Proliferation Of Human Stem Cells Research Articles

Preparation of gallic acid-containing PCL/gelatine scaffolds as an efficient drug delivery system for growth and proliferation of human stem cells

Preparation of gallic acid-containing PCL/gelatine scaffolds as an efficient drug delivery system for growth and proliferation of human stem cells

A Response to Article "Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells" [Letter

A Response to Article "Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells" [Letter

Reconstitution of a telomeric replicon organized by CST

Telomeres, the natural ends of linear chromosomes, comprise repeat-sequence DNA and associated proteins1. Replication of telomeres allows continued proliferation of human stem cells and immortality of cancer cells2. This replication requires telomerase3 extension of the single-stranded DNA (ssDNA) of the telomeric G-strand ((TTAGGG)n); the synthesis of the complementary C-strand ((CCCTAA)n) is much less well characterized. The CST (CTC1–STN1–TEN1) protein complex, a DNA polymerase α-primase accessory factor4,5, is known to be required for telomere replication in vivo6–9, and the molecular analysis presented here reveals key features of its mechanism. We find that human CST uses its ssDNA-binding activity to specify the origins for telomeric C-strand synthesis by bound Polα-primase. CST-organized DNA polymerization can copy a telomeric DNA template that folds into G-quadruplex structures, but the challenges presented by this template probably contribute to telomere replication problems observed in vivo. Combining telomerase, a short telomeric ssDNA primer and CST–Polα–primase gives complete telomeric DNA replication, resulting in the same sort of ssDNA 3′ overhang found naturally on human telomeres. We conclude that the CST complex not only terminates telomerase extension10,11 and recruits Polα–primase to telomeric ssDNA4,12,13 but also orchestrates C-strand synthesis. Because replication of the telomere has features distinct from replication of the rest of the genome, targeting telomere-replication components including CST holds promise for cancer therapeutics.

Rho-Associated Protein Kinase Inhibitor and Hypoxia Synergistically Enhance the Self-Renewal, Survival Rate, and Proliferation of Human Stem Cells.

IntroductionHigh-efficacy single-cell cloning of human-induced pluripotent cells (IPSCs) remains a major challenge. The development of a culture method that supports single-cell passaging while maintaining reproducibility, homogeneity, scalability, and cell expansion to clinically relevant numbers is necessary for clinical application.MethodsTo address this issue, we combined the use of the rho-associated protein kinase (ROCK) inhibitor Y-27632 and hypoxic conditions in culture to produce a novel, efficient single-cell culture method for human IPSCs and embryonic stem cells.ResultsThrough immunocytochemistry, alkaline phosphatase assays, and flow cytometry, we demonstrated that our method enabled high single-cell proliferation while maintaining self-renewal and pluripotency abilities.DiscussionWe showed the beneficial effect of the interaction between hypoxia and ROCK inhibition in regulating cell proliferation, pluripotency, and single-cell survival of pluripotent cells.

In vitro stimulation with radiofrequency currents promotes proliferation and migration in human keratinocytes and fibroblasts

ABSTRACT Capacitive-resistive electric transfer (CRET) therapies have been proposed as strategies for regeneration of cutaneous tissue lesions. Previous studies by our group have shown that intermittent stimulation with 448 kHz CRET currents at subthermal densities promotes in vitro proliferation of human stem cells involved in tissue regeneration. The present study investigates the effects of the in vitro exposure to these radiofrequency (RF) currents on the proliferation and migration of keratinocytes and fibroblasts, the main cell types involved in skin regeneration. The effects of the electric stimulation on cell proliferation and migration were studied through XTT and wound closure assays, respectively. The CRET effects on the expression and location of proteins involved in proliferation and migration were assessed by immunoblot and immunofluorescence. The obtained results reveal that electrostimulation promotes proliferation and/or migration in keratinocytes and fibroblasts. These effects would be mediated by changes observed in the expression and location of intercellular adhesion proteins such as β-catenin and E-cadherin, of proteins involved in cell-to-substrate adhesion such as vinculin, p-FAK and the metalloproteinase MMP-9, and of other proteins that control both processes: MAP kinases p-p38, p-JUNK and p-ERK1/2. These responses could represent a mechanism underlying the promotion of normotrophic wound regeneration induced by CRET. Indeed, electric stimulation would favor completion of granulation tissue formation prior to the closure of the outer tissue layers, thus preventing abnormal wound cicatrization or chronification.

