This preliminary study investigated the efficacy and safety of bone marrow-derived mesenchymal stem cell transplantation in a piglet Legg-Calve-Perthes disease (LCPD) model. The LCPD model was induced in two Landrace piglets (6- and 7-week-old, weighing 12 and 17 kg, respectively) by ligaturing the femoral neck. In the first piglet, the natural LCPD course was observed. In the second piglet, 4 weeks after ligaturing the femoral neck, simple medium and medium containing 2.44 × 10 7 bone marrow-derived mesenchymal stem cells were transplanted into the right and left femoral heads after core decompression, respectively. Plain radiographs were obtained every 4 weeks, and the epiphyseal quotient was calculated by dividing the maximum epiphysis height by the maximum epiphysis diameter. The piglets were sacrificed at 14 weeks postoperatively. The femoral heads were extracted and evaluated grossly, pathologically, and by using computed tomography. The transplanted cell characteristics were evaluated using flow cytometry. Flattening of the epiphysis was observed in both femoral heads of the first piglet and only in the right hip of the second piglet. The epiphyseal quotients immediately and at 14 weeks postoperatively in the right femoral head of the second piglet were 0.40 and 0.14, respectively, while those of the left femoral head were 0.30 and 0.42, respectively. Hematoxylin and eosin staining did not reveal physeal bar or tumor cell formation. The transplanted cells were 99.2%, 65.9%, 18.2%, and 0.16% positive for CD44, CD105, CD29, and CD31, respectively. Core decompression combined with bone marrow-derived mesenchymal stem cell transplantation prevented epiphyseal collapse.

Lymphedema is an intractable disease with few effective therapeutic options. Autologous mesenchymal stem cell (MSC) transplantation is a promising therapy for this disease. However, its use is limited by the cost and time for preparation. Recently, xenotransplantation of porcine MSCs has emerged as an alternative to autologous MSC transplantation. In this study, we aimed to clarify the usefulness of neonatal porcine bone marrow-derived MSC (NpBM-MSC) xenotransplantation for the treatment of lymphedema. One million NpBM-MSCs were xenotransplanted into the hind limbs of mice with severe lymphedema (MSC transplantation group). The therapeutic effects were assessed by measuring the femoral circumference, the volume of the hind limb, the number and diameter of lymphatic vessels in the hind limb, and lymphatic flow using a near-infrared fluorescence (NIRF) imaging system. We compared the effects using mice with lymphedema that did not undergo NpBM-MSC transplantation (negative control group). The condition of the transplanted NpBM-MSCs was also evaluated histologically. The femoral circumference and volume of the hind limb had been normalized by postoperative day (POD) 14 in the MSC transplantation group, but not in the negative control group (P = 0.041). NIRF imaging revealed that lymphatic flow had recovered in the MSC transplantation group by POD 14, as shown by an increase in luminance in the hind limb. Histological assessment also showed that the xenotransplantation of NpBM-MSC increased the proliferation of lymphatic vessels, but they had been rejected by POD 14. The xenotransplantation of NpBM-MSCs is an effective treatment for lymphedema, and this is mediated through the promotion of lymphangiogenesis.

Porcine tissues display a great potential as donor tissues in xenotransplantation, including cell therapy. Cryopreserving clinical grade porcine tissue and using it as a source for establishing therapeutic cells should be advantageous for transportation and scheduled manufacturing of MSCs. Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. It has been reported that co-transplantation of islets and Mesenchymal stem cells (MSCs) enhanced efficacy. We assume that co-transplantation of porcine islets and porcine islet-derived MSCs could improve the efficacy of clinical islet xenotransplantation.

MSCs were established from fresh and cryopreserved non-clinical grade neonatal porcine islets and bone marrow (termed non-clinical grade npISLET-MSCs and npBM-MSCs, respectively), as well as from cryopreserved clinical grade neonatal porcine islets (termed clinical grade npISLET-MSCs). Subsequently, the cell proliferation rate and diameter, surface marker expression, adipogenesis, osteogenesis, and colony-forming efficiency of the MSCs were assessed.

Cell proliferation rate and diameter did not differ between clinical grade and non-clinical grade npISLET-MSCs. However, non-clinical grade npBM-MSCs were significantly shorter and smaller than both npISLET-MSCs (p < 0.05). MSC markers (CD29, CD44, and CD90) were strongly expressed in clinical grade npISLET-MSCs and non-clinical grade npISLET-MSCs and npBM-MSCs. The expression of MSC-negative markers CD31, CD34, and SLA-DR was low in all MSCs. Clinical grade npISLET-MSCs derived from adipose and osteoid tissues were positive for Oil Red and alkaline phosphatase staining. The results of colony-forming assay were not significantly different between clinical grade npISLET-MSCs and non-clinical grade npBM-MSCs.

