Osteogenically differentiated mesenchymal stem cells promote the apoptosis of human umbilical vein endothelial cells in vitro.

The absence of blood vessels in tissue engineered bone often leads to necrosis of internal cells after implantation, ultimately affecting the process of bone repair. Herein, mesenchymal stem cells (MSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to induce osteogenesis and angiogenesis. Based on the findings, the number of HUVECs in the coculture system increased in the growth medium group, but decreased in the osteogenic induction medium (OIM) group. Considering that the paracrine effects of MSCs had changed, we tested the genes expression of osteogenically differentiated MSCs. The expression of osteogenic genes in MSCs increased during osteogenesis. Further, the expression levels of pigment epithelial-derived factor (PEDF) gene and protein, an antivascular factor, were also increased. To verify whether MSCs promote HUVECs apoptosis via PEDF, PEDF was silenced via siRNA. The conditioned medium of differentiated MSCs with PEDF silencing significantly improved the proliferation and apoptosis of HUVECs. Based on further experiments, PEDF mediated the apoptosis and proliferation of HUVECs through p53, BAX/BCL-2, FAS, and c-Caspase-3. However, when PEDF was silenced with siRNA, the osteogenic potential of MSCs was affected. The results of this study provide a theoretical basis for the construction of prevascularized bone tissues in vitro.

Human umbilical cord-derived mesenchymal stem cells affect urea synthesis and the cell apoptosis of human induced hepatocytes by secreting IL-6 in a serum-free co-culture system.

BACKGROUND: Bioartificial livers (BALs) are emerging as a potential supportive therapy for liver diseases. However, the maintenance of hepatocyte function and viability in vitro is a major challenge. Mesenchymal stem cells (MSCs) have attracted extensive attention for providing trophic support to hepatocytes, but only few studies have explored the interaction between human MSCs and human hepatocytes, and very little is known about the underlying molecular mechanisms whereby MSCs affect hepatocyte function, especially in serum-free medium (SFM). METHOD AND RESULTS: This study aims to explore the effects of human umbilical cord-derived MSCs (hUMSCs) on human-induced hepatocytes (hiHeps) function and viability, and know about the underlying molecular mechanism of interaction in SFM. The liver-specific function of hiHeps was evaluated by analysis of albumin secretion, urea synthesis, and metabolic enzyme activity. hiHeps apoptosis was mainly characterized by live/dead staining assay, JC-1 mitochondrial membrane potential assay, reactive oxygen species (ROS) generation, and cell apoptosis detection. The expression of related genes and proteins were measured by qRT-PCR and western blotting. The results indicate that co-culture with hUMSCs improved hiHep urea synthesis and reduced cell apoptosis compared to monoculture in SFM, and this effect was found to be mediated by secreted interleukin-6 (IL-6). Further, studies revealed that IL-6 reduced hiHep apoptosis via the activation of the JAK-Stat3-Ref-1 and JAK-Stat3-Bcl-2/Bax-Caspase3 pathways by binding to the IL-6 receptor. IL-6 also enhanced hiHep urea synthesis through the JAK-Akt-P53-ARG1 pathway. Finally, hiHep-specific functions were further prolonged and increased when co-cultured with hUMSCs on 3D polyethylene terephthalate (PET) fibrous scaffolds. CONCLUSION: The SFM co-culture strategy showed major advantages in maintaining hiHep function and viability in vitro, which is of great significance for the clinical application of hiHeps in BALs.

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1α.