Bovine pericardium membrane as new tool for mesenchymal stem cells commitment

Acellular matrices are widespread biomaterials used in surgical practice as tissue reinforcement and anatomical support to favor tissue regeneration. It is clear that a fundamental role in the regeneration of tissue is played by cell-material interaction. In this work, the interaction between a bovine pericardium membrane and human adult stem cells was investigated by microscopy analysis and gene expression analysis. Parallel cell cultures were prepared on the pericardium membrane or tissue culture plate. They were incubated in basal growth medium or in adipogenic differentiation medium to perform experiments on the seventh and the 14th day of culture. Results demonstrated that the membrane allows cell viability, adhesion, and proliferation of human stem cells. During adipogenic commitment on the membrane, the accumulation of cytoplasmatic lipid droplets and the expression of adipogenic gene PPARG, CEBPA, GLUT4, FABP4, and ADIPOQ were detected. Concurrently, a downregulation of mesenchymal stem cell gene CD29, CD90, and CD105 was detected. In basal medium, the adipogenic gene expression was upregulated, whereas the mesenchymal markers were indifferently expressed. These findings suggest that the bovine pericardium membrane is a biocompatible matrix and that their rough surface allows cell adhesion, spreading, and proliferation. The surface morphology activates mechanochemical signals that stimulate the adipogenic commitment of stem cells in basal medium and potentiate their commitment in adipogenic differentiation medium.

Modified calcium silicophosphate cements with improved properties

The synthesis and characterisation of inorganic phosphate cements based on calcium silicates (wollastonite and calcium disilicate) and phosphoric acid are presented in this paper. Nanopowders of barium titanate (BT) and biocellulose (BC) were used as additions (10 wt%) in order to improve their bioactivity for potential medical applications. The processing route of the precursors and the thermal treatment conditions had a high impact on the hardening processes and properties of calcium silicophosphate cements. The addition of BT and BC nanopowders determined the decrease of setting time (at the same liquid to powder ratio) but did not significantly influenced the mechanical properties of the resulted composites. In vitro biocompatibility tests showed no cellular oxidative stress, an increase of human stem cells proliferation on these materials and osteoconductive capacity by highlighting the presence of bone morphogenetic protein-1, osteopontin and alkaline phosphatase genes.

Research of Luteoloside in promoting proliferation and migration of human epidermal stem cells

Objective To investigate the effects of Luteoloside on the proliferation and migration of human epidermal stem cells (EpSCs) and the proliferation of epidermal cells in skin tissue in vitro. Methods EpSCs were isolated from human skin tissue by type Ⅳ collagenase digestion and collagen fast adherence. EpSCs were identified by immunofluorescence staining of CK19 and β1 integrin. The effects of Luteoloside (0 to 5.0 μmol/L) on the proliferation of EpSCs were measured by MTT assay. The effects of Luteoloside on EpSCs migration and wound healing were observed by scratch-wound model in vitro. The effects of Luteoloside on the thickness of epidermis in vitro were observed by HE staining. Results EpSCs formed a cobblestone-shaped cell monolayer when cultured on a collagen coated dish. Immunofluorescence staining showed that more than 90% cells were CK19 and β1 integrin positive. Luteoloside at 0.1 to 1.0 μmol/L could significantly promote the EpSCs proliferation; Luteoloside at 1.0 μmol/L could significantly enhance EpSCs migration and scratch healing. Culture of skin tissues with 1.0 μmol/L Luteoloside for 5 days could significantly increase the thickness of the epidermis. Conclusion Luteoloside can promote the proliferation and migration of EpSCs, promote the proliferation of epidermal tissue, and play an important role in skin self-renewal and injury repair. Key words: Cell proliferation; In vitro; Luteoloside; Epidermal stem cells; Migration

