Betaine regulates adipogenic and osteogenic differentiation of hAD-MSCs.

BACKGROUND: With an ageing population, the incidence of bone loss and obesity are increasing. Numerous studies emphasized the multidirectional differentiation ability of mesenchymal stem cells (MSCs), and reported betaine modulated the osteogenic differentiation and adipogenic differentiation of MSCs in vitro. We wondered how betaine affected the differentiation of hAD-MSCs and hUC-MSCs. METHODS AND RESULTS: ALP staining and alizarin red S (ARS) staining were proved 10 mM betaine significantly increased the number of ALP-positive cells and plaque calcified extracellular matrices, accompanying by the up-regulation of OPN, Runx-2 and OCN. Oil red O staining demonstrated the number and size of lipid droplets were reduced, the expression of adipogenic master genes such as PPARγ, CEBPα and FASN were down-regulated simultaneously. For further investigating the mechanism of betaine on hAD-MSCs, RNA-seq was performed in none-differentiation medium. The Gene Ontology (GO) analysis showed fat cell differentiation and bone mineralization function terms were enriched, and KEGG showed PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction and ECM-receptor interaction pathways were enriched in betaine treated hAD-MSCs, demonstrated betaine had a positive inducing effect on osteogenic of hAD-MSCs in the non-differentiation medium in vitro, which is opposite to the effect on adipogenic differentiation. CONCLUSIONS: Our study demonstrated that betaine promoted osteogenic and compromised adipogenic differentiation of hUC-MSCs and hAD-MSCs upon low concentration administration. PI3K-Akt signaling pathway, cytokine-cytokine receptor interaction and ECM-receptor interaction were significantly enriched under betaine-treated. We showed hAD-MSCs were more sensitive to betaine stimulation and have a better differentiation ability than hUC-MSCs. Our results contributed to the exploration of betaine as an aiding agent for MSCs therapy.

Effects of Low-Intensity Pulsed Ultrasound on the Migration and Homing of Human Amnion-Derived Mesenchymal Stem Cells to Ovaries in Rats With Premature Ovarian Insufficiency.

Premature ovarian insufficiency (POI) can cause multiple sequelae and is currently incurable. Mesenchymal stem cell (MSC) transplantation might provide an effective treatment method for POI. However, the clinical application of systemic MSC transplantation is limited by the low efficiency of cell homing to target tissue in vivo, including systemic MSC transplantation for POI treatment. Thus, exploration of methods to promote MSC homing is necessary. This study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the migration and homing of transplanted human amnion-derived MSCs (hAD-MSCs) to ovaries in rats with chemotherapy-induced POI. For LIPUS treatment, hAD-MSCs were exposed to LIPUS or sham irradiation. Chemokine receptor expressions in hAD-MSCs were detected by polymerase chain reaction (PCR), Western blot, and immunofluorescence assays. hAD-MSC migration was detected by wound healing and transwell migration assays. Cyclophosphamide-induced POI rat models were established to evaluate the effects of LIPUS on the homing of systemically transplanted hAD-MSCs to chemotherapy-induced POI ovaries in vivo. We found that hAD-MSCs expressed chemokine receptors. The LIPUS promoted the expression of chemokine receptors, especially CXCR4, in hAD-MSCs. SDF-1 induced hAD-MSC migration. The LIPUS promoted hAD-MSC migration induced by SDF-1 through SDF-1/CXCR4 axis. SDF-1 levels significantly increased in ovaries induced by chemotherapy in POI rats. Pretreating hAD-MSCs with LIPUS increased the number of hAD-MSCs homing to ovaries in rats with chemotherapy-induced POI to some extent. However, the difference was not significant. Both hAD-MSC and LIPUS-pretreated hAD-MSC transplantation reduced ovarian injuries and improved ovarian function in rats with chemotherapy-induced POI. CXCR4 antagonist significantly reduced the number of hAD-MSCs- and LIPUS-pretreated hAD-MSCs homing to POI ovaries, and further reduced their efficacy in POI treatment. According to these findings, pretreating MSCs with LIPUS before transplantation might provide a novel, convenient, and safe technique to explore for improving the homing of systemically transplanted MSCs to target tissue.

