Cell Source For Tissue Engineering Research Articles

Effects of glucose, lactate and basic FGF as limiting factors on the expansion of human induced pluripotent stem cells

Pluripotent stem cells (PSCs) are one of the promising cell sources for tissue engineering and drug screening. However, mass production of induced pluripotent stem cells (iPSCs) is still developing. Especially, a huge amount of culture medium usage causes expensive cost in the mass production process. In this report, we reduced culture medium usage by extending interval of changing culture medium. In parallel, we also increased glucose concentration and supplied heparan sulfate to avoid depletion of glucose and bFGF, respectively. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses showed that reducing medium change frequency increased differentiation marker expressions but high glucose concentration downregulated these expressions. In contrast, heparan sulfate did not prevent differentiation marker expressions. According to analyses of growth rate, cell growth with extended medium change interval was decreased in later stage of log growth phase despite the existence of high glucose concentration and heparan sulfate. This result and culturing iPSCs with lactate showed that the accumulation of excreted lactate decreased the growth rate regardless of pH control. Conclusively, these experiments show that adding glucose and removing lactate are important to expand iPSCs with reduced culture medium usage. This knowledge should be useful to design economical iPSC mass production and differentiation system.

In vitrodifferentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β 1 and vascular endothelial growth factor

To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblasts in vitro. The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β 1 (TGF-β 1) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days ( P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, and Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture ( P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group ( P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points ( P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days ( P>0.05). The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after induction in vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β 1 and VEGF could be used as growth factors sources in constructing tissue engineered ligament.

SFRP2 enhanced the adipogenic and neuronal differentiation potentials of stem cells from apical papilla.

Dental tissue-derived mesenchymal stem cells (MSCs) are easily obtained and considered as a favorable cell source for tissue engineering, but the regulation of direct differentiation is unknown, which restricts their application. The present study investigated the effect of SFRP2, a Wnt signaling modulator, on MSC differentiation using stem cells from apical papilla (SCAPs). The cells were cultured in specific inducing medium for adipogenic, neurogenic, or chondrogenic differentiation. Over-expression of SFRP2 via retroviral infection enhanced the adipogenic and neurogenic differentiation of SCAPs. While inhibit of Wnt pathway by IWR1-endo could enhance the neurogenic differentiation potentials of SCAPs, similar with the function of SFRP2. In addition, over-expression of SFRP2 up-regulated the expression of stemness-related genes SOX2 and OCT4. Furthermore, SOX2 and OCT4 expression was significantly inhibited after lentiviral silencing of SFRP2 in SCAPs. Therefore, our results suggest that SFRP2 enhances the adipogenic and neurogenic differentiation potentials of SCAPs by up-regulating SOX2 and OCT4. Moreover, the effect of SFRP2 in neurogenic differentiation of SCAPs maybe also associated with Wnt inhibition. Our results provided useful information about the molecular mechanism underlying directed differentiation in dental tissue-derived MSCs.

Dental pulp stem cells express tendon markers under mechanical loading and are a potential cell source for tissue engineering of tendon-like tissue.

Postnatal mesenchymal stem cells have the capacity to differentiate into multiple cell lineages. This study explored the possibility of dental pulp stem cells (DPSCs) for potential application in tendon tissue engineering. The expression of tendon-related markers such as scleraxis, tenascin-C, tenomodulin, eye absent homologue 2, collagens I and VI was detected in dental pulp tissue. Interestingly, under mechanical stimulation, these tendon-related markers were significantly enhanced when DPSCs were seeded in aligned polyglycolic acid (PGA) fibre scaffolds. Furthermore, mature tendon-like tissue was formed after transplantation of DPSC-PGA constructs under mechanical loading conditions in a mouse model. This study demonstrates that DPSCs could be a potential stem cell source for tissue engineering of tendon-like tissue.

Differentiation of rat dermal mesenchymal cells and calcification in three-dimensional cultures.

