropolitan Taipei.

Prevalence of cervical human papillomavirus in Taiwanese women.

The aim of this study was to evaluate the percentage of pregnant women with negative Group B Streptococcus (GBS) screening results by culture at 35 weeks' gestation, who subsequently had positive GBS test results after 39 weeks' gestation.

Prenatal Group B Streptococcus test using real-time polymerase chain reaction.

Neuroplasticity induced by neonatal inflammation is the consequence of a combination of activity-dependent changes in neurons. We investigated neuronal sensitivity to a noxious stimulus in a rat model of neonatal hind-paw peripheral inflammation and assessed changes in pain behaviour at the physiological and molecular levels after peripheral reinflammation in adulthood.

Increased spinal prodynorphin gene expression in reinflammation-associated hyperalgesia after neonatal inflammatory insult.

We evaluated the higher levels of carcinoembryonic antigen (CEA) secreted by the LoVo human colon carcinoma cells in a medium containing anticancer drugs. Drug-resistant LoVo cells were analyzed by subcutaneously xenotransplanting them into mice. The aim of this study was to evaluate whether the drug-resistant cells isolated in this study were cancer-initiating cells, known also as cancer stem cells (CSCs).

Drug-resistant colon cancer cells produce high carcinoembryonic antigen and might not be cancer-initiating cells.

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.

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

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.

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

Human adipose-derived stem cells (hADSCs) exhibit heterogeneous characteristics, indicating various genotypes and differentiation abilities. The isolated hADSCs can possess different purity levels and divergent properties depending on the purification methods used. We developed a hybrid-membrane migration method that purifies hADSCs from a fat tissue solution with extremely high purity and pluripotency. A primary fat-tissue solution was permeated through the porous membranes with a pore size from 8 to 25 μm, and the membranes were incubated in cell culture medium for 15-18 days. The hADSCs that migrated from the membranes contained an extremely high percentage (e.g., >98%) of cells positive for mesenchymal stem cell markers and showed almost one order of magnitude higher expression of some pluripotency genes (Oct4, Sox2, Klf4 and Nanog) compared with cells isolated using the conventional culture method.

A hybrid-membrane migration method to isolate high-purity adipose-derived stem cells from fat tissues.

Stem cell culture is typically based on batch-type culture, which is laborious and expensive. Here, we propose a continuous harvest method for stem cells cultured on thermoresponsive nanobrush surfaces. In this method, stem cells are partially detached from the nanobrush surface by reducing the temperature of the culture medium below the critical solution temperature needed for thermoresponse. The detached stem cells are harvested by exchange into fresh culture medium. Following this, the remaining cells are continuously cultured by expansion in fresh culture medium at 37 °C. Thermoresponsive nanobrush surfaces were prepared by coating block copolymers containing polystyrene (for hydrophobic anchoring onto culture dishes) with three types of polymers: (a) polyacrylic acid with cell-binding oligopeptides, (b) thermoresponsive poly-N-isopropylacrylamide, and (c) hydrophilic poly(ethyleneglycol)methacrylate. The optimal coating durations and compositions for these copolymers to facilitate adequate attachment and detachment of human adipose-derived stem cells (hADSCs) and embryonic stem cells (hESCs) were determined. hADSCs and hESCs were continuously harvested for 5 and 3 cycles, respectively, via the partial detachment of cells from thermoresponsive nanobrush surfaces.

Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces.

The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database creates networks from interrelations between molecular biology and underlying chemical elements. This allows for analysis of biologic networks, genomic information, and higher-order functional information at a system level. Through high throughput experiments and system biology analysis, we investigated the genes and pathways associated with NGF induced neuronal differentiation. We performed microarray experiments and used the KEGG database, system biology analysis, and annotation of pathway functions to study NGF-induced differentiation in PC12 cells. We identified 2020 NGF-induced genes with altered expressions over time. Cross-matching with the KEGG database revealed 830 genes; among which, 395 altered genes were found to have a 2-fold increase in gene expression over a two-hour period. We then identified 191 associated biologic pathways in the KEGG database; the top 15 pathways showed correlation with neural differentiation. These included the neurotrophin pathways, mitogen-activated protein kinase (MAPK) pathways, genes associated with axonal guidance and the Wnt pathways. The activation of these pathways synchronized with nerve growth factor (NGF)-induced differentiation in PC12 cells. In summary, we have established a model system that allows one to systematically characterize the functional pathway changes in a group of neuronal population after an external stimulus.

Chemogenomic analysis of neuronal differentiation with pathway changes in PC12 cells.

The tentative clinical application of human pluripotent stem cells (hPSCs), such as human embryonic stem cells and human induced pluripotent stem cells, is restricted by the possibility of xenogenic contamination resulting from the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. Therefore, we investigated hPSC cultures on biomaterials with different elasticities that were grafted with different nanosegments. We prepared dishes coated with polyvinylalcohol-co-itaconic acid hydrogels grafted with an oligopeptide derived from vitronectin (KGGPQVTRGDVFTMP) with elasticities ranging from 10.3 to 30.4 kPa storage moduli by controlling the crosslinking time. The hPSCs cultured on the stiffest substrates (30.4 kPa) tended to differentiate after five days of culture, whereas the hPSCs cultured on the optimal elastic substrates (25 kPa) maintained their pluripotency for over 20 passages under xeno-free conditions. These results indicate that cell culture matrices with optimal elasticity can maintain the pluripotency of hPSCs in culture.

Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity.

