Petaloid recombinant peptide enhances in vitro cartilage formation by synovial mesenchymal stem cells.

In vitro chondrogenesis of mesenchymal stem cells (MSCs) mimics in vivo chondrogenesis of MSCs. However, the size of the cartilage pellets that can be attained in vitro is limited by current methods; therefore, some modifications are required to obtain larger pellets. Petaloid pieces of recombinant peptide (petaloid RCP) have the advantage of creating spaces between cells in culture. The RCP used here is based on the alpha-1 sequence of human collagen type I and contains 12 Arg-Gly-Asp motifs. We examined the effect and mechanisms of adding petaloid RCP on the in vitro chondrogenesis of human synovial MSCs by culturing 125k cells with or without 0.125 mg petaloid RCP in chondrogenic medium for 21 days. The cartilage pellets were sequentially analyzed by weight, sulfated glycosaminoglycan content, DNA retention, and histology. Petaloid RCP significantly increased the weight of the cartilage pellets: The petaloid RCP group weighed 7.7 ± 1.2 mg (n = 108), whereas the control group weighed 5.3 ± 1.6 mg. Sulfated glycosaminoglycan and DNA contents were significantly higher in the petaloid RCP group than in the control group. Light and transmission electron microscopy images showed that the petaloid RCP formed the framework of the pellet at day 1, the framework was broken by production of cartilage matrix by the synovial MSCs at day 7, and the cartilage pellet grew larger, with diffuse petaloid RCP remaining, at day 21. Therefore, petaloid RCP formed a framework for the pellet, maintained a higher cell number, and promoted in vitro cartilage formation of synovial MSCs. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. J Orthop Res 37:1350-1357, 2019.

Specific markers and properties of synovial mesenchymal stem cells in the surface, stromal, and perivascular regions.

BACKGROUND: Synovial mesenchymal stem cells (MSCs) are an attractive cell source for cartilage and meniscus regeneration. Synovial tissue can be histologically classified into three regions; surface, stromal and perivascular region, but the localization of synovial MSCs has not been fully investigated. We identified markers specific for each region, and compared properties of MSCs derived from each region in the synovium. METHODS: The intensity of immunostaining with 19 antibodies was examined for surface, stromal, and perivascular regions of human synovium from six osteoarthritis patients. Specific markers were identified and synovial cells derived from each region were sorted. Proliferation, surface marker expression, chondrogenesis, calcification and adipogenesis potentials were compared in synovial MSCs derived from the three regions. RESULTS: We selected CD55+ CD271- for synovial cells in the surface region, CD55- CD271- in the stromal region, and CD55- CD271+ in the perivascular region. The ratio of the sorted cells to non-hematopoietic lineage cells was 5% in the surface region, 70% in the stromal region and 15% in the perivascular region. Synovial cells in the perivascular fraction had the greatest proliferation potential. After expansion, surface marker expression profiles and adipogenesis potentials were similar but chondrogenic and calcification potentials were higher in synovial MSCs derived from the perivascular region than in those derived from the surface and stromal regions. CONCLUSIONS: We identified specific markers to isolate synovial cells from the surface, stromal, and perivascular regions of the synovium. Synovial MSCs in the perivascular region had the highest proliferative and chondrogenic potentials among the three regions.

Complete human serum maintains viability and chondrogenic potential of human synovial stem cells: suitable conditions for transplantation.

BACKGROUND: In our clinical practice, we perform transplantations of autologous synovial mesenchymal stem cells (MSCs) for cartilage and meniscus regenerative medicine. One of the most important issues to ensuring clinical efficacy involves the transport of synovial MSCs from the processing facility to the clinic. Complete human serum (100% human serum) is an attractive candidate material in which to suspend synovial MSCs for their preservation during transport. The purpose of this study was to investigate whether complete human serum maintained MSC viability and chondrogenic potential and to examine the optimal temperature conditions for the preservation of human synovial MSCs. METHODS: Human synovium was harvested from the knees of 14 donors with osteoarthritis during total knee arthroplasty. Passage 2 synovial MSCs were suspended at 2 million cells/100 µL in Ringer's solution or complete human serum at 4, 13, and 37 °C for 48 h. These cells were analyzed for live cell rates, cell surface marker expression, metabolic activity, proliferation, and adipogenic, calcification, and chondrogenic differentiation potentials before and after preservation. RESULTS: After preservation, synovial MSCs maintained higher live cell rates in human serum than in Ringer's solution at 4 and 13 °C. Synovial MSCs preserved in human serum at 4 and 13 °C also maintained high ratios of propidium iodide- and annexin V- cells. MSC surface marker expression was not altered in cells preserved at 4 and 13 °C. The metabolic activities of cells preserved in human serum at 4 and 13 °C was maintained, while significantly reduced in other conditions. Replated MSCs retained their proliferation ability when preserved in human serum at 4 and 13 °C. Adipogenesis and calcification potential could be observed in cells preserved in each condition, whereas chondrogenic potential was retained only in cells preserved in human serum at 4 and 13 °C. CONCLUSION: The viability and chondrogenic potential of synovial MSCs were maintained when the cells were suspended in human serum at 4 and 13 °C.