Three-dimensional (3D) cultures are known to promote cell differentiation. Previously, we investigated the differentiation of rat dermal fibroblasts to α-smooth muscle actin (α-SMA)-positive myofibroblasts through transforming growth factor (TGF)-ß production using a 3D culture model. Here, we investigated the phenotypic change from dermal mesenchymal cells (mostly fibroblasts) to osteoblast-like cells, being inspired by the roles of smooth muscle cells or fibroblasts during vascular calcification. Spindle-shaped cells that grew in heterologous populations out of dermal explants from 2-day-old Wistar rats were cultured within a collagen matrix. α-SMA and alkaline phosphatase (ALP) meßsenger RNA (mRNA) levels initially increased, followed by a rise in Runx2 and osteocalcin (OCN) mRNA levels without calcification. Calcium deposits were produced in the presence of a high concentration of inorganic phosphate (2.1 mM) or ß-glycerophosphate (ßGP, 10 mM) after 2 weeks of culture, and both were sensitive to an inhibitor of type III phosphate transporters. An ALP inhibitor decreased only ßGP-induced calcification. Inhibition of TGF-ß type-I receptors attenuated ALP mRNA levels and ßGP-induced calcification, suggesting that endogenous TGF-ß stimulates ALP activity and then ßGP breakdown. An increase in the number of cells embedded in the collagen gel enhanced the mRNA levels of Runx2 and OCN, but not of ALP. Collectively, several factors are likely to promote the differentiation of dermal mesenchymal cells into osteoblast-like cells and ectopic calcification in a 3D collagen matrix, implying the utility of these cells as a potential autologous cell source for tissue engineering.

Human odontogenic epithelial cells derived from epithelial rests of Malassez possess stem cell properties

Epithelial cell rests of Malassez (ERM) are quiescent epithelial remnants of the Hertwig's epithelial root sheath (HERS) that are involved in the formation of tooth roots. ERM cells are unique epithelial cells that remain in periodontal tissues throughout adult life. They have a functional role in the repair/regeneration of cement or enamel. Here, we isolated odontogenic epithelial cells from ERM in the periodontal ligament, and the cells were spontaneously immortalized. Immortalized odontogenic epithelial (iOdE) cells had the ability to form spheroids and expressed stem cell-related genes. Interestingly, iOdE cells underwent osteogenic differentiation, as demonstrated by the mineralization activity in vitro in mineralization-inducing media and formation of calcification foci in iOdE cells transplanted into immunocompromised mice. These findings suggest that a cell population with features similar to stem cells exists in ERM and that this cell population has a differentiation capacity for producing calcifications in a particular microenvironment. In summary, iOdE cells will provide a convenient cell source for tissue engineering and experimental models to investigate tooth growth, differentiation, and tumorigenesis.

Contextual Control of Adipose-Derived Stem Cell Function: Implications for Engineered Tumor Models.

Adipose-derived stem cells (ASCs) are multipotent and, thus, are an attractive cell source for tissue engineering and regenerative medicine. However, ASCs can also differentiate into myofibroblasts, a cell type known to support tumorigenesis, yet the mechanisms underlying the myofibroblastic differentiation of ASCs and the resulting consequences on tumor pathogenesis are not well understood. This knowledge deficit is due, in part, to a lack of 3D culture models that capture the biological and physical heterogeneity of the microenvironment that influences ASC fate under both physiological and pathological conditions. Advanced biomanufacturing strategies offer new opportunities toward the generation of in vivo-like 3D culture environments that can help study how some of these conditions regulate ASC fate. This review discusses the pro-tumorigenic plasticity of ASCs in the tumor microenvironment as well as other disease contexts (e.g., obesity and aging) and highlights advanced biomanufacturing technologies that could be used to integrate stromal parameters into the next generation of engineered 3D tumor models. Such models will enable new insights into ASC-dependent microenvironmental aspects contributing to tumor initiation and progression that may ultimately be targeted for improved anticancer therapies.

Angiogenic Synergistic Effect of Adipose-Derived Stromal Cell Spheroids with Low-Level Light Therapy in a Model of Acute Skin Flap Ischemia

Human adipose-derived mesenchymal stem cells (hASCs) are an attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted hASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted spheroid hASCs in skin flaps of mice. hASCs were cultured in monolayers or spheroids. LLLT, hASCs, spheroids and spheroids transplanted with LLLT were applied to the skin flaps. Healing of the skin flaps was assessed by gross evaluation and by hematoxylin and eosin staining and elastin van Gieson staining. Compared with the spheroid group, skin flap healing was enhanced in the spheroid + LLLT group, including the neovascularization and regeneration of skin appendages. The survival of hASCs was enhanced by decreased apoptosis of hASCs in the skin flaps of the spheroid + LLLT group. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASC and spheroid groups. These data suggest that LLLT was an effective biostimulator of spheroid hASCs in the skin flaps, enhancing the survival of hASCs and stimulating the secretion of growth factors.