Cowries derived aragonite as raw biomaterials for bone regenerative medicine

Carbonate apatites are sought as a bone substitute due to their biocompatibility and excellent resorbability. The present study deals with Cowrie's shell derived powder (CSDP) as natural biomaterial for bone regenerative medicine. Structural and physicochemical analysis showed that Cowrie's shells, presenting brick and mortar microstructures, were mainly composed of aragonite crystals, which were converted into poorly crystalline B-type carbonate apatite once soaked, at 37 °C, in simulated body fluid for 7 days, reflecting bioactive features. Cytotoxic assays showed that CSDP boosted human stem cell proliferation over the study time compared to nacre derived powder (NDP), used as positive control. Human stem cells adopted a flattened morphology and established physical contact with CSDP, signature of a good biocompatibility. Thus, these results suggested that CSDP presents a great interest for bone regenerative medicine, and could be a useful and versatile carrier/scaffold for bone tissue engineering or a raw material for 3D printed orthopedic devices.

Hydrogen Sulfide-Releasing Fibrous Membranes: Potential Patches for Stimulating Human Stem Cells Proliferation and Viability under Oxidative Stress.

The design of biomaterial platforms able to release bioactive molecules is mandatory in tissue repair and regenerative medicine. In this context, electrospinning is a user-friendly, versatile and low-cost technique, able to process different kinds of materials in micro- and nano-fibers with a large surface area-to-volume ratio for an optimal release of gaseous signaling molecules. Recently, the antioxidant and anti-inflammatory properties of the endogenous gasotramsmitter hydrogen sulfide (H2S), as well as its ability to stimulate relevant biochemical processes on the growth of mesenchymal stem cells (MSC), have been investigated. Therefore, in this work, new poly(lactic) acid fibrous membranes (PFM), doped and functionalized with H2S slow-releasing donors extracted from garlic, were synthetized. These innovative H2S-releasing mats were characterized for their morphological, thermal, mechanical, and biological properties. Their antimicrobial activity and effects on the in vitro human cardiac MSC growth, either in the presence or in the absence of oxidative stress, were here assessed. On the basis of the results here presented, these new H2S-releasing PFM could represent promising and low-cost scaffolds or patches for biomedical applications in tissue repair.

Evaluation of astaxanthin effects on the survival and proliferation of human adipose derived stem cells

Evaluation of astaxanthin effects on the survival and proliferation of human adipose derived stem cells

Effects of Bambusa tulda on the proliferation of human stem cells.

To date, the effects of Bambusa tulda on stem cells have not been thoroughly assessed. The present study aimed to evaluate the effects of Bambusa tulda extract on the morphology and proliferative potential of human mesenchymal stem cells derived from the gingiva. The stem cells were cultured in a growth medium in the presence of Bambusa tulda methanolic extract (BBT) at concentrations ranging from 0.001 to 1%. Evaluation of cell morphology and cellular proliferation as well as immunofluorescent assays for collagen I were performed on days 1, 3, 5 and 7. Stem cells in the control group displayed a fibroblast-like morphology, and BBT treatment did not produce any noticeable morphological changes. However, application of 1% BBT produced a significant increase in cell proliferation. BBT, particularly at the concentration of 1%, also caused a noticeable increase of collagen I expression at day 1 and day 3. Based on these findings, it was concluded that BBT exerted beneficial effects on the proliferation of mesenchymal stem cells and enhanced collagen I expression at early time points.

Real-time regulatory effects of IFN-γ and programed death ligand 2 (PDL2) on adherence, proliferation and migration of human placenta-derived mesenchymal stem cells

