Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Estrogen Regulates the Expression and Localization of YAP in the Uterus of Mice.

The dynamics of uterine endometrium is important for successful establishment and maintenance of embryonic implantation and development, along with extensive cell differentiation and proliferation. The tissue event is precisely and complicatedly regulated as several signaling pathways are involved including two main hormones, estrogen and progesterone signaling. We previously showed a novel signaling molecule, Serine/threonine protein kinase 3/4 (STK3/4), which is responded to hormone in the mouse uterine epithelium. However, the role and regulation of its target, YES-associated protein (YAP) remains unknown. In this study, we investigated the expression and regulation of YAP in mouse endometrium. We found that YAP was periodically expressed in the endometrium during the estrous cycle. Furthermore, periodic expression of YAP was shown to be related to the pathway under hormone treatment. Interestingly, estrogen was shown to positively modulate YAP via endometrial epithelial receptors. In addition, the knockdown of YAP showed that YAP regulated various target genes in endometrial cells. The knockdown of YAP down-regulated numerous targets including ADAMTS1, AMOT, AMOTL1, ANKRD1, CTNNA1, MCL1. On the other hand, the expressions of AREG and AXL were increased by its knockdown. These findings imply that YAP responds via Hippo signaling under various intrauterine signals and is considered to play a role in the expression of factors important for uterine endometrium dynamic regulation.

Toll-like receptors: A pathway alluding to cancer control.

Toll-like receptors (TLRs) are usually expressed on immune cells such as macrophages, dendritic cells, mast cells, as well as on eosinophils and some epithelial cells. They play a central role in the recognition of harmful molecules that belong to invading microorganisms or internal damaged tissues, which lead to inflammation. Among the hallmarks of cancer, there is immune evasion and inflammation. Summing this with the discovery that a majority of cancers also seem to express TLRs, made researchers realize these receptors might also be linked with cancer progression. This review will cover some of the effects of TLR engagement in cancer cells that might induce the promotion or inhibition of cancer with mechanisms involved. The differences of TLR expression in cancer progression and its possible relation with patient prognosis, TLR genetic disorders found in cancer, and new strategies to cancer therapy will be discussed to target TLRs in cancer cells.

A novel calcium-accumulating peptide/gelatin in situ forming hydrogel for enhanced bone regeneration.

Bioactive agents, including proteins and peptides, can be loaded into hydrogels to improve bone regenerative capacity with their controlled release. However, the current loading method has focused on physical mixing, which has limited release control. Therefore, alternative conjugation of bioactive agents with hydrogels is highly recommended. Direct chemical conjugation of synthetic peptides containing a functional moiety with a hydrogel would be ideal. Here, we synthesized a bioactive calcium accumulating peptide (CAP) containing a collagen binding motif, which can induce osteogenic differentiation. A tyrosine residue in CAP was used to directly chemically conjugate the peptide with a gelatin-based enzymatically crosslinked hydroxyphenyl propionic acid hydrogel under H2 O2 /Horse radish peroxidase conditions. To test the acceleration of bone formation, human periodontal ligament stem cells (PDLSCs) were loaded into a chemically conjugated CAP hydrogel. The CAP hydrogel induced bone mineralization around the PDLSCs and increased osteogenic marker expressions in vitro. It also recovered a bone layer in a calvarial defect 4 weeks postimplantation. In summary, an injectable CAP hydrogel scaffold system was developed as a potentially useful engineered microenvironment to enhance bone restoration, and it could be utilized as a vehicle for bioactive delivery of stem cells in tissue regenerative therapy. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 531-542, 2018.

Current treatments for advanced melanoma and introduction of a promising novel gene therapy for melanoma (Review).