Important role of the SDF-1/CXCR4 axis in the homing of systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) to ovaries in rats with chemotherapy-induced premature ovarian insufficiency (POI).

BACKGROUND: Chemotherapy can induce premature ovarian insufficiency (POI). POI causes multiple sequelae and is currently incurable. As shown in our previous studies, systemically transplanted human amnion-derived mesenchymal stem cells (hAD-MSCs) home to ovaries with chemotherapy-induced POI and subsequently reduce ovarian injury and improve ovarian function in rats with POI. However, the cellular mechanisms that direct the migration and homing of hAD-MSCs to ovaries with chemotherapy-induced POI are incompletely understood. This study investigated the role of the SDF-1/CXCR4 axis in the migration and homing of systemically transplanted hAD-MSCs to ovaries with chemotherapy-induced POI and its relevant downstream signalling pathways. METHODS: CXCR4 expression in hAD-MSCs was assessed using Western blotting and immunofluorescence staining. hAD-MSC migration was tested using Transwell migration assays. SDF-1 levels were detected using ELISA. Seventy-two female SD rats were randomly divided into the control, POI, hAD-MSCs and hAD-MSCs + AMD3100 groups. Cyclophosphamide was used to establish rat POI models. For inhibitor treatment, hAD-MSCs were pretreated with AMD3100 before transplantation. PKH26-labeled hAD-MSCs were injected into the tail vein of POI rats 24 h after chemotherapy. After hAD-MSC transplantation, the homing of hAD-MSCs to ovaries and ovarian function and pathological changes were examined. We further investigated the molecular mechanisms by detecting the PI3K/Akt and ERK1/2 signalling pathways. RESULTS: hAD-MSCs expressed CXCR4. SDF-1 induced hAD-MSC migration in vitro. SDF-1 levels in ovaries and serum were significantly increased in rats with chemotherapy-induced POI, and ovaries with POI induced the homing of hAD-MSCs expressing CXCR4. Blocking the SDF-1/CXCR4 axis with AMD3100 significantly reduced the number of hAD-MSCs homing to ovaries with POI and further reduced their efficacy in POI treatment. The binding of SDF-1 to CXCR4 activated the PI3K/Akt signalling pathway, and LY294002 significantly inhibited hAD-MSC migration induced by SDF-1 in vitro. Moreover, inhibition of the PI3K/Akt signalling pathway significantly reduced the number of systemically transplanted hAD-MSCs homing to chemotherapy-induced ovaries in rats with POI. CONCLUSIONS: SDF-1/CXCR4 axis partially mediates the migration and homing of systemically transplanted hAD-MSCs to the ovaries of rats with chemotherapy-induced POI, and the PI3K/Akt signalling pathway might be involved in the migration and homing of hAD-MSCs mediated by the SDF-1/CXCR4 axis.

TIPE2-modified human amnion-derived mesenchymal stem cells promote the efficacy of allogeneic heart transplantation through inducing immune tolerance.

BACKGROUND: Immune rejection of heart transplantation has been regarded as the biggest challenge encountered by a patient suffering from end-stage heart disease. The transplantation of human amnion-derived mesenchymal stem cells (hAD-MSCs) has exhibited promising application prospects in organ transplantation. However, its persistent unsatisfactory tolerance has limited the widespread application of this technology. We aim to investigate the role of tumor necrosis factor-α-induced protein-8 like-2 (TIPE2)-mediated hAD-MSCs in immune tolerance in heart transplantation and its molecular regulatory mechanisms. METHODS: This project detected the effect of TIPE2 on immune tolerance by constructing an allogeneic heart transplantation mouse model through which TIPE2-overexpressed hAD-MSCs were injected into recipients. The fluorescence distribution of TIPE2-hAD-MSCs in mice was observed by a small animal in vivo imaging system. Pathological changes of the transplanted heart were detected by hematoxylin and eosin (HE) staining. Flow cytometry was performed to detect the content of cardiac lymphocytes. The expression of immune-induced related factors was measured by quantitative real-time PCR (qRT-PCR) and western blot assays. RESULTS: TIPE2-hAD-MSCs protected myocardial tissue structures, reduced the spleen and thymus indexes in recipient mice, minimized the content of cardiac lymphocytes, reduced expressions of ERK, p38, and IFN-γ, and elevated expressions of both IL-10 and TGF-ß, markedly improving the survival time and survival rates of recipient mice. CONCLUSIONS: TIPE2-hAD-MSCs induce immune tolerance and improve the survival rates of allogeneic heart transplantation in mice. This study is expected to offer an ideal source and target of cells for organ transplantation.