BACKGROUND AND AIM: Inadequate vascularization is a challenge in bone tissue engineering because internal cells are prone to necrosis due to a lack of nutrient supply. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to construct prevascularized bone tissue in osteogenic induction medium (OIM) in vitro. The angiogenic capacity of HUVECs was limited in the coculture system. In this study, the effects of the components in the medium on HUVEC angiogenesis were analyzed. METHODS: The coculture system was established in OIM. Alizarin red staining and alkaline phosphatase staining were used to assess the osteogenic ability of MSCs. A Matrigel tube assay was used to assess the angiogenic ability of HUVECs in vitro. The proliferation of HUVECs was evaluated by cell counting and CCK-8 assays, and migration was evaluated by the streaked plate assay. The expression levels of angiogenesis-associated genes and proteins in HUVECs were measured by qRT-PCR and Western blotting, respectively. RESULTS: Dexamethasone in the OIM suppressed the proliferation and migration of HUVECs, inhibiting the formation of capillary-like structures. Our research showed that dexamethasone stimulated HUVECs to secrete tissue inhibitor of metalloproteinase (TIMP-3), which competed with vascular endothelial growth factor (VEGF-A) to bind to vascular endothelial growth factor receptor 2 (VEGFR2, KDR). This effect was related to inhibiting the phosphorylation of ERK and AKT, which are two downstream targets of KDR. However, under hypoxia, the enhanced expression of hypoxia-inducible factor-1α (HIF-1α) decreased the expression of TIMP-3 and promoted the phosphorylation of KDR, improving HUVEC angiogenesis in the coculture system. CONCLUSION: Coculture of hypoxia-preconditioned HUVECs and MSCs showed robust angiogenesis and osteogenesis in OIM, which has important implications for prevascularization in bone tissue engineering in the future.

Fabrication and evaluation of modified poly(ethylene terephthalate) microfibrous scaffolds for hepatocyte growth and functionality maintenance.

For hepatocyte culture in vitro, the surface feature of utilized scaffolds exerts a direct impact on cell adhesion, growth and differentiated functionality. Herein, to regulate hepatocyte growth and differentiated functionality, modified microfibrous scaffolds were fabricated by surface grafting monoamine terminated lactobionic lactone (L-NH2) and gelatin onto non-woven poly(ethylene terephthalate) (PET) fibrous substrate (PET-Gal and PET-Gel), respectively. The physicochemical properties of PET scaffolds before and after modification were characterized. Upon 15-day culture, the effects of modified PET scaffolds on growth and differentiated functionality of human induced hepatocytes (hiHeps) were evaluated, compared with that of control without modification. Results demonstrated that both L-NH2 and gelatin modifications improved scaffold properties including hydrophilicity, water uptake ratio, stiffness and roughness, resulting in efficient cell adhesion, ~20-fold cell expansion and enhanced differentiated functionality. After culture for 15 days, PET-Gal cultured cells formed aggregates, displaying better cell viability and significantly higher differentiated functionality regarding albumin secretion, urea synthesis, phases I (cytochrome P450, CYP1A1/2 and CYP3A4) and II (uridine 5'-diphosphate glucuronosyltransferases, UGT) enzyme activity, biliary excretion and detoxification ability (ammonia elimination and bilirubin conjugation), compared with PET and PET-Gel cultured ones. Hence, as a three-dimensional (3D) microfibrous scaffold, PET-Gal promotes hiHeps growth and differentiated functionality maintenance, which is promisingly utilized in bioartificial liver (BAL) bioreactors.

Expansion of Transdifferentiated Human Hepatocytes in a Serum-Free Microcarrier Culture System.

BACKGROUND AND AIMS: Bioartificial livers (BALs) have attracted much attention as potential supportive therapies for liver diseases. A serum-free microcarrier culture strategy for the in vitro high-density expansion of human-induced hepatocyte-like cells (hiHeps) suitable for BALs was studied in this article. METHODS: hiHeps were transdifferentiated from human fibroblasts by the lentiviral overexpression of FOXA3, HNF1A, and HNF4A. Cells were cultured on microcarriers, their proliferation was evaluated by cell count and CCK-8 assays, and their function was evaluated by detecting liver function parameters in the supernatant, including urea secretion, albumin synthesis, and lactate dehydrogenase levels. The expressions of hepatocyte function-associated genes of hiHeps were measured by qRT-PCR in 2D and 3D conditions. The expression of related proteins during fibronectin promotes cell adhesion, and proliferation on microcarrier was detected by western blotting. RESULTS: During microcarrier culture, the optimal culture conditions during the adherence period were the use of half-volume high-density inoculation, Cytodex 3 at a concentration of 3 mg/mL, a cell seeding density of 2.0 × 105 cells/mL, and a stirring speed of 45 rpm. The final cell density in self-developed, chemically defined serum-free medium (SFM) reached 2.53 × 106 cells/mL, and the maximum increase in expansion was 12.61-fold. In addition, we found that fibronectin (FN) can promote hiHep attachment and proliferation on Cytodex 3 microcarriers and that this pro-proliferative effect was mediated by the integrin-ß1/FAK/ERK/CyclinD1 signaling pathway. Finally, the growth and function of hiHeps on Cytodex 3 in SFM were close to those of hiHeps on Cytodex 3 in hepatocyte maintenance medium (HMM), and cells maintained their morphology and function after harvest on microcarriers. CONCLUSIONS: Serum-free microcarrier culture has important implications for the expansion of a sufficient number of hiHeps prior to the clinical application of BALs.