Metastatic melanoma is a fatal form of skin cancer that has a tendency to proliferate more rapidly than any other solid tumor. Since 2010, treatment options for metastatic melanoma have been developed including chemotherapies, checkpoint inhibition immunotherapies, e.g., anti­cytotoxic T­lymphocyte antigen­4 (CTLA­4) and anti­programmed death­1 (PD­1), and molecular-targeted therapies, e.g., BRAF and MEK inhibitors. These treatments have shown not only high response rates yet also side­effects and limitations. Notwithstanding its limitations, stem cell therapy has emerged as a new auspicious therapy for various tumor types. Since stem cells possess the ability to serve as a novel vehicle for delivering therapeutic or suicide genes to primary or metastatic cancer sites, these cells can function as part of gene­directed enzyme prodrug therapy (GDEPT). This review focuses on introducing engineered neural stem cells (NSCs), which have tumor­tropic behavior that allows NSCs to selectively approach primary and invasive tumor foci, as a potential gene therapy for melanoma. Therapy using engineered NSCs with cytotoxic agents resulted in markedly reduced tumor volumes and significantly prolonged survival rates in preclinical models of various tumor types. This review elucidates current treatment options for metastatic melanoma and introduces a promising NSC therapy.

Postural stability in patients with anterior cruciate ligament tears with and without medial meniscus tears.

PURPOSE: To compare postural stability in patients with isolated anterior cruciate ligament (ACL) tears and ACL tears with associated meniscal tears. METHODS: Quadriceps and hamstring muscle strength and their ratio, as well as the relationships of these parameters with postural stability, were compared in 23 patients with isolated ACL tears and 27 with combined ACL and medial meniscus tears. Postural stability was determined from the anterior-posterior, medial-lateral, and overall stability indices using the Biodex Stability System. RESULTS: On both the involved and uninvolved sides, there were no differences in mean stability indices, including anterior-posterior, medial-lateral, and overall stability indices, in patients with isolated and combined ACL tears. In patients with isolated ACL tears, both overall (2.3 ± 1.2 vs. 1.8 ± 1.4, p = 0.033) and medial-lateral (1.2 ± 0.6 vs. 1.0 ± 0.5, p = 0.031) stability indices were significantly higher on the involved compared to the uninvolved side. These differences, however, were not observed in the combined ACL tear group. CONCLUSION: No significant differences in postural instability on the affected and unaffected sides were observed in patients with isolated ACL tears and those with combined ACL and medial meniscus tears. These findings indicate that there is no need to reduce the goal of restoring proprioception in patients with combined compared with isolated ACL tears. LEVEL OF EVIDENCE: III.

Paradoxical effects of human adipose tissue-derived mesenchymal stem cells on progression of experimental arthritis in SKG mice.

We evaluated the therapeutic effect of human adipose tissue-derived mesenchymal stem cells (hAd-MSCs) in a SKG arthritis model, a relevant animal model for human rheumatoid arthritis. hAd-MSCs were administered intraperitoneally into the mice for five consecutive days from on day 12 or 34 after arthritis induction, when the average clinical score was 0.5 or 5, respectively. They remarkably suppressed arthritis when administered on day 12. Disease suppression was correlated with reduction of pro-inflammatory cytokines and with increased levels of TGF-ß and IL-10 from splenocytes. However, when hAd-MSCs were administered on day 34, the clinical scores were not improved, the histopathological scores were aggravated, and cytokine profiles were differed. Thus, hAd-MSCs showed paradoxical effects, according to the disease phase when they were administered. These suggest that the same cells acted differently depending on the disease progress, and cautions should be paid for safe and effective use of MSCs.

Phenotypic characterization and in vivo localization of human adipose-derived mesenchymal stem cells.