Yields and chondrogenic potential of primary synovial mesenchymal stem cells are comparable between rheumatoid arthritis and osteoarthritis patients.

BACKGROUND: Mesenchymal stem cells derived from the synovial membrane (synovial MSCs) are a candidate cell source for regenerative medicine of cartilage and menisci due to their high chondrogenic ability. Regenerative medicine can be expected for RA patients with the inflammation well-controlled as well as OA patients and transplantation of synovial MSCs would also be a possible therapeutic treatment. Some properties of synovial MSCs vary dependent on the diseases patients have, and whether or not the pathological condition of RA affects the chondrogenesis of synovial MSCs remains controversial. The purpose of this study was to compare the properties of primary synovial MSCs between RA and OA patients. METHODS: Human synovial tissue was harvested during total knee arthroplasty from the knee joints of eight patients with RA and OA respectively. Synovial nucleated cells were cultured for 14 days. Total cell yields, surface markers, and differentiation potentials were analyzed for primary synovial MSCs. RESULTS: Nucleated cell number per 1 mg synovium was 8.4 ± 3.9 thousand in RA and 8.0 ± 0.9 thousand in OA. Total cell number after 14-day culture/1 mg synovium was 0.7 ± 0.4 million in RA and 0.5 ± 0.3 million in OA, showing no significant difference between in RA and OA. Cells after 14-day culture were mostly positive for CD44, CD73, CD90, CD105, negative for CD45 both in RA and OA. There was no significant difference for the cartilage pellet weight and sGAG content per pellet between in RA and OA. Both oil red O-positive colony rate and alizarin red-positive colony rate were similar in RA and OA. CONCLUSIONS: Yields, surface markers and chondrogenic potential of primary synovial MSCs in RA were comparable to those in OA. Synovium derived from RA patients can be the cell source of MSCs for cartilage and meniscus regeneration.

Generation of kidney tubular organoids from human pluripotent stem cells.

Recent advances in stem cell research have resulted in methods to generate kidney organoids from human pluripotent stem cells (hPSCs), which contain cells of multiple lineages including nephron epithelial cells. Methods to purify specific types of cells from differentiated hPSCs, however, have not been established well. For bioengineering, cell transplantation, and disease modeling, it would be useful to establish those methods to obtain pure populations of specific types of kidney cells. Here, we report a simple two-step differentiation protocol to generate kidney tubular organoids from hPSCs with direct purification of KSP (kidney specific protein)-positive cells using anti-KSP antibody. We first differentiated hPSCs into mesoderm cells using a glycogen synthase kinase-3ß inhibitor for 3 days, then cultured cells in renal epithelial growth medium to induce KSP+ cells. We purified KSP+ cells using flow cytometry with anti-KSP antibody, which exhibited characteristics of all segments of kidney tubular cells and cultured KSP+ cells in 3D Matrigel, which formed tubular organoids in vitro. The formation of tubular organoids by KSP+ cells induced the acquisition of functional kidney tubules. KSP+ cells also allowed for the generation of chimeric kidney cultures in which human cells self-assembled into 3D tubular structures in combination with mouse embryonic kidney cells.

miR-363 induces transdifferentiation of human kidney tubular cells to mesenchymal phenotype.

BACKGROUND: microRNAs (miRNAs) are non-coding small RNAs that regulate embryonic development, cell differentiation and pathological processes via interaction with mRNA. Epithelial-mesenchymal transition (EMT) is pathological process that involves in a variety of diseases such as cancer or fibrosis. METHODS: In this study, we identified miR-363 as a potent inducer of EMT by microarray analysis in human kidney tubular cells, and analyzed the function and mechanisms of miR-363. RESULTS: Overexpression of miR-363 induced mesenchymal phenotypes with loss of epithelial phenotypes in human kidney tubular cells. In addition, in vitro scratch assay demonstrated that miR-363 promotes cell migration of primary culture of human kidney tubular cells. We identified TWIST/canonical WNT pathway as the downstream effecter of miR-363, and inhibition of canonical WNT by small molecule, IWR-1, attenuated EMT induced by miR-363. CONCLUSION: miR-363 induces transdifferentiation of human kidney tubular cells via upregulation of TWIST/canonical WNT pathway.