Effects of Human Amnion-Derived Mesenchymal Stem Cell (hAD-MSC) Transplantation In Situ on Primary Ovarian Insufficiency in SD Rats.

Human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced primary ovarian insufficiency (POI). However, ensuring that stem cells home to the ovary to improve their effects on ovarian injury is challenging. This research aimed to directly inject ovarian tissue with hAD-MSCs and improve the homing of stem cells to the ovary. The animals were divided into POI, hAD-MSC (tail vein) treatment, hAD-MSC (in situ) treatment, and control groups. POI rat models were established by intraperitoneal injection of cyclophosphamide (CTX) and busulfan (BUS). The hAD-MSCs isolated from the amnion were injected into the tail vein or ovary of POI rats. The estrous cycle, serum sex hormone levels, follicle counts, ovarian pathological changes, and proteome of the ovaries were evaluated. hAD-MSCs were successfully isolated and cultured from the amnion. Both hAD-MSC (tail vein) and hAD-MSC (in situ) transplantation increased body weight, improved the AMH levels and follicle numbers, and reduced reproductive organ injuries in POI rats. Transplantation of hAD-MSCs (in situ) upregulated 24 proteins and downregulated 4 proteins. Both hAD-MSC (tail vein) and hAD-MSC (in situ) transplantations can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced POI. The paracrine proteome of hAD-MSCs in the ovarian microenvironment can protect against chemotherapy-induced damage by reducing apoptosis and promoting angiogenesis, cell proliferation, and gene expression.

Mineral-Doped Poly(L-lactide) Acid Scaffolds Enriched with Exosomes Improve Osteogenic Commitment of Human Adipose-Derived Mesenchymal Stem Cells.

Exosomes derived from mesenchymal stem cells are extracellular vesicles released to facilitate cell communication and function. Recently, polylactic acid (PLA), calcium silicates (CaSi), and dicalcium phosphate dihydrate (DCPD) have been used to produce bioresorbable functional mineral-doped porous scaffolds-through thermally induced phase separation technique, as materials for bone regeneration. The aim of this study was to investigate the effect of mineral-doped PLA-based porous scaffolds enriched with exosome vesicles (EVs) on osteogenic commitment of human adipose mesenchymal stem cells (hAD-MSCs). Two different mineral-doped scaffolds were produced: PLA-10CaSi-10DCPD and PLA-5CaSi-5DCPD. Scaffolds surface micromorphology was investigated by ESEM-EDX before and after 28 days immersion in simulated body fluid (HBSS). Exosomes were deposited on the surface of the scaffolds and the effect of exosome-enriched scaffolds on osteogenic commitment of hAD-MSCs cultured in proximity of the scaffolds has been evaluated by real time PCR. In addition, the biocompatibility was evaluated by direct-contact seeding hAD-MSCs on scaffolds surface-using MTT viability test. In both formulations, ESEM showed pores similar in shape (circular and elliptic) and size (from 10-30 µm diameter). The porosity of the scaffolds decreased after 28 days immersion in simulated body fluid. Mineral-doped scaffolds showed a dynamic surface and created a suitable bone-forming microenvironment. The presence of the mineral fillers increased the osteogenic commitment of hAD-MSCs. Exosomes were easily entrapped on the surface of the scaffolds and their presence improved gene expression of major markers of osteogenesis such as collagen type I, osteopontin, osteonectin, osteocalcin. The experimental scaffolds enriched with exosomes, in particular PLA-10CaSi-10DCPD, increased the osteogenic commitment of MSCs. In conclusion, the enrichment of bioresorbable functional scaffolds with exosomes is confirmed as a potential strategy to improve bone regeneration procedures.

Xeno-Free In Vitro Cultivation and Osteogenic Differentiation of hAD-MSCs on Resorbable 3D Printed RESOMER®.