Human adipose-derived mesenchymal stem cells (hADMSCs) are a potential cell source for autologous cell therapy due to their regenerative ability. However, detailed cytological or phenotypic characteristics of these cells are still unclear. Therefore, we determined and compared cell size, morphology, ultrastructure, and immunohistochemical (IHC) expression profiles of isolated hADMSCs and cells located in human adipose tissues. We also characterized the localization of these cells in vivo. Light microscopy examination at low power revealed that hADMSCs acquired a spindle-shaped morphology after four passages. Additionally, high power views showed that these cells had various sizes, nuclear contours, and cytoplasmic textures. To further evaluate cell morphology, transmission electron microscopy was performed. hADMSCs typically had ultrastructural characteristics similar to those of primitive mesenchymal cells including a relatively high nuclear/cytosol ratio, prominent nucleoli, immature cytoplasmic organelles, and numerous filipodia. Some cells contained various numbers of lamellar bodies and lipid droplets. IHC staining demonstrated that PDGFR and CD10 were constitutively expressed in most hADMSCs regardless of passage number but expression levels of α-SMA, CD68, Oct4 and c-kit varied. IHC staining of adipose tissue showed that cells with immunophenotypic characteristics identical to those of hADMSCs were located mainly in the perivascular adventitia not in smooth muscle area. In summary, hADMSCs were found to represent a heterogeneous cell population with primitive mesenchymal cells that were mainly found in the perivascular adventitia. Furthermore, the cell surface markers would be CD10/PDGFR. To obtain defined cell populations for therapeutic purposes, further studies will be required to establish more specific isolation methods.

Comparison of the ultrastructural and immunophenotypic characteristics of human umbilical cord-derived mesenchymal stromal cells and in situ cells in Wharton's jelly.

The umbilical cord contains mucinous connective tissue, called Wharton's jelly. It consists of stromal cells, collagen fibers, and amorphous ground substances composed of proteoglycan. Recently, these stromal cells have been redefined as a new cell therapy source, named human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). However, there are few studies on the ultrastructural features and immune-phenotypic characteristics of isolated hUCMSCs and comparisons with the cells found in original cord tissues. In this study, the authors describe and compare the phenotypic characteristics of hUCMSCs with cells in the umbilical cord in order to know the kinds of cells and ultrastructural changes. Isolated hUCMSCs showed similar ultrastructure with few structural differences from in situ stromal cells, and they are relatively homogenous and well-developed mesenchymal cells that demonstrate a myofibroblastic phenotype.

Chondrogenesis of periodontal ligament stem cells by transforming growth factor-ß3 and bone morphogenetic protein-6 in a normal healthy impacted third molar.

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-ß3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-ß3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-ß3 and 220% by BMP-6. The synergetic effect of TGF-ß3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-ß3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

Is the critical pathway effective for the treatment of gastric cancer?

PURPOSE: The present study was conducted to investigate the low compliance rate of the critical pathway (CP) and whether CP is effective for treatment of gastric cancer in radical gastrectomy. METHODS: The medical records of 631 patients who had undergone radical gastrectomy with D2 lymph node dissection were reviewed. This study compared data from patients in early gastric cancer (EGC) and advanced gastric cancer (AGC) groups, which were further subdivided into general care (non-CP) and CP groups. RESULTS: The mean length of preoperative hospital stays were significantly different between the EGC and AGC patients (P < 0.05). However, there was no difference in the mean length of postoperative hospital stays between non-CP and CP groups among either EGC patients or AGC patients (P > 0.05). The postoperative and total cost of hospitalization was not statistically different between either of the groups (P > 0.05); however, the mean preoperative costs were significantly different (P < 0.05). CONCLUSION: We conclude that use of the CP following gastrectomy is unnecessary. To decrease the length of hospital stay and associated costs, preoperative examination and consultation should be performed before admission.

Prealbumin levels as a useful marker for predicting infectious complications after gastric surgery.

BACKGROUND/OBJECTIVES: Preoperative nutritional status is associated with postoperative complications. Prealbumin, a visceral protein, is sensitive to protein malnutrition. The objective of this study is to evaluate the role of preoperative prealbumin levels as a marker for predicting complications after gastric surgery. METHODS: An observational study was performed on 183 patients who underwent gastric surgery due to benign or malignant gastric disease at Seoul National University Hospital (SNUH) between August 2009 and October 2010. Preoperative prealbumin levels were also measured. Nutritional variables such as prealbumin (cutoff value, 18 mg/dL), albumin, body mass index (BMI), and clinicopathologic data were collected. Postoperative hospital stay, 30-day complications and mortality rate were obtained to investigate outcomes. RESULTS: The complication rate was 52% in the abnormal prealbumin group (n = 23) and 24% in the normal prealbumin group (n = 160; p = 0.005). The complication rate was higher in patients with low preoperative albumin levels (<3.5 g/dL) and abnormal BMI (<18.5 kg/m(2)), but the differences were not statistically significant. Comorbidity of diabetes mellitus (DM), resection extent, combined resection, TNM stage and prealbumin levels were associated with complications. In multivariate analysis, DM and combined resection were significantly correlated with complications (p = 0.001 for each). In subgroup analysis, resection extent, approach, combined resection, TNM stage, and prealbumin levels were significantly associated with infectious complications. Multivariate analysis identified combined resection (p = 0.001) and prealbumin levels (p = 0.032) as independent variables. CONCLUSIONS: Preoperative prealbumin levels could be a useful marker for predicting complications, especially infectious complications, after gastric surgery.