The role of insulin in transdifferentiated hepatocyte proliferation and function in serum-free medium.

Transdifferentiated hepatocytes are potential seeding cells for bioartificial liver (BAL) treatment, and it is important to obtain a sufficient number of functional hepatocytes in serum-free medium (SFM). Although insulin plays an essential role in promoting cell proliferation and metabolism, the functions of insulin in transdifferentiated cells remain poorly understood. Here, we found that 1.0 mg/L insulin significantly increased human-induced hepatocyte-like cells (hiHeps) proliferation and viability in SFM. The pro-proliferative effect of insulin on these cells occurred via augmented cyclin D1 expression that was mediated by activation of the Akt1/mTOR/p70S6K and Akt1/P53 pathways. Further studies revealed that insulin also enhanced the specific liver function of hiHeps in SFM. Additionally, Western blotting and siHNF1A transfection analysis showed that insulin increased the protein expression of Albumin (ALB) and UDP-glucuronosyltransferase1A1 （UGT1A1 ） in hiHeps via HNF1A. Finally, hiHep proliferation and the expression of specific genes were maintained during long-term passaging in SFM supplemented with 1.0 mg/L insulin. Collectively, our findings show that insulin promotes transdifferentiated hiHep proliferation and specific functional expression. These findings have important implications for the expansion of functional hiHeps prior to clinical applications of BALs.

A serum-free medium suitable for maintaining cell morphology and liver-specific function in induced human hepatocytes.

hiHep is a new type of hepatocyte-like cell that is predicted to be a potential unlimited source of hepatocytes for a bioartificial liver. However, hiHep cannot currently be used in clinical settings because serum must be added during the culture process. Thus, a defined medium is required. Because serum is complex, an efficient statistical approach based on the Plackett-Burman design was used. In this manner, an original medium and several significant cell growth factors were identified. These factors include insulin, VH, and VE, and the original medium was optimized based on these significant factors. Additionally, hiHep liver-specific functions and metabolism in the optimized serum-free medium were measured. Results showed that hiHep functions, such as glycogen storage, albumin secretion, and urea production, were well maintained in our optimized serum-free medium. In summary, we created a chemically defined, serum-free medium in which cell growth, proliferation, metabolism, and function were well maintained. This medium has the potential to support the clinical use of hiHep.

Fabrication and in vitro evaluation of a packed-bed bioreactor based on an optimum two-stage culture strategy.

A packed-bed (PB) bioreactor for bioartificial liver (BAL) was fabricated based on an optimum two-stage culture strategy and evaluated in vitro in this research. Human induced hepatocytes (hiHeps) were first expanded using Cytodex 3 microcarriers and the choice of microcarrier concentration and fetal bovine serum (FBS) content was optimized. Then, the cells expanded under the optimum expansion condition were perfused into a perfusion system containing Fibra-Cel (FC) disks to fabricate a PB bioreactor. Operating parameters including flow rate and seeding density for perfusion culture were optimized, respectively. Results indicated that during suspension culture, rapid cell proliferation and favorable amino acid metabolism were achieved at 3 mg/mL microcarriers combined with 1% FBS. While for the perfusion culture, the most effective flow rate and seeding density were 2 mL/min and 1 × 106 cells/mL, respectively. Under this optimum perfusion condition, hiHeps showed good proliferation ability, high viability, homogeneous distribution, high metabolism activities and efficient albumin secretion as well as high liver-specific genes expression. Therefore, the two-stage culture strategy based on operating parameters optimization provides a new method for the development of PB bioreactors.