Deriving Osteogenic Cells from Induced Pluripotent Stem Cells for Bone Tissue Engineering.

Induced pluripotent stem cells (iPSCs), reprogrammed from adult somatic cells using defined transcription factors, are regarded as a promising cell source for tissue engineering. For the purpose of bone tissue regeneration, efficient in vitro differentiation of iPSCs into downstream cells, such as mesenchymal stem cells (MSCs), osteoblasts, or osteocyte-like cells, before use is necessary to limit undesired tumorogenesis associated with the pluripotency of iPSCs. Until recently numerous techniques on the production of iPSC-derived osteogenic progenitors have been introduced. We reviewed these protocols and provided a perspective on the comparisons of osteogenic potentials of (1) iPSC-derived osteogenic cells produced by different protocols, (2) iPSCs from different somatic origins, and (3) iPSC-derived MSC-like cells and bone marrow stem cells. Finally, we discussed the potential application of the diseased iPSCs for systematic bone disorders.

Isolation and characterization of human gingiva-derived mesenchymal stem cells using limiting dilution method

Background/purposeGingiva-derived mesenchymal stem cells (GMSCs) are attractive alternative MSC sources because of their relative abundance of sources and ease of accessibility. However, the isolation method for harboring GMSCs remains under discussion. The aim of the study was to isolate and explore in vitro characterization of human GMSCs, and compare stem cell properties with bulk-cultured gingival fibroblasts (GFs). Materials and methodsGMSCs were isolated with limiting dilution method. Tissue-matched bulk-cultured GFs and GMSCs were evaluated in terms of their colony-forming abilities, population doubling capacities, cell surface epitopes, and multilineage differentiation potentials. ResultsGMSCs showed a significantly higher number of colony-forming units-fibroblast (P < 0.001) than bulk-cultured GFs, while the population doubling capacity of GMSCs reduced. Both types of cells were uniformly positive for MSC-associated makers CD44, CD73, CD90, CD105, and CD166, and were negative for hematopoietic markers CD14, CD34, and CD45. The only distinct marker was STRO-1, which was more highly expressed in GMSCs (13.4%) than in bulk-cultured GFs (0.02%). Upon induction, GMSCs displayed the capacity to undergo osteogenic, adipogenic, and chondrogenic differentiation. Real-time polymerase chain reaction showed related gene levels were significantly upregulated (P < 0.001). By contrast, bulk-cultured GFs lacked the capacity to undergo multilineage differentiation, and related gene levels showed no significant difference when compared with control groups. ConclusionThe data validate the effectiveness of limiting dilution method for GMSCs isolation. GMSCs, in contrast to bulk-cultured GFs, harbor stem cell characteristics and can act as alternative cell sources for tissue engineering.

The feasibility of adenoviral co-transduction of BMP2 and BMP7 for the expression of recombinant human BMP2/7 heterodimer in rat bone marrow mesenchymal stem cells

To investigate the feasibility of rat bone marrow mesenchymal stem cells (BMSCs) as the target cell of adenovirus-mediated co-transduction of BMP2 and BMP7 genes and then facilitate the expression of recombinant BMP2/7 heterodimer protein. 3 adult male Fischer 344 rats of about 10 weeks of age were used for harvest and in vitro culture of rat BMSCs. Recombinant adenovirus vector carrying BMP2 or BMP7 target genes were constructed with AdMax vector system, and production of high-titer adenoviruses were packaged with HEK293T cells and then concentrated with CSCl2 density-gradient ultra-centrifugation. Rat BMSCs from passage 3 were seeded in 6-well plates at the concentration of 10 000 cells/cm2.After overnight pre-culture, BMSCs were allowed to culture in 200 μl serum-free alpha MEM containing both Ad-BMP2 and Ad-BMP7 adenovirus (100 MOI of each virus). After 7 days in vitro culture, conditioned cell culture supernatants were collected and followed by immunoprecipitation through immune protein G columns pre-loaded with mouse anti-human BMP7 antibody. The resulted protein immune-precipitates were used to assay the expression of BMP2/7 heterodimers via Western Blot and ELISA assay. As a negative control, Rat BMSCs were also genetically transduced with Ad-GFP virus at a concentration of 200 MOI. Our data demonstrated that recombinant adenoviruses carrying BMP2 or BMP7 target gene was successfully reconstructed, packaged, and confirmed via Western Blot assay, which as respected, presented as an unique band at 55 000 size for BMP2 or 49 000 size for BMP7.Adenovirus Ad-GFP was used to verify the integrity of recombinant virus and its transfection efficiency in rat BMSCs, which showed well cell attachment to culture plate and had no cytotoxicity. Green fluorescent protein in BMSCs was also noted eminently under fluorescent microscope. Combined transduction with AdBMP2 plus Ad-BMP7 resulted in the formation of BMP2/7 heterodimers from rat BMSCs. Analysis of conditioned medium via Western Blot assay showed a single protein band of about 47 000 size, just as expected. Quantitative ELISA analysis presented a prominent expression of about (4.33 ± 0.42) ng/ml for recombinant BMP2/7 heterodimers. A paired t test showed significant difference (P ＜ 0.05),when compare to control groups of (0.08 ± 0.02) ng/ml. As an ideal cell source for tissue engineering, rat BMSCs can be genetically modified with Ad-BMP2 plus Ad-BMP7 mediated co-transduction strategy, and efficiently produce recombinant human BMP2/7 heterodimers in vitro, which should facilitate further studies on the beneficial effect of BMP2/7 heterodimers to ex vivo osteogenesis of BMSCs

Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan.

Mesenchymal stem cells (MSCs) are known to be an attractive cell source for tissue engineering and regenerative medicine. One of the main limiting steps for clinical use or biotechnological purposes is the expansion step. The research of compatible biomaterials for MSCs expansion is recently regarded as an attractive topic. The aim of this study was to create new functional biomaterial for MSCs expansion by evaluating the impact of chitosan derivative films modified by enzymatic approach. First, chitosan particles were enzymatically modified with ferulic acid (FA) or ethyl ferulate (EF) under an eco-friendly procedure. Then, films of chitosan and its modified derivatives were prepared and evaluated by physicochemical and biological properties. Results showed that the enzymatic grafting of FA or EF onto chitosan significantly increased hydrophobic and antioxidant properties of chitosan films. The MSCs cell viability on chitosan derivative films also increased depending on the film thickness and the quantity of grafted phenols. Furthermore, the cytotoxicity test showed the absence of toxic effect of chitosan derivative films towards MSCs cells. Cell morphology showed a well attached and spread phenotype of MSCs cells on chitosan derivative films. On the other hand, due to the higher phenol content of FA-chitosan films, their hydrophobic, antioxidant properties and cell adhesion were improved in comparison with those of EF-chitosan films. Finally, this enzymatic process can be considered as a promising process to favor MSCs cell growth as well as to create useful biomaterials for biomedical applications especially for tissue engineering. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:491-500, 2016.

Obesity inhibits the osteogenic differentiation of human adipose-derived stem cells.

BackgroundCraniomaxillofacial defects secondary to trauma, tumor resection, or congenital malformations are frequent unmet challenges, due to suboptimal alloplastic options and limited autologous tissues such as bone. Significant advances have been made in the application of adipose-derived stem/stromal cells (ASCs) in the pre-clinical and clinical settings as a cell source for tissue engineering approaches. To fully realize the translational potential of ASCs, the identification of optimal donors for ASCs will ensure the successful implementation of these cells for tissue engineering approaches. In the current study, the impact of obesity on the osteogenic differentiation of ASCs was investigated.MethodsASCs isolated from lean donors (body mass index <25; lnASCs) and obese donors (body mass index >30; obASCs) were induced with osteogenic differentiation medium as monolayers in an estrogen-depleted culture system and on three-dimensional scaffolds. Critical size calvarial defects were generated in male nude mice and treated with scaffolds implanted with lnASCs or obASCs.ResultslnASCs demonstrated enhanced osteogenic differentiation in monolayer culture system, on three-dimensional scaffolds, and for the treatment of calvarial defects, whereas obASCs were unable to induce similar levels of osteogenic differentiation in vitro and in vivo. Gene expression analysis of lnASCs and obASCs during osteogenic differentiation demonstrated higher levels of osteogenic genes in lnASCs compared to obASCs.ConclusionCollectively, these results indicate that obesity reduces the osteogenic differentiation capacity of ASCs such that they may have a limited suitability as a cell source for tissue engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0776-1) contains supplementary material, which is available to authorized users.