The Porcine Aortic Tissue Culture System in vitro for Stem Cell Research.

BACKGROUND AND OBJECTIVES: Due to the shortage of human donors for transplantation, the use of animal organs for xenotransplantation has come into great interest. Xeno-derived vessels and cardiac valves would be possible alternatives for the patient suffering from cardiovascular diseases. Therefore, we established in vitro culture system of a porcine vessel that could be helpful for the research of xenograft and stem cell research. METHODS AND RESULTS: We primarily isolated porcine thoracic aorta, cultured square-shaped pieces up to 17 days and analyzed its morphology and characters. The endothelial cells were primarily isolated from cultured porcine aortic pieces and their morphology, function and character were analyzed in order to confirm them as endothelial cells at day 3, 4, 8, 10 and 17. Even at day 17, the morphology exhibited the intact endothelial layer as well as specifically expressed CD31 and von Willebrand factor. The morphology of primarily isolated cells from cultured tissues was identical as an endothelial cell. By flow cytometry analysis, more than 80% of the isolated cells expressed CD31 and up to 80% took up acetyl low density lipoprotein (ac-LDL) until day 10 of tissue culture period even though it decreased to about 50% at day 17 that means they not only showed typical endothelial cell characters but also functioned properly. CONCLUSIONS: We successfully established and optimized a porcine vascular tissue in vitro culture system that could be a valuable model for in vitro study of xenotransplantation and stem cell research.

Human feeder layer system derived from umbilical cord stromal cells for human embryonic stem cells.

OBJECTIVE: To evaluate the potential of human umbilical cord-derived stromal cells (hUCSCs) as a human feeder for human embryonic stem cells (ESCs). DESIGN: Prospective study. SETTING: Laboratory of Molecular Genetics and Stem Cell Differentiation, Dental Research Institute, School of Dentistry, Seoul National University. INTERVENTION(S): The hUCSCs were established, and human ESCs were cultured on established hUCSCs without serum. MAIN OUTCOME MEASURE(S): Cell-surface markers, karyotyping, and teratoma formation. RESULT(S): Primary cultures of hUCSCs from individual umbilical cords were maintained by an established protocol. Human ESCs on hUCSC layers were successfully maintained in serum-free culture medium past passage 30. Compared with hESCs on mouse feeder cells, the hESCs on hUCSCs showed similar levels of pluripotency-related cell-surface markers, self-renewal capacity, and teratoma formation in immune-deficient mice. These ESCs cultured on hUCSCs had a normal karyotype, even after long-term culture. CONCLUSION(S): The hUCSCs supported self-renewal of hESCs in serum-free conditions. This culture system has the potential to facilitate the development of clinical-grade hESCs for regenerative medicine.

Increased morbidity rates in patients with heart disease or chronic liver disease following radical gastric surgery.