The development of alloplastic resorbable materials can revolutionize the field of implantation technology in regenerative medicine. Additional opportunities to colonize the three-dimensionally (3D) printed constructs with the patient's own cells prior to implantation can improve the regeneration process but requires optimization of cultivation protocols. Human platelet lysate (hPL) has already proven to be a suitable replacement for fetal calf serum (FCS) in 2D and 3D cell cultures. In this study, we investigated the in vitro biocompatibility of the printed RESOMER® Filament LG D1.75 materials as well as the osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultivated on 3D printed constructs under the influence of different medium supplements (FCS, human serum (HS) and hPL). Additionally, the in vitro degradation of the material was studied over six months. We demonstrated that LG D1.75 is biocompatible and has no in vitro cytotoxic effects on hMSCs. Furthermore, hMSCs grown on the constructs could be differentiated into osteoblasts, especially supported by supplementation with hPL. Over six months under physiological in vitro conditions, a distinct degradation was observed, which, however, had no influence on the biocompatibility of the material. Thus, the overall suitability of the material LG D1.75 to produce 3D printed, resorbable bone implants and the promising use of hPL in the xeno-free cultivation of human MSCs on such implants for autologous transplantation have been demonstrated.

Human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation improves ovarian function in rats with premature ovarian insufficiency (POI) at least partly through a paracrine mechanism.

BACKGROUND: Chemotherapy can induce premature ovarian insufficiency (POI) and reduce fertility in young female patients. Currently, there is no effective therapy for POI. Human amnion-derived mesenchymal stem cells (hAD-MSCs) may be a promising seed cell for regenerative medicine. This study investigated the effects and mechanisms of hAD-MSC transplantation on chemotherapy-induced POI in rats. METHODS: Chemotherapy-induced POI rat models were established by intraperitoneal injection of cyclophosphamide. Seventy-two female SD rats were randomly divided into control, POI, and hAD-MSC-treated groups. hAD-MSCs were labeled with PKH26 and injected into the tail veins of POI rats. To examine the underlying mechanisms, the differentiation of transplanted hAD-MSCs in the POI ovaries was analyzed by immunofluorescent staining. The in vitro expression of growth factors secreted by hAD-MSCs in hAD-MSC-conditioned media (hAD-MSC-CM) was analyzed by ELISA. Sixty female SD rats were divided into control, POI, and hAD-MSC-CM-treated groups, and hAD-MSC-CM was injected into the bilateral ovaries of POI rats. After hAD-MSC transplantation or hAD-MSC-CM injection, serum sex hormone levels, estrous cycles, ovarian pathological changes, follicle counts, granulosa cell (GC) apoptosis, and Bcl-2, Bax, and VEGF expression in ovaries were examined. RESULTS: PKH26-labeled hAD-MSCs mainly homed to ovaries after transplantation. hAD-MSC transplantation reduced ovarian injury and improved ovarian function in rats with POI. Transplanted hAD-MSCs were only located in the interstitium of ovaries, rather than in follicles, and did not express the typical markers of oocytes and GCs, which are ZP3 and FSHR, respectively. hAD-MSCs secreted FGF2, IGF-1, HGF, and VEGF, and those growth factors were detected in the hAD-MSC-CM. hAD-MSC-CM injection improved the local microenvironment of POI ovaries, leading to a decrease in Bax expression and an increase in Bcl-2 and endogenous VEGF expression in ovarian cells, which inhibited chemotherapy-induced GC apoptosis, promoted angiogenesis and regulated follicular development, thus partly reducing ovarian injury and improving ovarian function in rats with POI. CONCLUSIONS: hAD-MSC transplantation can improve ovarian function in rats with chemotherapy-induced POI at least partly through a paracrine mechanism. The presence of a paracrine mechanism accounting for hAD-MSC-mediated recovery of ovarian function might be attributed to the growth factors secreted by hAD-MSCs.

Effects of low-intensity pulsed ultrasound (LIPUS)-pretreated human amnion-derived mesenchymal stem cell (hAD-MSC) transplantation on primary ovarian insufficiency in rats.