Oxidative stress response of human fibroblasts and endometrial mesenchymal stem cells

Human mesenchymal stem cells are a promising cell source for tissue engineering. During transplantation, they may be subjected to oxidative stress due to unfavorable cellular microenvironment characterized by an increased level of reactive oxygen species. Recently, we have demonstrated that oxidative stress response of human mesenchymal stem cells derived from endometrium (hMESCs) depends on the oxidizer concentration. The duration of cell treatment with an oxidizer also may play an important role. In this study, we investigated the dependence of the cell response on H2O2 treatment duration. The effects of high H2O2 doses on hMESCs and human lung embryonic fibroblasts were compared. In both cell types, H2O2 treatment for 60 min caused multiphase cell cycle arrest, with dose-dependent cell death occurring equally in all phases of the cell cycle. However, the cell death dynamics in hMESCs and fibroblasts were different. Interestingly, in both cell types, shortening of H2O2 treatment from 60 to 10 min induced growth retardation, G1-phase cell accumulation, and cell size increase. Collectively, these findings suggest that there is induction of premature senescence. Thus, shortening of oxidative stress induced in human endometrial stem cells and embryonic fibroblasts by high H2O2 doses enables one to modulate cellular response as both cell death and premature senescence.

Adenosine Signaling Mediates Osteogenic Differentiation of Human Embryonic Stem Cells on Mineralized Matrices.

Human embryonic stem cells (hESCs) are attractive cell sources for tissue engineering and regenerative medicine due to their self-renewal and differentiation ability. Design of biomaterials with an intrinsic ability that promotes hESC differentiation to the targeted cell type boasts significant advantages for tissue regeneration. We have previously developed biomineralized calcium phosphate (CaP) matrices that inherently direct osteogenic differentiation of hESCs without the need of osteogenic-inducing chemicals or growth factors. Here, we show that CaP matrix-driven osteogenic differentiation of hESCs occurs through A2b adenosine receptor (A2bR). The inhibition of the receptor with an A2bR-specific antagonist attenuated mineralized matrix-mediated osteogenic differentiation of hESCs. In addition, when cultured on matrices in an environment deficient of CaP minerals, exogenous adenosine promoted osteogenic differentiation of hESCs, but was attenuated by the inhibition of A2bR. Such synthetic matrices that intrinsically support osteogenic commitment of hESCs are not only beneficial for bone tissue engineering but can also be used as a platform to study the effect of the physical and chemical cues to the extracellular milieu on stem cell commitment. Insights into the cell signaling during matrix-induced differentiation of stem cells will also help define the key processes and enable discovery of new targets that promote differentiation of pluripotent stem cells for bone tissue engineering.

Formation of vascular network structures within cardiac cell sheets from mouse embryonic stem cells.

Bioengineered cardiac tissues represent a promising strategy for regenerative medicine. However, methods of vascularization and suitable cell sources for tissue engineering and regenerative medicine have not yet been established. In this study, we developed methods for the induction of vascular endothelial cells from mouse embryonic stem (ES) cells using three-dimensional (3D) suspension culture, and fabricated cardiac cell sheets with a pre-vascularized structure by co-culture of mouse ES cell-derived endothelial cells. After induction, isolated CD31+ cells expressed several endothelial cell marker genes and exhibited the ability to form vascular network structures similar to CD31+ cells from neonatal mouse heart. Co-culture of ES cell-derived CD31+ cells with ES cell-derived cardiomyocytes and dermal fibroblasts resulted in the formation of cardiac cell sheets with microvascular network formation. In contrast, microvascular network formation was reduced in co-cultures without cardiomyocytes, suggesting that cardiomyocytes within the cell sheet might enhance vascular endothelial cell sprouting. Polymerase chain reaction array analysis revealed that the expression levels of several angiogenesis-related genes, including fibroblast growth factor 1 (FGF1), were up-regulated in co-culture with cardiomyocytes compared with cultures without cardiomyocytes. The microvascular network in the cardiac sheets was attenuated by treatment with anti-FGF1 antibody. These results indicate that 3D suspension culture methods may be used to prepare functional vascular endothelial cells from mouse ES cells, and that cardiomyocyte-mediated paracrine effects might be important for fabricating pre-vascularized cardiac cell sheets.

Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

IntroductionCollagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils.MethodsSpecifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces.ResultsCollagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space.ConclusionThese findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0191-1) contains supplementary material, which is available to authorized users.

Differentiation of Bone Marrow: Derived Mesenchymal Stem Cells into Hepatocyte-like Cells.