The method described herein was successful in of developing clinical grade npISLET-MSCs from cryopreserved islets. Cryopreserved clinical grade porcine islets could be an excellent stable source of MSCs for cell therapy.

Mesenchymal stem cells (MSCs) can be used as a cell source for cultivated meat production due to their adipose differentiation potential, but MSCs lose their stemness and undergo replicative senescence during expansion in vitro. Autophagy is an important mechanism for senescent cells to remove toxic substances. However, the role of autophagy in the replicative senescence of MSCs is controversial. Here, we evaluated the changes in autophagy in porcine MSCs (pMSCs) during long-term culture in vitro and identified a natural phytochemical, ginsenoside Rg2, that could stimulate pMSC proliferation. First, some typical senescence characteristics were observed in aged pMSCs, including decreased EdU-positive cells, increased senescence-associated beta-galactosidase activity, declined stemness-associated marker OCT4 expression, and enhanced P53 expression. Importantly, autophagic flux was impaired in aged pMSCs, suggesting deficient substrate clearance in aged pMSCs. Rg2 was found to promote the proliferation of pMSCs using MTT assay and EdU staining. In addition, Rg2 inhibited D-galactose-induced senescence and oxidative stress in pMSCs. Rg2 increased autophagic activity via the AMPK signaling pathway. Furthermore, long-term culture with Rg2 promoted the proliferation, inhibited the replicative senescence, and maintained the stemness of pMSCs. These results provide a potential strategy for porcine MSC expansion in vitro.

The goal of this study was to evaluate how glucose and fructose affected the adipose differentiation of pig newborn mesenchymal stem cells (MSCs). Cells were grown with or without inosine in 7.5 mM glucose (substituted with 1.5 or 6 mM fructose). MSCs displayed adipose morphology after 70 days of differentiation. Fructose stimulated the highest levels of PPARγ and C/EBPβ. Fructose at 6 mM, but not glucose at 7.5 mM or fructose at 1.5 mM, promotes differentiation of MSCs into adipocytes and increases 11-hydroxysteroid dehydrogenase (11β-HSD1) and NADPH oxidase 4 (NOX4) mRNA in the absence of hepatic effects (as simulated by the inosine). Fructose and glucose increased xanthine oxide-reductase (XOR) catalytic activity almost 10-fold and elevated their products: intracellular reactive oxygen species (ROS) pool, extracellular H₂ O₂ pool by 4 orders of magnitude, and uric acid by a factor of 10. Therefore, in our experimental model, differentiation of MSCs into adipocytes occurs exclusively at the blood concentration of fructose detected after ingestion by people on a high fructose diet. PRACTICAL APPLICATIONS: The results of this study provide new evidence for fructose's adipogenic potential in mesenchymal stem cells, a model in which its effects on XOR activity had not been studied. The increased expression of genes such as C/EBPβ, PPARγ, and NOX4, as well as the increased XOR activity and high production of ROS during the differentiation process in the presence of fructose, coincides in pointing to this hexose as an important factor in the development of adipogenesis in young animals, which could have a great impact on the development of future obesity.

Glucocorticoid-induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis. Although many studies related to GIOP have been published, there was no bibliometric analysis in this field. This study aimed to investigate the research trends on GIOP by using bibliometric analysis.

All data were collected from the Web of Science Core Collection (WoSCC). All original research articles regarding GIOP from 2012 to 2021 were retrieved. CiteSpace was used to analyze the distribution of countries, institutions, journals, authors, and keywords. We revealed hotspots and trends in the field by drawing co-occurrence keyword maps and identifying burst keywords.

From 2012 to 2021, 685 relevant articles were published, with a peak in 2018 in the annual number of publications. China and McMaster University were the leading country and institution in this field with 208 and 12 publications, respectively. Osteoporosis International was the journal with the most studies, while Journal of Bone and Mineral Research was the most cited journal. "Bone mineral density", "fracture", "postmenopausal women", "prevention" and "therapy" were the most high-frequency keywords, while "bone mineral density", "bisphosphonate" and "metabolism" were the top high-centrality keywords.

The results from this bibliometric study provided insight into the status and research trends in GIOP of the past decade, which could help researchers quickly determine the current hotspots and frontier trends in this field.

We demonstrated that neonatal porcine bone marrow-derived mesenchymal stem cell (npBM-MSCs) could improve a critical ischemic limb disease in rat model more efficiently compared with human MSCs. However, since porcine MSC presents galactosyl-alpha 1,3-galactose antigen (Gal antigen), MSC could be eliminated by the xenogeneic rejection. Recently, we established Gal knockout (KO) pigs by a technique of the electroporation of the CRISPR/Cas9 system into vitro-fertilized zygotes. In this study, we hypothesized that MSC from the established Gal KO pigs could further improve the efficacy. Before examining the hypothesis, in this study, we have established and characterized bone marrow-derived MSC from the Gal KO adult pigs (apBM-MSCs).

