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.

Parathyroid-on-a-chip simulating parathyroid hormone secretion in response to calcium concentration

“…The second reason is that under conditions of high cell density, PTH mRNA expression in parathyroid-like cells increases. 17 Maintaining a high cell density in a 3D cultured spheroid enhances intercellular communication, thereby inducing cell function. 43 Although both types of organoids secreted similar levels of PTH, it was important that parathyroid organoids secreted PTH within the range of normal serum PTH levels (10–65 pg ml −1 ).…”

“…TMSCs were isolated and cultured using the well-established protocol. 17–20 Tonsil tissues were chopped and digested in Dulbecco's Modified Eagle Medium with high glucose (DMEM-HG, WelGENE Inc., Daegu, Korea) containing 205 U mL −1 collagenase type I and 10 μg mL −1 DNase for 30 min at 37 °C. Digested tissues were filtered by a mesh for removing debris, and cells were collected by centrifugation at 1300 rpm for 3 min.…”

“…TMSCs were differentiated into parathyroid-like cells using the modified Bingham protocol, 22 as a well-established protocol. 17–20 TMSCs were incubated in DMEM-HG for over 90% confluency on a 2D culture dish, and then they were cultured with a differentiation medium containing 100 ng ml −1 activin A and 100 ng ml −1 soluble sonic hedgehog (Shh) for 7 days. The differentiation medium was changed every other day during the differentiation period.…”

Preclinical Efficacy of Peripheral Nerve Regeneration by Schwann Cell-like Cells Differentiated from Human Tonsil-Derived Mesenchymal Stem Cells in C22 Mice

“…Tonsil tissue is readily obtained from a tonsillectomy, the most frequently performed minimally invasive surgery for patients aged 5 to 19 years [15,16]. TMSCs have the potential to differentiate into mesodermal lineages (bone, cartilage, fat, muscle, and tendon), endoderm (hepatocytes, PTH/insulin-releasing cells), and ectodermal lineages (neuron-like cells and glial or Schwann cell-like cells) [15][16][17][18][19][20].…”

New Sources, Differentiation, and Therapeutic Uses of Mesenchymal Stem Cells 2.0

“…Tonsil-derived mesenchymal stem cells (T-MSCs) can be differentiated into endoderm lineages, especially parathyroid-hormone (PTH)-releasing cells [ 4 , 7 ]. Kim et al performed quality optimization by standardizing the differentiation rate for a better clinical application of human T-MSCs differentiated into PTH-releasing cells to overcome the donor-dependent variation of T-MSCs [ 8 ]. A standardized efficiency of differentiation into PTH-releasing cells was achieved by initiating differentiation at a high cell density [ 9 , 10 , 11 ].…”