Cirrhosis is the end-stage liver fibrosis, whereby normal liver architecture is disrupted by fibrotic bands, parenchymal nodules and vascular distortion. Portal hypertension and hepatocyte dysfunction are the end results and give rise to major systemic complications and premature death. Mesenchymal stem cells (MSC) have the capacity of self-renew and to give rise to cells of various lineages, so MSC can be isolated from bone marrow (BM) and induced to differentiate into hepatocyte-like cells. MSC were induced to differentiate into hepatocyte-like cells by hepatotic growth factor (HGF) and fibroblast growth factor-4 (FGF-4). Differentiated cells were examined for the expression of hepatocyte-specific markers and hepatocyte functions. MSC were isolated. Flow cytometry analysis showed that they expressed the MSC-specific markers, reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that MSC expressed the hepatocyte-specific marker cytokeratin 18 (CK-18) following hepatocyte induction. This study demonstrates that BM-derived-MSC can differentiate into functional hepatocyte-like cells following the induction of HGF and FGF-4. MSC can serve as a favorable cell source for tissue engineering in the treatment of liver disease.

Therapeutic possibility of human fetal cartilage-derived progenitor cells in rat arthritis model

Fetal cartilage-derived progenitor cells (FCPCs) are a novel cell source for tissue engineering and cell therapy. Previously, cells from various fetal tissues were shown to have immune-modulatory activity just like mesenchymal stem cells. In this study, we investigated the cytotoxicity and therapeutic possibility of FCPCs as a potential cell therapy for arthritis in vivo. To evaluate the acute toxicity and safety, FCPCs were labeled with PKH-26 and subjected to intra-articular injection in rats. When examined at 1 and 2 weeks, the PKH-26 labeled FCPCs were observed only in the synovial membrane but not in other organs. There were no changes either in the white blood cells, red blood cells, and platelets in the hematological analysis and in the weight of the internal organs at 1 week. Therapeutic effect of FCPCs was examined on the complete Freund’s adjuvant (CFA)-induced knee arthritis in rats in comparison with triamcinolone, a representative anti-arthritis drug. When inflammation-related edema of the arthritic knee joint was measured by the knee circumference, it increased significantly from the beginning and maintained until the end of measurement in the CFA group, which decreased back rapidly from 1 day in the triamcinolone group and slowly after 7 days in the FCPCs group. In the histological analysis, both triamcinolone and FCPCs decreased the infiltration of neutrophils and lymphocytes into the arthritic synovial membrane gradually at 3 and 7 days. These results suggest that FCPCs shows low toxicity or immune rejection when injected in the synovial cavity and have a therapeutic potential on the synovial arthritis in the rat model.

Human Urine Derived Stem Cells in Combination with β-TCP Can Be Applied for Bone Regeneration.

Bone tissue engineering requires highly proliferative stem cells that are easy to isolate. Human urine stem cells (USCs) are abundant and can be easily harvested without using an invasive procedure. In addition, in our previous studies, USCs have been proved to be able to differentiate into osteoblasts, chondrocytes, and adipocytes. Therefore, USCs may have great potential and advantages to be applied as a cell source for tissue engineering. However, there are no published studies that describe the interactions between USCs and biomaterials and applications of USCs for bone tissue engineering. Therefore, the objective of the present study was to evaluate the interactions between USCs with a typical bone tissue engineering scaffold, beta-Tricalcium Phosphate (β-TCP), and to determine whether the USCs seeded onto β-TCP scaffold can promote bone regeneration in a segmental femoral defect of rats. Primary USCs were isolated from urine and seeded on β-TCP scaffolds. Results showed that USCs remained viable and proliferated within β-TCP. The osteogenic differentiation of USCs within the scaffolds was demonstrated by increased alkaline phosphatase activity and calcium content. Furthermore, β-TCP with adherent USCs (USCs/β-TCP) were implanted in a 6-mm critical size femoral defect of rats for 12 weeks. Bone regeneration was determined using X-ray, micro-CT, and histologic analyses. Results further demonstrated that USCs in the scaffolds could enhance new bone formation, which spanned bone defects in 5 out of 11 rats while β-TCP scaffold alone induced modest bone formation. The current study indicated that the USCs can be used as a cell source for bone tissue engineering as they are compatible with bone tissue engineering scaffolds and can stimulate the regeneration of bone in a critical size bone defect.