Objective To investigate the real-time regulatory effects of IFN-γ, programed death ligand 2(PDL2) and janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway on the adherence, proliferation and migration of human placenta-derived mesenchymal stem cells(hPMSCs) based on a finding that IFN-γ could enhance the expression of PDL2 in hPMSCs through JAK/STAT signaling pathway. Methods hPMSCs were isolated by using enzyme digestion method and then co-cultured with IFN-γ, anti-PDL2 monoclonal antibody (anti-PDL2 McAb) and JAK inhibitor, respectively. Real-time cell analysis (RTCA) was used to detect the dynamic changes in the adherence, proliferation and migration of hPMSCs following various interventions. Results IFN-γ remarkably suppressed hPMSCs proliferation during the period from 40 hours to 80 hours after intervention and also inhibited the non-targeted migration of hPMSCs. However, hPMSCs adherence was not affected by IFN-γ. Co-culturing hPMSCs with anti-PDL2 McAb significantly enhanced hPMSCs adhesion and inhibited their non-targeted migration, but had no significant effect on hPMSCs proliferation. Furthermore, the proliferation of hPMSCs co-cultured with IFN-γ and anti-PDL2 McAb was significantly inhibited as compared with that of anti-PDL2McAb treatment group. The adhesion, migration and proliferation of hPMSCs were significantly inhibited after co-culturing them with JAK inhibitor. Conclusion IFN-γ can remarkably suppress the proliferation and migration of hPMSCs. PDL2 can enhance the migration and inhibit the adhesion of hPMSCs. JAK/STAT signaling pathway is involved in regulating the adhesion, migration and proliferation of hPMSCs. Key words: IFN-γ; Mesenchymal stem cell; Adherence; Proliferation; Migration; PDL2

Palmitic Acid Methyl Ester Regulates Proliferation of Human Mesenchymal Stem Cells

BACKGROUNDHuman mesenchymal stem cell (hMSCs) have unique multilineage differentiation and self‐renewal capabilities that can be used for cell therapy and regenerative medicine. Although MSCs can be easily isolated from many tissues such as bone marrow, adipose tissue, and umbilical cord etc., it is still challenging to obtain sufficient numbers of cells for clinical use. In vitro environments, chemical cues such as fatty acid affect stem cell proliferation. Palmitic acid methyl ester (PAME) is released from adipose tissue and synthesized through transesterification of palmitic acid(PA) with methanol. PAME was shown to possess potent anti‐inflammatory and anti‐fibrotic. However, the effect of PAME on MSCs proliferation is unknown.PURPOSEIn this study, we investigated effects and mechanisms of PAME on proliferation of the cultured hMSCs.METHODShMSCs were cultured in αMEM for 1 day, and then treated with PAME for 2 days. We performed MTT and BrdU assays to determine the proliferate capacity of these cells, and evaluated cell cycle and mitochondrial reactive oxygen species (ROS) using flow cytometry. The action of PAME on signaling pathway was analyzed by using Western Blots and Quantitative real time‐PCR.RESULTSOur results show that PAME inhibited the proliferation of hMSCs in a dose‐dependent manner (10, 30, 50 and 100 μM) through G2/M phase arrest, which is mediated by down‐regulation of G2/M‐related mRNA and proteins (CDK1 and Cyclin B1). In addition, we found that PAME treatment (2 days) increased p53 and p21 expression, but did not affect the mitochondrial ROS level.CONCLUSIONPAME inhibits the proliferation of hMSCs through G2/M phase arrest by regulating the p53–p21 signaling pathway.

Effects of SP600125 at various concentrations on proliferation and osteogenesis of human adipose-derived stem cells in vitro

Objective To elucidate the effects of SP600125 at different concentrations on the proliferation and osteo-differentiation of human adipose-derived stem cells (hASCs). Methods The hASCs harvested were cocultured with SP600125 at concentrations of 0 μmol/L, 1 μmol/L, 5 μmol/L and 10 μmol/L in growth medium (OM group) and in osteogenesis medium (OM group), respectively. The DNA quantitative assay was carried out to evaluate proliferation of the hASCs; flow cytometry was used to determine the effect of SP600125 on the cell cycles of hASCs; Alkaline phosphatase level (ALP) and calcium deposition tests were conducted to observe the effects of SP600125 at different concentrations on osteogenic differentiation of the hASCs. Results The proliferation of hASCs was inhibited by 42.1% when the cells were cocultured with SP600125 at the concentration of 10 μmol/L; the suppression decreased with decreased concentration of SP600125. The hASCs of phase G0/G1 in GM cocultured with SP600125 at the concentration of 10 μmol/L were more than those in GM cocultured with dimethylsulfoxide at the same concentration. ALP test revealed that after 10 days of culture in vitro the staining was more and more weakened and scattered and the ALP activity was more and more decreased with the increased concentration of SP600125. The extracellular calcium deposition of hASCs after 14 days of culture in vitro showed that the size and number of calcium nodules decreased with the increased concentration of SP600125. Conclusion SP600125 can suppress the proliferation and osteogenic differentiation of hASCs in vitro. Key words: Human; Adipocytes; Cell proliferation; Osteogenic differentiation; SP600125