BACKGROUND: Human amnion-derived mesenchymal stem cells (hAD-MSCs) have the features of mesenchymal stem cells (MSCs). Low-intensity pulsed ultrasound (LIPUS) can promote the expression of various growth factors and anti-inflammatory molecules that are necessary to keep the follicle growing and to reduce granulosa cell (GC) apoptosis in the ovary. This study aims to explore the effects of LIPUS-pretreated hAD-MSC transplantation on chemotherapy-induced primary ovarian insufficiency (POI) in rats. METHODS: The animals were divided into control, POI, hAD-MSC treatment, and LIPUS-pretreated hAD-MSC treatment groups. POI rat models were established by intraperitoneal injection of cyclophosphamide (CTX). The hAD-MSCs isolated from the amnion were exposed to LIPUS or sham irradiation for 5 consecutive days and injected into the tail vein of POI rats. Expression and secretion of growth factors promoted by LIPUS in hAD-MSCs were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) in vitro. Estrous cycle, serum sex hormone levels, follicle counts, ovarian pathological changes, GC apoptosis, Bcl2 and Bax expression, and pro-inflammatory cytokine levels in ovaries were examined. RESULTS: Primary hAD-MSCs were successfully isolated from the amnion. LIPUS promoted the expression and secretion of growth factors in hAD-MSCs in vitro. Both hAD-MSC and LIPUS-pretreated hAD-MSC transplantation increased the body and reproductive organ weights, improved ovarian function, and reduced reproductive organ injuries in POI rats. Transplantation of hAD-MSCs increased the Bcl-2/Bax ratio and reduced GC apoptosis and ovarian inflammation induced by chemotherapy in ovaries. These effects could be improved by pretreatment with LIPUS on hAD-MSCs. CONCLUSION: Both hAD-MSC transplantation and LIPUS-pretreated hAD-MSC transplantation can repair ovarian injury and improve ovarian function in rats with chemotherapy-induced POI. LIPUS-pretreated hAD-MSC transplantation is more advantageous for reducing inflammation, improving the local microenvironment, and inhibiting GC apoptosis induced by chemotherapy in ovarian tissue of POI rats.

The Extracellular Matrix Affected Proliferation and Cell Adhesion of Human Adipose Tissue Derived Mesenchymal Stem Cells in vitro

PURPOSE: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. METHODS: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. RESULTS: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin beta1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. CONCLUSION: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.

The Extracellular Matrix Affected Proliferation and Cell Adhesion of Human Adipose Tissue Derived Mesenchymal Stem Cells in vitro

PURPOSE: Human mesenchymal stem cells (hMSCs) have the potency for self-renewal and differentiation into various kinds of cells. The hMSCs are obtained from the various tissues, including adipose tissue, bone marrow and cord blood. The extracellular matrix (ECM) is an important factor that affects cell adherence, growth, migration, apoptosis and differentiation both in vitro and vivo. The adipose-derived mesenchymal stem cells (AD-MSCs) have CD29 (integrin) on the cell surface, which is the receptor for fibronectin. The aim of this study is to validate the efficacy of ECM, and especially fibronectin, for cell expansion. METHODS: The AD-MSCs were obtained from the abdominal fat of humans. These cells were seeded onto culture plates coated with fibronectin-Human (FN) and plates without ECM (control). The cells were incubated for 3 passages and the cellular morphology was simultaneously observed with microscopy. CCK-8 assay was performed to compare the proliferation ability in each condition at the same passage. Immunocytochemistry staining for integrin-beta1 was performed to observe the cell to cell interaction. RESULTS: The hAD-MSCs in the FN-coated and non-coated plates exhibited cytoplasm staining for integrin-beta1. In all the cultures, extended fibroblastic-shaped cells that turned into rhomboid cells were most frequently observed. The cell growth rates for the non coated culture plate were lower than those for the FN coated plates. After 72 hour culture under the different coated concentrations of FN and the non coated condition (control), the control group had a lower growth rate. In the culture with a FN coated plate, a significant change was observed as compared with that of the control group. We observed an increase in cell proliferation, with a maximum of 140%, on the FN coated plate by performing CCK-8 assay. In comparison, integrin beta1 on the cells was more expressed in the FN-coated plates than that in the non-coated plates. CONCLUSION: The cell morphology can be changed faster in the FN coated culture plates than that in the non coated culture plates. Because proliferation and adhesion with FN can enhance the expansion, the culture within a FN coated plate is needed to encourage hAD-MSCs to proliferate in vitro.