Mononuclear cells (MNCs) were isolated from bone marrow cells of both Gal KO adult pigs and wild-type (WT) adult pigs. MNCs were further manipulated to create Gal KO apBM-MSCs and WT apBM-MSCs. Both MSCs were assessed by their surface markers, the capability of differentiation into adipocytes, osteocytes and chondrocytes, grow speed and colony-forming assay. To assess the efficacy of Gal KO apBM-MSCs, angiogenesis-related genes and immunosuppression-related genes were assessed by cytokine stimulation.

Gal KO apBM-MSC showed no Gal antigen on their cell surfaces. Both Gal KO apBM-MSCs and WT apBM-MSCs, presented little or no negative surface markers of MSCs, while they presented positive surface markers of MSCs. Furthermore, Gal KO apBM-MSCs were able to differentiate into adipocytes, osteocytes, and chondrocytes as well as WT apBM-MSCs. There was no difference in doubling time between Gal KO apBM-MSCs and WT apBM-MSCs. Interestingly, the colony-forming efficiency of Gal KO apBM-MSCs was about half that of WT apBM-MSC. However, angiogenesis and immunosuppression-related genes were equally upregulated in both Gal KO apBM-MSCs and WT apBM-MSCs by cytokine stimulation.

We created and characterized Gal KO apBM-MSCs which showed similar characteristics and cytokine-induced gene upregulation to the WT apBM-MSCs.

Organ transplantation is the ultimate treatment for end-stage diseases such as heart and liver failure. However, the severe shortage of donor organs has limited the organ transplantation progress. Xenogeneic stem cell transplantation provides a new strategy to solve this problem. Researchers have shown that xenogeneic stem cell transplantation has significant therapeutic effects and broad application prospects in treating liver failure, myocardial infarction, advanced type 1 diabetes mellitus, myelosuppression, and other end-stage diseases by replacing the dysfunctional cells directly or improving the endogenous regenerative milieu. In this review, the sources, problems and solutions, and potential clinical applications of xenogeneic stem cell transplantation will be discussed.

The clinical utility of stem cell therapy for peripheral artery disease has not been fully discussed, and one obstacle is limited donor supplies. In this study, we attempted to rescue mouse ischemic hind limb by xenotransplantation of neonatal porcine bone marrow-derived mesenchymal stem cells (npBM-MSCs).

Neonatal porcine bone marrow-derived mesenchymal stem cells were transplanted to ischemic hind limbs of male C57BL/6J mice (npBM-MSCs group). Mice with syngeneic transplantation of mouse BM-MSCs (mBM-MSCs group) were also prepared for comparison. The angiogenic effects were evaluated by recovery of blood flow on laser Doppler imaging, histologic findings, and genetic and protein levels of angiogenic factors.

Regarding laser Doppler assessments, blood flow in the hind limb was rapidly recovered in the npBM-MSCs group, compared with that in the mBM-MSCs group (P = .016). Compared with the mBM-MSCs group, the npBM-MSCs group had early and prominent lymphangiogenesis [P < .05 on both post-operative days (PODs) 3 and 7] but had similar angiogenesis. Regarding genomic assessments, xenotransplantation of npBM-MSCs enhanced the expressions of both porcine and murine Vegfc in the hind limbs by POD 3. Interestingly, the level of murine Vegfc expression was significantly higher in the npBM-MSCs group than in the mBM-MSCs group on PODs 3 and 7 (P < .001 for both). Furthermore, the secreted VEGFC protein level was higher from npBM-MSCs than from mBM-MSCs (P < .001).

Xenotransplantation of npBM-MSCs contributed to the improvement of hind limb ischemia by both angiogenesis and lymphangiogenesis, especially promotion of the latter. npBM-MSCs may provide an alternative to autologous and allogeneic MSCs for stem cell therapy of critical limb ischemia.

