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Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells.

ABSTRACT: Mesenchymal stem cells (MSCs) can differentiate into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells. We have previously reported that tonsil-derived MSC (T-MSC) can differentiate into PTH-releasing cells (T-MSC-PTHCs), which restored the parathyroid functions in parathyroidectomy (PTX) rats. In this study, we demonstrate quality optimization by standardizing the differentiation rate for a better clinical application of T-MSC-PTHCs to overcome donor-dependent variation of T-MSCs. Quantitation results of PTH mRNA copy number in the differentiated cells and the PTH concentration in the conditioned medium confirmed that the differentiation efficiency largely varied depending on the cells from each donor. In addition, the differentiation rate of the cells from all the donors greatly improved when differentiation was started at a high cell density (100% confluence). The large-scale expression profiling of T-MSC-PTHCs by RNA sequencing indicated that those genes involved in exiting the differentiation and the cell cycle were the major pathways for the differentiation of T-MSC-PTHCs. Furthermore, the implantation of the T-MSC-PTHCs, which were differentiated at a high cell density embedded in hyaluronic acid, resulted in a higher serum PTH in the PTX model. This standardized efficiency of differentiation into PTHC was achieved by initiating differentiation at a high cell density. Our findings provide a potential solution to overcome the limitations due to donor-dependent variation by establishing a standardized differentiation protocol for the clinical application of T-MSC therapy in treating hypoparathyroidism.

SUBMITTER: Kim JY

PROVIDER: S-EPMC8775366 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with parathyroid hormone 1 receptor (PTH1R) and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1(+) CD45(-) CD11b(-) MSCs, showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone led to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMP receptor type II (BMPRII) significantly in C2C12 cells, and PTH treatment significantly enhanced cell-surface binding of (125) I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted green fluorescent protein (GFP)-labeled Sca-1(+) CD45(-) CD11b(-) MSCs into the bone marrow cavity of Rag2(-/-) immunodeficient mice showed that PTH enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH enhancement of MSC differentiation to the osteoblast lineage occurs through a PTH- and LRP6-dependent pathway by endocytosis of the PTH1R/LRp6 complex, allowing enhancement of BMP signaling.

Project description:BackgroundStem cell therapies can be a new therapeutic strategy that may rebalance anabolic and anti-resorptive effects in osteoporosis patients. Tonsil-derived mesenchymal stem cells (TMSCs) can be an alternative therapeutic source for chronic degenerative diseases including osteoporosis. MSCs acquire immune regulatory function under the inflammatory cytokines. Since interleukin (IL) 1β is known to be one of inflammatory cytokines involved in osteoporosis progression, treatment of IL1β with TMSCs may enhance immunomodulatory function and therapeutic effects of TMSCs in osteoporosis.MethodsFor IL1β priming, TMSCs were cultured in the presence of the medium containing IL1β for 1 day. Characteristics of IL1β priming TMSCs such as multipotent differentiation properties, anti-inflammatory potential, and suppression of osteoclast differentiation were assessed in vitro. For in vivo efficacy study, IL1β priming TMSCs were intravenously infused twice with ovariectomized (OVX) osteoporosis mouse model, and blood serum and bone parameters from micro computed tomography images were analyzed.ResultsIL1β priming TMSCs had an enhanced osteogenic differentiation and secreted factors that regulate both osteoclastogenesis and osteoblastogenesis. IL1β priming TMSCs also suppressed proliferation of peripheral blood mononuclear cells (PBMCs) and decreased expression of Receptor activator of nuclear factor kappa-Β ligand (RANKL) in PHA-stimulated PBMCs. Furthermore, osteoclast specific genes such as Nuclear factor of activated T cells c1 (NFATc1) were effectively down regulated when co-cultured with IL1β priming TMSCs in RANKL induced osteoclasts. In OVX mice, IL1β priming TMSCs induced low level of serum RANKL/osteoprotegerin (OPG) ratio on the first day of the last administration. Four weeks after the last administration, bone mineral density and serum Gla-osteocalcin were increased in IL1β priming TMSC-treated OVX mice. Furthermore, bone formation and bone resorption markers that had been decreased in OVX mice with low calcium diet were recovered by infusion of IL1β priming TMSCs.ConclusionIL1β priming can endow constant therapeutic efficacy with TMSCs, which may contribute to improve bone density and maintain bone homeostasis in postmenopausal osteoporosis. Therefore, IL1β priming TMSCs can be a new therapeutic option for treating postmenopausal osteoporosis.

Project description:BackgroundCo-transplantation of bone marrow cells (BMCs) and mesenchymal stem cells (MSCs) is used as a strategy to improve the outcomes of bone marrow transplantation. Tonsil-derived MSCs (TMSCs) are a promising source of MSCs for co-transplantation. Previous studies have shown that TMSCs or conditioned media from TMSCs (TMSC-CM) enhance BMC engraftment. However, the factors in TMSCs that promote better engraftment have not yet been identified.MethodsMice were subjected to a myeloablative regimen of busulfan and cyclophosphamide, and the mRNA expression in the bone marrow was analyzed using an extracellular matrix (ECM) and adhesion molecule-targeted polymerase chain reaction (PCR) array. Nano-liquid chromatography with tandem mass spectrometry, real-time quantitative PCR, western blots, and enzyme-linked immunosorbent assays were used to compare the expression levels of metalloproteinase 3 (MMP3) in MSCs derived from various tissues, including the tonsils, bone marrow, adipose tissue, and umbilical cord. Recipient mice were conditioned with busulfan and cyclophosphamide, and BMCs, either as a sole population or with control or MMP3-knockdown TMSCs, were co-transplanted into these mice. The effects of TMSC-expressed MMP3 were investigated. Additionally, Enzchek collagenase and Transwell migration assays were used to confirm that the collagenase activity of TMSC-expressed MMP3 enhanced BMC migration.ResultsMice subjected to the myeloablative regimen exhibited increased mRNA expression of collagen type IV alpha 1/2 (Col4a1 and Col4a2). Among the various extracellular matrix-modulating proteins secreted by TMSCs, MMP3 was expressed at higher levels in TMSCs than in other MSCs. Mice co-transplanted with BMCs and control TMSCs exhibited a higher survival rate, weight recovery, and bone marrow cellularity compared with mice co-transplanted with BMCs and MMP3-knockdown TMSCs. Control TMSC-CM possessed higher collagenase activity against collagen IV than MMP3-knockdown TMSC-CM. TMSC-CM also accelerated BMC migration by degrading collagen IV in vitro.ConclusionsCollectively, these results indicate that TMSCs enhance BMC engraftment by the secretion of MMP3 for the modulation of the bone marrow extracellular matrix.

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Density-Dependent Differentiation of Tonsil-Derived Mesenchymal Stem Cells into Parathyroid-Hormone-Releasing Cells.

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