Abstract 1711: Notch and mTORC1 regulate human gastric stem cell function during normal tissue homeostasis and gastric cancer

Abstract Notch and mTOR pathway activation have been observed in human gastric cancers. We have recently shown that chronic Notch activation induces gastric stem cell proliferation and tissue expansion in mouse via gland fission, which can be attenuated by mTORC1 inhibition. These findings suggest that these two pathways may synergize to promote gastric tumorigenesis. Here we tested the requirement of Notch and mTOR signaling for maintenance of human stem cells from control and gastric cancer tissues. Gastric glands were isolated from control (non-cancer) and human gastric cancer tissues, as well as mouse antrum to initiate organoid cultures. Established organoids were treated with vehicle, the Notch inhibitor DAPT, the mTORC1 inhibitor rapamycin, or both, every other day for 5 days, to test epithelial-specific requirements of each pathway for stem cell maintenance. Growth was determined by measuring organoid size, and proliferation was assessed by EdU incorporation. Gastric cancer cell lines AGS, MKN45 and NCI-N87 were treated with pathway inhibitors once per day for 5 days, with cell growth measured each day. Gene expression analysis of Notch pathway components was performed by qRT-PCR. In both human and mouse stomach, Notch1 and Notch2 were the main Notch receptors expressed in tissue and organoids. Notch inhibition with DAPT significantly inhibited growth of both human and mouse gastric organoids; however organoids derived from human gastric cancer tissue required 10-fold increased DAPT for significant growth inhibition. Surprisingly, mTORC1 inhibition with rapamycin significantly increased organoid size and epithelial proliferation of mouse organoids, as well as both control and tumor-derived human organoids, suggesting that active mTORC1 signaling restricts human stem cell proliferation. Combination treatment of human organoid cultures with both pathway inhibitors attenuated the rapamycin-induced increase in organoid size and proliferation. In contrast, treatment of gastric cancer cell lines with DAPT, rapamycin or both inhibitors significantly reduced cell growth compared to vehicle, suggesting that blocking Notch or mTORC1 in gastric cancer cells can be used to reduce cancer cell growth. In conclusion, Notch and mTORC1 signaling regulate mouse and human gastric stem cell function although through opposite mechanisms, with Notch stimulating human stem cell proliferation and mTORC1 inhibiting proliferation. Additionally, mTORC1 may regulate human gastric stem cell proliferation via a different mechanism than gastric cancer cells. Further studies testing the mechanism of Notch and mTORC1 interactions to regulate human gastric stem cell maintenance will inform our understanding of therapeutic strategies for gastric cancer. NIH F32-DK093349; NIH UL1TR000433; NIH P01-DK062041 and NCI P50-CA130810 Citation Format: Elise S. Demitrack, Gail B. Gifford, Gabriela Wong, Linda C. Samuelson. Notch and mTORC1 regulate human gastric stem cell function during normal tissue homeostasis and gastric cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1711.

Selective enhancement of human stem cell proliferation by mussel inspired surface coating

The biocompatibility and cell adhesion properties of mussel inspired polydopamine and polynorepinephrine surface coatings on PCL fibers for human mesenchymal and human induced pluripotent stem cell derived mesenchymal stem cells were investigated.