This data article contains two figures and one table supporting the research article entitled: "Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surface" [1]. The table shows coating conditions of three copolymers, poly(styrene-co-acrylic acid) grafted with oligovitronectin, poly(styrene-co-N-isopropylacrylamide) and poly(styrene-co-polyethylene glycol methacrylate) to prepare thermoresponsive surface. XPS spectra show the nitrogen peak of the polystyrene surface coated with poly(styrene-co-acrylic acid) grafted with oligovitronectin. The surface coating density analyzed from sorption of poly(styrene-co-acrylic acid) grafted with oligovitronectin by UV-vis spectroscopy is also presented.

Data of continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces.

Establishing cultures of human embryonic (ES) and induced pluripotent (iPS) stem cells in xeno-free conditions is essential for producing clinical-grade cells. Development of cell culture biomaterials for human ES and iPS cells is critical for this purpose. We designed several structures of oligopeptide-grafted poly (vinyl alcohol-co-itaconic acid) hydrogels with optimal elasticity, and prepared them in formations of single chain, single chain with joint segment, dual chain with joint segment, and branched-type chain. Oligopeptide sequences were selected from integrin- and glycosaminoglycan-binding domains of the extracellular matrix. The hydrogels grafted with vitronectin-derived oligopeptides having a joint segment or a dual chain, which has a storage modulus of 25 kPa, supported the long-term culture of human ES and iPS cells for over 10 passages. The dual chain and/or joint segment with cell adhesion molecules on the hydrogels facilitated the proliferation and pluripotency of human ES and iPS cells.

Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs.

Cardiovascular disease remains the leading cause of death and disability in advanced countries. Stem cell transplantation has emerged as a promising therapeutic strategy for acute and chronic ischemic cardiomyopathy. The current status of stem cell therapies for patients with myocardial infarction is discussed from a bioengineering and biomaterial perspective in this review. We describe (a) the current status of clinical trials of human pluripotent stem cells (hPSCs) compared with clinical trials of human adult or fetal stem cells, (b) the gap between fundamental research and application of human stem cells, (c) the use of biomaterials in clinical and pre-clinical studies of stem cells, and finally (d) trends in bioengineering to promote stem cell therapies for patients with myocardial infarction. We explain why the number of clinical trials using hPSCs is so limited compared with clinical trials using human adult and fetal stem cells such as bone marrow-derived stem cells.

Stem cell therapies for myocardial infarction in clinical trials: bioengineering and biomaterial aspects.

This study investigates peptide components of L-selectin ligand (LSL) and their gene expressions in human endometrium during the natural menstrual cycle. We recruited 41 endometrial samples from reproductive-aged women with leiomyoma and undergoing hysterectomy and 11 endometrial samples from menopausal women as controls. Immunohistochemistry revealed strong MECA-79 expression from the early through the mid-secretory phase and low expression in menopausal endometrium. Five peptide components of LSL were detected in reproductive and menopausal endometrium by one-step quantitative RT-PCR: podocalyxin, endomucin, nepmucin, GlyCAM-1, and CD34. Endomucin differed significantly between the proliferative and early-secretory phases. CHST2 and CHST4 genes (which are involved in the generation of LSL epitopes) were expressed without significant differences among phases. The gene expression of progesterone receptor decreased from the proliferative to the late-secretory phase, and the difference was significant. However, estrogen receptor α expression showed stability among phases. The significant expression of endomucin between the proliferative and early-secretory phases might play a vital role in endometrial receptivity. Further studies are needed to investigate the factors that regulate the expression of endomucin and other LSL peptide components in different phases of the menstrual cycle.

Article	Year
Lower limb wrapping prevents hypotension, but not hypothermia or shivering, after the introduction of epidural anesthesia for cesarean delivery. Anesthesia and analgesia\| PubMed ID: 15281537	2004
Prevalence of cervical human papillomavirus in Taiwanese women. Clinical and investigative medicine. Medecine clinique et experimentale\| PubMed ID: 16265998	2005
Prenatal Group B Streptococcus test using real-time polymerase chain reaction. Taiwanese journal of obstetrics & gynecology\| PubMed ID: 19574170	2009
Increased spinal prodynorphin gene expression in reinflammation-associated hyperalgesia after neonatal inflammatory insult. BMC neuroscience\| PubMed ID: 20973986	2010
Drug-resistant colon cancer cells produce high carcinoembryonic antigen and might not be cancer-initiating cells. Drug design, development and therapy\| PubMed ID: 23818760	2013
Purification of human adipose-derived stem cells from fat tissues using PLGA/silk screen hybrid membranes. Biomaterials\| PubMed ID: 24565521	2014
Generation of pluripotent stem cells without the use of genetic material. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 25365202	2015
A hybrid-membrane migration method to isolate high-purity adipose-derived stem cells from fat tissues. Scientific reports\| PubMed ID: 25970301	2015
Continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces. Biomaterials\| PubMed ID: 26519650	2016
Chemogenomic analysis of neuronal differentiation with pathway changes in PC12 cells. Molecular bioSystems\| PubMed ID: 26595144	2016
Long-term xeno-free culture of human pluripotent stem cells on hydrogels with optimal elasticity. Scientific reports\| PubMed ID: 26656754	2015
Data of continuous harvest of stem cells via partial detachment from thermoresponsive nanobrush surfaces. Data in brief\| PubMed ID: 26909373	2016
Xeno-free culture of human pluripotent stem cells on oligopeptide-grafted hydrogels with various molecular designs. Scientific reports\| PubMed ID: 28332572	2017
Stem cell therapies for myocardial infarction in clinical trials: bioengineering and biomaterial aspects. Laboratory investigation; a journal of technical methods and pathology\| PubMed ID: 28869589	2017
Gene expression of human endometrial L-selectin ligand in relation to the phases of the natural menstrual cycle. Scientific reports\| PubMed ID: 29362381	2018

Qing-Dong Ling

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