Neonatal and juvenile porcine islet cell clusters (ICC) present an unlimited source for islet xenotransplantation to treat type 1 diabetes patients. We isolated ICC from pancreata of 14 days old juvenile piglets and characterized their maturation by immunofluorescence and insulin secretion assays. Multipotent mesenchymal stromal cells derived from exocrine tissue of same pancreata (pMSC) were characterized for their differentiation potential and ability to sustain ICC insulin secretion in vitro and in vivo. Isolation of ICC resulted in 142 ± 50 × 103 IEQ per pancreas. Immunofluorescence staining revealed increasing presence of insulin-positive beta cells between day 9 and 21 in culture and insulin content per 500IEC of ICC increased progressively over time from 1178.4 ± 450 µg/L to 4479.7 ± 1954.2 µg/L from day 7 to 14, P < .001. Highest glucose-induced insulin secretion by ICC was obtained at day 7 of culture and reached a fold increase of 2.9 ± 0.4 compared to basal. Expansion of adherent cells from the pig exocrine tissue resulted in a homogenous CD90+ , CD34- , and CD45- fibroblast-like cell population and differentiation into adipocytes and chondrocytes demonstrated their multipotency. Insulin release from ICC was increased in the presence of pMSC and dependent on cell-cell contact (glucose-induced fold increase: ICC alone: 1.6 ± 0.2; ICC + pMSC + contact: 3.2 ± 0.5, P = .0057; ICC + pMSC no-contact: 1.9 ± 0.3; theophylline stimulation: alone: 5.4 ± 0.7; pMSC + contact: 8.4 ± 0.9, P = .013; pMSC no-contact: 5.2 ± 0.7). After transplantation of encapsulated ICC using Ca2+ -alginate (alg) microcapsules into streptozotocin-induced diabetic and immunocompetent mice, transient normalization of glycemia was obtained up to day 7 post-transplant, whereas ICC co-encapsulated with pMSC did not improve glycemia and showed increased pericapsular fibrosis. We conclude that pMSC derived from juvenile porcine exocrine pancreas improves insulin secretion of ICC by direct cell-cell contact. For transplantation purposes, the use of pMSC to support beta-cell function will depend on the development of new anti-fibrotic polymers and/or on genetically modified pigs with lower immunogenicity.

We tested the efficacy of lactated Ringer's solution with 3% trehalose and 5% dextran 40 (LR-3T-5D) as a vehicle solution for cryopreservation using human adipose-derived mesenchymal stromal cells (hADSCs) with dimethyl sulfoxide (Me2SO). We also tested the effect of the Me2SO concentration in the cryopreservation solution, and the effect of washing with lactated Ringer's solution with 3% trehalose (LR-3T) and replacement with LR-3T or LR-3T-5D. LR-3T-5D was more effective for cell viability, viable cell recovery ratio, annexin V-positive ratio, and colony-forming capacity as a vehicle solution for cryopreservation with 10% Me2SO than LR. The additive effects as cryoprotectants of trehalose and dextran 40 were confirmed to be dose dependent. The cell viability, cell proliferation ability, cell differentiation ability, and the ratio of cell surface positive/negative markers of hADSCs were well maintained after cryopreservation with LR-3T-5D containing 10% Me2SO in liquid nitrogen or in a -80 °C freezer. The cell viability and the proliferation curve in LR-3T-5D with 5% Me2SO were comparable to those with 10% Me2SO. LR-3T-5D was superior to LR-3T as a replacement solution in terms of viability and annexin V positivity. Our data showed that LR-3T-5D is effective as a vehicle solution for cryopreservation. Reducing Me2SO concentration to 5%, and washing and replacement with fresh LR-3T and LR-3T-5D after thawing, are feasible approaches to maintain cryopreservation efficacy.

It is commonly accepted that xenogeneic stem cell transplantation for tissue engineering is faced with host immune rejection. Using a rat critical-size mandibular defect model, this study examined whether the immune rejection can be evaded by diminishing T-cell immunity.

To examine donor cell survival and host immune reaction, pig bone marrow-derived mesenchymal stem cells (BM-MSCs) were labeled with CM-DiI, loaded onto gelatin sponge (5 × 10⁶ cells/scaffold), and transplanted into 5-mm mandibular defects of immunocompetent and T cell-deficient athymic rats. To examine the effects of xenogeneic BM-MSCs on bone regeneration, athymic rats undergone the same surgeries were terminated at post-operative weeks 1, 3, and 6. Control rats underwent the same jaw surgery without BM-MSC transplantation.

The density of CM-DiI-labeled BM-MSCs decreased with time in both strains of rats. Although it was substantially higher in athymic rats than in immunocompetent rats at post-operative day 1, by day 3-7 the density became comparable between the two strains of rats. Apoptosis reflected by cleaved Caspase-3 staining was low in both strains. Stronger infiltration of neutrophils, macrophages, B cells and CD8⁺ T cells was found in MSC-treated animals. In athymic rats, infiltration of neutrophils and macrophages was strong, but it occurred later than that in immunocompetent rats. While bone volume fraction significantly increased with time (P < .001), no difference was found between MSC-treated and control groups.

Even in hosts with deficient T-cell immunity, xenogeneic BM-MSC transplantation into mandibular critical-sized defects still faces challenges from host innate immunity, which compromises their regenerative efficacy.