Bioactive glass 45S5-silk fibroin membrane supports proliferation and differentiation of human dental pulp stem cells

To investigate the effect of bioactivity glass 45S5- silk fibroin(BG45S5- SF) membrane on growth, proliferation and differentiation of human dental pulp stem cells(hDPSC), and to provide new ideas and method for the regeneration of pulp-dentine complex. hDPSC seed on pure silk fibroin membrane (protein membrane group) and BG45S5-SF membrane with different concentrations(1 000, 5 000 mg/L, composite membrane group A and B, respectively) were prepared, and the materials were incubated in cell culture fluid for 24 h. No material membrane orifice plate was used as blank control group. Contact angle meter was used to measure surface contact angle of protein membrane and composite membrane group(each group had three repeated holes). Cell proliferation was assessed by cell counting kit- 8 on the 4, 7, 14, and 21 days. The state of adhesion and growth of hDPSC on the materials surface was evaluated by scanning electron microscopy and cytoskeleton staining; and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation potential. The expression of odontoblastic differentiation-related genes was measured by real-time PCR. Surface contact angle of the protein membrane group and composite membrane group A and group B were 89.51° ± 0.12°, 70.32° ± 0.07° and 71.31° ± 0.09° respectively. hDPSC adhered well on each materials surface on the 7, 14, 21 days, ALP activity and differentiation genes of composite membrane group A and B rised more significantly than the blank control group and protein membrane group did (P<0.05). Dentin matrix protein1(DMP- 1), dentin sialoprotein(DSP), ALP, osteocalcin(OC) mRNA expression reached peak on the 14 days in group A, and in group B on the 21 days. Bone sialoprotein(BSP) mRNA expression in both group A and B reached peak on the 21 days. BG45S5- SF membrane is able to support the proliferation and showed the potential of odontoblastic differentiation for hDPSC. This finding suggests that BG45S5-SF membrane was a kind of tissue engineering film material with the regeneration potential for pulp-dentine complex.

Copolyesters from Soybean Oil for Use as Resorbable Biomaterials

A family of soybean oil (SO) based biodegradable cross-linked copolyesters sourced from renewable resources was developed for use as resorbable biomaterials. The polyesters were prepared by a melt condensation of epoxidized soybean oil polyol and sebacic acid with citric acid (CA) as a cross-linker. D-Mannitol (M) was added as an additional reactant to improve mechanical properties. Differential scanning calorimetry revealed that the polyester synthesized using only CA as the cross-linker was semicrystalline and elastomeric at physiological temperature. The polymers were hydrophobic in nature. The water wettability, elongation at break and the degradation rate of the polyesters decreased with increase in M content or curing time. Modeling of release kinetics of dyes showed a diffusion controlled mechanism underlies the observed sustained release from these polymers. The polyesters supported attachment and proliferation of human stem cells and were thus cytocompatible. Porous scaffolds induced osteogenic differentiation of the stern cells suggesting that these polymers are well suited for bone tissue engineering. Thus, this family of polyesters offers a low cost and green alternative as biocompatible, bioresobable polymers for potential use as resorbable biomaterials for tissue engineering and controlled release.

Effect of high-expression HOXB4 on human cartilage endplate stem cells and its significance

Objective To observe the effect and significance of high-expression HOXB4 in controlling proliferation and cycle of human cartilage endplate stem cells (CESCs).Methods CESCs were divided into adenovirus-mediated HOXB4 delivery group (Group A),empty virus delivery group (Group B) and blank control group.Gene and protein expressions of HOXB4 in Group A were detected by PCR and Western blot respectively; cell proliferation among those groups were determined using cell counting kit 8 (CCK8) technique; cell cycle among those groups was measured by propidium iodide (PI) assay and flow cytometry.Results (1) Over-expressed HOXB4 virus was transferred to CESCs successfully; (2) Real-time quantitative PCR results showed 3.6 times higher expression of HOXB4 in Group A than in blank control group.Western blotting indicated HOXB4 protein in Group A was 3 times the level in control group; (3) HOXB4 promoted CESCs proliferation (P ＜ 0.05) and blocked the cells at phase S.Cells at phase S in Group A was increased from 29.27 to 30.28 (P ＜ 0.05).Conclusion Over-expressed HOXB4 accelerates proliferation of CESCs and increases cell population at phase S,indicating that HOXB4 hindering CESCs degeneration may be an approach to treat lumbar intervertebral disc protrusion. Key words: Intervertebral disc; Cartilage; Stem cells