BACKGROUND: The aim of this study was to investigate possible associations between (i) comorbid disease and (ii) perioperative risk factors and morbidity following radical surgery for gastric cancer. MATERIALS AND METHODS: Consecutive patients (759) undergoing radical gastrectomy and D2 level lymph node dissection for gastric cancer were included. Clinical data concerning patient characteristics, operative methods, and complications were collected prospectively. RESULTS: The morbidity rate for radical gastrectomy was 14.2% (108/759). The most significant comorbid risk factors for postoperative morbidity were heart disease [anticoagulant medication: OR = 1.5 (95% CI = 0.35-6.6, P = 0.53); history without medication: OR = 4.0 (95% CI = 1.1-14.6, P = 0.03); history with current medication: OR = 6.7 (95% CI = 1.5-29.9, P = 0.01)] and chronic liver disease [chronic hepatitis: OR = 2.4 (95% CI = 0.9-6.5, P = 0.07); liver cirrhosis class A: OR = 8.4 (95% CI = 2.8-25.3, P = 0.00); liver cirrhosis class B: OR = 9.38 (95% CI = 0.7-115.5, P = 0.08)]. The most significant perioperative risk factors for postoperative morbidity were high TNM stage and combined organ resection (P < 0.05), and there was no association between increased postoperative morbidity and well controlled hypertension, anticoagulant therapy, diabetes mellitus, pulmonary disease, tuberculosis, or thyroid disease (P > 0.05). CONCLUSION: Patients with heart disease or chronic liver disease are at a higher risk of morbidity following radical surgery for gastric cancer.

Novel embryoid body-based method to derive mesenchymal stem cells from human embryonic stem cells.

Application of human embryonic stem cells (hESCs) to stem-cell therapy is not feasible because of the risk of tumorigenicity and rejection. In contrast, human mesenchymal stem cells (hMSCs) are free from the risk of tumorigenicity and also have immune privilege. However, hMSCs obtained from adults have infinite variety in terms of the biological characteristics and functionality. We report here a new derivation method of hMSCs from hESCs. The derivation of hMSCs from three different hESC lines (SNUhES3, CHA3-hESC, and H9) was performed by embryoid bodies formation and subsequent culture with stage-different media without using inductive xenogenic feeder and mechanical selection procedure. The derived cells were morphologically similar to the unique fingerprint-like pattern of hMSCs and grew stably for at least 35 passages in vitro. These cells had hMSCs-like immunophenotypes: negative for CD34 and CD45; positive for CD29, CD44, CD73, CD90, and CD105. They could be differentiated into multiple lineages including osteocytes, chondrocytes, adipocytes, and myocytes. They maintained normal karyotype during the long-term cultivation and did not show tumorigenicity when transplanted into the immunodeficient mice. In conclusion, the new embryoid body-based derivation method of hMSCs from hESCs is simple, safe, and reproducible in three different hESC lines. We expect that this method will provide a more effective and powerful tool to derive hMSCs from various hESC lines.

Effect of the polyvinylpyrrolidone concentration of cryoprotectant on mouse embryo development and production of pups: 7.5% of PVP is beneficial for in vitro and in vivo development of frozen-thawed mouse embryos.

In this study, we investigated the effect of polyvinylpyrrolidone (PVP) concentration on in vitro and in vivo development of 2 cell stage, vitrified ICR mouse embryos using a cryoprotectant consisting of ethylene glycol (EG) and sucrose. M2 was selected as the basic medium for vitrification and thawing. After equilibration with 4% (v/v) EG at 37 C for 15 min, the embryos were vitrified with 35% EG, 5, 6 or 7.5% (w/v) PVP and 0.4 M sucrose at 37 C for 30 sec. One week later, the cryotubes of cryopreserved embryos in liquid nitrogen were directly immersed into a 37 C water bath for 1 min and transferred serially into 300 microl of 0.5 or 0.3 M sucrose at room temperature for 5 min and M2 medium at 37 C for 10 min. The surviving embryos were cultured in KSOM (potassium simplex optimized medium) for 96-120 h in an atmosphere of 5% CO(2) in humidified air. Survival was evaluated by morphological appearance, including membrane integrity and presence of apoptotic blastomeres after thawing. For in vivo evaluation, blastocysts were transferred to the uteri of pseudopregnant mice. The survival rates of the 5 and 7.5% PVP concentration groups showed a significantly higher difference compared with that of the 6% PVP group (85.5 and 86.5 vs. 71.2%), respectively. Each pup in the of 5 and 6% groups was cannibalized immediately after parturition. A litter of live pups was obtained from only the 7.5% PVP groups. Our study indicated that supplementation of EG and sucrose cryoprotectant solution with 7.5% PVP is optimal for successful vitrification of 2-cell stage ICR mouse embryos.