Platelet-derived growth factor (PDGF)-AA/AB in human serum are potential indicators of the proliferative capacity of human synovial mesenchymal stem cells.

INTRODUCTION: For expansion of human mesenchymal stem cells (MSCs), autologous human serum is safer than fetal bovine serum in clinical situations. One of the problems with the use of autologous human serum is that its proliferative effect on MSCs varies widely between donors. The threefold goals of this study were: (1) to demonstrate an improved method for preparing human serum; (2) to identify growth factors predictive of proliferative potential; and (3) to identify a cytokine to promote MSC proliferation in human serum. METHODS: Fresh blood was collected using a closed bag system containing glass beads. The bag was shaken at 20 °C for 30 minutes for rapid preparation, or kept stationary at 4 °C for 24 hours for slow preparation. Passage 0 synovial MSCs derived from four donors were cultured with 10 % conventional rapid preparation serum or modified slow preparation serum from four different donors. To perform the colony-forming unit assay, synovial MSCs were cultured in these serums. The protein expression profile in serum was analyzed using cytokine array. The candidate proteins were speculated from the correlation between the colony-forming ability and protein expression. As an evaluation of the candidate proteins, proliferation ability, surface marker phenotype and differentiation capability of synovial MSCs were examined. RESULTS: Compared with rapid preparation serum, slow preparation serum resulted in a significantly higher total colony number and twofold higher expression levels of nine proteins (angiopoietin-1, BDNF, EGF, ENA-78, IGFBP-2, platelet-derived growth factor (PDGF)-AA, PDGF-AB/BB, RANTES and TfR). Colony number was positively correlated with PDGF-AA/AB concentrations. Exogenous PDGF-AA significantly promoted proliferation of synovial MSCs, whereas PDGF receptor (PDGFR) inhibitor decreased it. Addition of PDGFs or PDGFR inhibitor did not affect surface epitopes of synovial MSCs. Pretreatment with PDGFs or PDGFR inhibitor did not affect chondrogenic, adipogenic, or calcification potentials of synovial MSCs. CONCLUSION: Slow preparation serum contained higher concentrations of PDGF-AA/AB and increased the colony formation number of synovial MSCs. PDGF-AA/AB were indicators of the proliferative potential of human serum. Exogenous PDGF-AA increased proliferation of synovial MSCs without alteration of surface epitopes and differentiation potentials.

miR-34c attenuates epithelial-mesenchymal transition and kidney fibrosis with ureteral obstruction.

micro RNAs (miRNAs) are small non-coding RNAs that act as posttranscriptional repressors by binding to the 3'-UTR of target mRNAs. On the other hand, mesenchymal-epithelial transition (EMT) and kidney fibrosis is a pathological process of chronic kidney disease (CKD), and its relationship to miRNAs is becoming recognized as a potential target for CKD therapies. To find new miRNAs involved in EMT, we examined miRNA expression in experimental models of EMT and renal epithelialization using microarray, and found that miR-34c attenuates EMT induced by TGF-ß in a mouse tubular cell line. To confirm the effects of miR-34c in vivo, we administered the precursor of miR-34c to mice with unilateral ureteral obstruction, and miR-34c decreased kidney fibrosis area and the expression of connective tissue growth factor, α-SMA, collagen type 1, collagen type 3 and fibronectin. In conclusion, our study showed miR-34c attenuates EMT and kidney fibrosis of mice with ureteral obstruction.

Kidney specific protein-positive cells derived from embryonic stem cells reproduce tubular structures in vitro and differentiate into renal tubular cells.

Embryonic stem cells and induced pluripotent stem cells have the ability to differentiate into various organs and tissues, and are regarded as new tools for the elucidation of disease mechanisms as well as sources for regenerative therapies. However, a method of inducing organ-specific cells from pluripotent stem cells is urgently needed. Although many scientists have been developing methods to induce various organ-specific cells from pluripotent stem cells, renal lineage cells have yet to be induced in vitro because of the complexity of kidney structures and the diversity of kidney-component cells. Here, we describe a method of inducing renal tubular cells from mouse embryonic stem cells via the cell purification of kidney specific protein (KSP)-positive cells using an anti-KSP antibody. The global gene expression profiles of KSP-positive cells derived from ES cells exhibited characteristics similar to those of cells in the developing kidney, and KSP-positive cells had the capacity to form tubular structures resembling renal tubular cells when grown in a 3D culture in Matrigel. Moreover, our results indicated that KSP-positive cells acquired the characteristics of each segment of renal tubular cells through tubular formation when stimulated with Wnt4. This method is an important step toward kidney disease research using pluripotent stem cells, and the development of kidney regeneration therapies.

The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells.

BACKGROUND: Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs. METHODS AND RESULTS: Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase. CONCLUSION: We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.