Role of complement regulatory proteins in the survival of murine allo-transplanted Sertoli cells.

Sertoli cells (SC) are known to contain immunoprotective properties, which allow them to survive as allografts without the use of immunosuppressive drugs. Experiments were designed to determine which factors are related to prolonged survival of allogeneic SC. Balb/c derived Sertoli (TM4) and colon cancer (CT-26) cell lines were implanted beneath the kidney capsule of non-immunosuppressed C57BL/6 mice and compared their survival as allografts. Compared to TM4 graft, which survived more than 7 days after transplantation, CT-26 showed massive infiltration of polymorphonuclear cells, necrosis and enlargement of draining lymph nodes. Cultured cell lines showed no differences in their expression patterns of FasL, TGF beta1, clusterin and two complement regulatory proteins (CRP, i.e., membrane cofactor protein, MCP; decay accelerating factor, DAF), but protectin (CD59), another member of CRP was expressed only on TM4. These results suggest that CD59 and unknown factors may contribute to the prolonged survival of SC in non-immunoprivileged sites.

Establishment and characterization of porcine Sertoli cell line for the study of xenotransplantation.

BACKGROUND: An understanding of the main mechanism that determines the ability of immune privilege related to Sertoli cells (SC) will provide clues for promoting a local tolerogenic environment. In this report, we established neonatal porcine SC line and evaluated their characteristics. METHODS: SC line was established following the transfection of primary SC (NPSC) from the testis of neonatal pig with plasmid pRNS-1 carrying genes for neomycin resistance and the SV40 large T antigen. Immunohistochemistry and RT-PCR were performed to evaluate the character of immortalized SC lines. RESULTS: Our immortalized SC line (iPS) proliferated stably and had a phenotype similar to NPSC, as indicated by the immunoexpression of follicle stimulating hormone receptor (FSHR), and mRNA expression of androgen receptor (AR), and Wilms' tumor antigen (WT1). Interestingly, NPSC and iPS expressed mRNA of complement regulatory proteins (CRP) such as membrane cofactor protein (CD46), decay accelerating factor (DAF or CD55), and protectin (CD59), but CD59 mRNA expression was negligible in iPS. CONCLUSION: These results suggest that iPS, immortalized by the introduction of SV40 T, retain their original characteristics, except for the relatively low expression of CD59, and that they may be useful for future in vitro and in vivo studies of immune privilege mechanisms related to SC.

Age-dependent expression of immune-privilege and proliferation-related molecules on porcine Sertoli cells.

BACKGROUND: The immunoprotective nature of the testes has prompted numerous investigations into their supportive roles during allogeneic or xenogeneic cellular grafts. However, the optimal developmental stage of these cells in terms of maximum efficacy for cellular grafts has not been elucidated. In this study, the time-dependent expressions of immune-privilege- and proliferation-related molecules in Sertoli cells were determined. METHODS: To investigate the time course of the expression of proteins related to immune privilege and proliferation, immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed using testes of 18 Yorkshire pigs from birth to 20 weeks of age. We included fas ligand (FasL), transforming growth factor beta1 (TGF-beta1) and clusterin as the immune-protective molecules, and follicle stimulating hormone receptor (FSHR), p27Kip1, GATA-4 and Wilm's tumor antigen (WT1) as Sertoli cell proliferation markers. RT-PCR was used to complement histological assessment. RESULTS: The expression of FasL in Sertoli cells gradually increased with age, whereas TGF-beta1 showed the reverse pattern, and clusterin expression showed no age-related difference. p27Kip1 showed a gradual increase in its expression from week 12, but GATA-4 showed earlier postnatal expression from birth to week 12. WT1 expression gradually increased from week 16. CONCLUSION: We confirmed the age-dependent expression of immune-privilege- and proliferation-related molecules in porcine Sertoli cells. These data may be useful for determining the optimal time for harvesting Sertoli cells with respect to maximal immunoprotection and proliferative activity.