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Background: Articular cartilage has limited healing capacity, owing in part to poor vascularity and innervation. Once injured, it cannot be repaired, typically leading to high risk for developing osteoarthritis. Thus, cell-based and/or tissue-engineered approaches have been investigated; however, no approach has yet achieved safety and regenerative repair capacity via a simple implantation procedure. Purpose: To assess the safety and efficacy of using a scaffold-free tissue-engineered construct (TEC) derived from autologous synovial membrane mesenchymal stem cells (MSCs) for effective cartilage repair. Study Design: Case series; Level of evidence, 4. Methods: Five patients with symptomatic knee chondral lesions (1.5-3.0 cm2) on the medial femoral condyle, lateral femoral condyle, or femoral groove were included. Synovial MSCs were isolated from arthroscopic biopsy specimens and cultured to develop a TEC that matched the lesion size. The TECs were then implanted into chondral defects without fixation and assessed up to 24 months postoperatively. The primary outcome was the safety of the procedure. Secondary outcomes were self-assessed clinical scores, arthroscopy, tissue biopsy, and magnetic resonance image–based estimation of morphologic and compositional quality of the repair tissue. Results: No adverse events were recorded, and self-assessed clinical scores for pain, symptoms, activities of daily living, sports activity, and quality of life were significantly improved at 24 months after surgery. Secure defect filling was confirmed by second-look arthroscopy and magnetic resonance imaging in all cases. Histology of biopsy specimens indicated repair tissue approaching the composition and structure of hyaline cartilage. Conclusion: Autologous scaffold-free TEC derived from synovial MSCs may be used for regenerative cartilage repair via a sutureless and simple implantation procedure. Registration: 000008266 (UMIN Clinical Trials Registry number).

Mesenchymal stem cell- (MSC-) based therapy is a promising treatment for cartilage. However, repair tissue in general fails to regenerate an original hyaline-like tissue. In this study, we focused on increasing the expression levels for insulin-like growth factor-1 (IGF-1) to improve repair tissue quality. The IGF-1 gene was introduced into human synovial MSCs with a lentiviral vector and examined the levels of gene expression and morphological status of MSCs under chondrogenic differentiation condition using pellet cultures. The size of the pellets derived from IGF-1-MSCs were significantly larger than those of the control group. The abundance of glycosaminoglycan (GAG) was also significantly higher in the IGF-1-MSC group. The histology of the IGF-1-induced pellets demonstrated similarities to hyaline cartilage without exhibiting features of a hypertrophic chondrocyte phenotype. Expression levels for the Col2A1 gene and protein were significantly higher in the IGF-1 pellets than in the control pellets, but expression levels for Col10, MMP-13, ALP, and Osterix were not higher. Thus, IGF-1 gene transfer to human synovial MSCs led to an improved chondrogenic differentiation capacity without the detectable induction of a hypertrophic or osteogenic phenotype.

Supplemental material, DS_10.1177_0363546518781825 for First-in-Human Pilot Study of Implantation of a Scaffold-Free Tissue-Engineered Construct Generated From Autologous Synovial Mesenchymal Stem Cells for Repair of Knee Chondral Lesions by Kazunori Shimomura, Yukihiko Yasui, Kota Koizumi, Ryota Chijimatsu, David A. Hart, Yasukazu Yonetani, Wataru Ando, Takashi Nishii, Takashi Kanamoto, Shuji Horibe, Hideki Yoshikawa and Norimasa Nakamura in The American Journal of Sports Medicine

DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b cooperatively regulate cytosine methylation in CpG dinucleotides in mammalian genomes, providing an epigenetic basis for gene silencing and maintenance of genome integrity. Proper CpG methylation is required for the normal growth of various somatic cell types, indicating its essential role in the basic cellular function of mammalian cells. Previous studies using Dnmt1(-/-) or Dnmt3a(-/-)Dnmt3b(-/-) ES cells, however, have shown that undifferentiated embryonic stem (ES) cells can tolerate hypomethylation for their proliferation. In an attempt to investigate the effects of the complete loss of CpG DNA methyltransferase function, we established mouse ES cells lacking all three of these enzymes by gene targeting. Despite the absence of CpG methylation, as demonstrated by genome-wide methylation analysis, these triple knockout (TKO) ES cells grew robustly and maintained their undifferentiated characteristics. TKO ES cells retained pericentromeric heterochromatin domains marked with methylation at Lys9 of histone H3 and heterochromatin protein-1, and maintained their normal chromosome numbers. Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA methyltransferases.

Background. One major concern about adenoviral vectors for repetitive gene delivery is the induction of an immune response to the vector, thus impeding effective gene transduction. Methods. To assess the immune response to the adenoviral vector, repetitive gene dosing was performed into rhesus monkey cervix and C3H mouse skin using the adenoviral vector carrying the lacZ gene. Three repetitive intracervical injections of adenovirus- lacZ were done in the rhesus monkey at the intervals of 4 weeks. Gene expression on the second and third injection was completely suppressed. Results. Anti-adenovirus IgG levels and neutralizing antibody titers in the rhesus monkey significantly increased after the first injection of adenovirus. In the C3H mouse, neutralizing antibody titers significantly increased after the first injection of adenovirus- lacZ at more than 10 8 plaque-forming unit (PFU). The repetitive expression of lacZ gene in the mouse skin markedly decreased when the second injection is done more than 2 weeks after the first injection. Chronic low-dose treatment with cyclophosphamide or etoposide markedly suppressed neutralizing antibody titers in the mouse serum and restored the gene expression in the mouse skin on the second and third injection. Conclusions. It is suggested that repetitive gene expression by adenovirus-mediated transfer may be reduced by circulating neutralizing antibodies and could be restored by chronic low-dose treatment with cyclophosphamide or etoposide.

The novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) inhibits cell proliferation and is a very effective inducer of apoptosis in a variety of carcinoma cell lines. In order to obtain greater insight into the mechanism of AHPN-induced growth arrest and apoptosis, we began to examine AHPN-induced changes in gene expression by cDNA array screening using human lung carcinoma H460 cells. This analysis identified several AHPN-inducible genes, including the immediate-early genes Egr-1 and Nur77. AHPN was able to increase Egr-1 and Nur77 mRNA expression and protein in a variety of carcinoma cell lines. This induction appeared to be regulated at the transcriptional level and was specific for AHPN since an RAR- and an RXR-selective retinoid were inactive. These results suggest that the induction of Egr-1 and Nur77 by AHPN is independent of nuclear retinoid receptors and involves a novel mechanism. Overexpression of Bcl-2, which inhibits AHPN-induced apoptosis but not growth arrest in human T cell lymphoma Molt-4 cells, did not block the induction of immediate-early gene expression. Treatment of H460 cells with AHPN induced activation of the p38 MAP-kinase but not the ERK1/2 signaling pathway. However, inhibition of the ERK1/2 signaling pathway by PD98059 blocked the induction of Egr-1 and Nur77 mRNA while the p38 MAPK inhibitor PD169316 had little effect. Expression of a dominant-negative ERK1 completely abolished the increase in Egr-1 mRNA. Treatment with MAPK inhibitors or expression of dnERK1 reduced but did not block AHPN-induced apoptosis. Our results suggest that the induction of Egr-1 in H460 by AHPN requires active ERK1/2 and is independent of p38 activation. Egr-1, in cooperation with several other growth-suppressor proteins, is likely involved in AHPN-induced inhibition of cell growth and cell death. Cell Death and Differentiation (2001) 8, 411–424

Treatment of normal human epidermal keratinocytes (NHEK) with interferon-γ (IFN-γ) causes a 9-fold increase in the level of cyclooxygenase-2 (COX-2) mRNA expression. Nuclear run-off assays indicate that this induction is at least partly due to increased transcription. Activation of the epidermal growth factor receptor (EGFR) signaling pathway due to the enhanced transforming growth factor α (TGFα) expression plays an important role in the induction of COX-2 by IFN-γ. This is supported by the ability of TGFα to rapidly induce COX-2 and the inhibition of the IFN-γ-mediated COX-2 mRNA induction by an EGFR antibody and EGFR-selective kinase inhibitors. Deletion and mutation analysis indicates the importance of the proximal cAMP-response element/ATF site in the transcriptional control of this gene by TGFα. The increase in COX-2 mRNA by TGFα requires activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK) pathways. Inhibition of p38 MAPK decreases the stability of COX-2 mRNA, while inhibition of MAPK/ERK kinase (MEK) does not. These results suggest that the p38 MAPK signaling pathway controls COX-2 at the level of mRNA stability, while the ERK signaling pathway regulates COX-2 at the level of transcription. In contrast to NHEK, IFN-γ and TGFα are not very effective in inducing TGFα or COX-2 expression in several squamous carcinoma cell lines, indicating alterations in both IFN-γ and TGFα response pathways.

To explore the potential of an adenoviral antisense RNA transcript for gene therapy of cervical cancer, we introduced the antisense RNA transcript of E6 and E7 genes of human papillomavirus (HPV) 16 into cervical cancer cells harboring HPV 16 via a recombinant adenoviral vector, Ad5CMV-HPV 16 AS and analyzed the effects of expression of these genes on cell growth and tumor growth. Ad5CMV-HPV 16 AS contains the cytomegalovirus-promoter, E6 and E7 genes of HPV 16 in antisense orientation, and the SV40 polyadenylation signal in a mini-gene cassette, which is inserted into the E1-deleted region of modified adenovirus 5. The entire E6/E7 region of HPV 16 was amplified by polymerase chain reaction (PCR) before cloning into the mini-gene cassette. By reverse transcriptase-PCR, HPV 16 E6/E7 antisense RNA was detected in SiHa cells infected with Ad5CMV-HPV 16 AS. The growth of the Ad5CMV-HPV 16 AS-infected cells was greatly suppressed, as evidenced by a decrease in cell count. The growth inhibitory effect of Ad5CMV-HPV 16 AS was significantly enhanced by an adenoviral p53 construct, Ad5CMV-p53. In an ex vivo study in nude mice, tumorigenicity was completely inhibited in mice injected with Ad5CMV-HPV 16 AS-infected SiHa cells. These data suggest that transfection of cervical cancer cells with HPV 16 E6/E7 antisense RNA in a form such as Ad5CMV-HPV 16 AS is a potential novel approach to the therapy of HPV 16-positive cervical cancer.

To clarify the role of TGF-β1 in myocardial healing process after virus-induced myocarditis, we have examined the time course of TGF-β1 expression and its localization in myocardial healing process after Coxsackie B3-induced murine myocarditis. TGF-β1 immunoreactivity increased in parallel with its mRNA level and reached a peak at 10 days after Coxsackie B3 virus inoculation. Immunohistochemically, TGF-β1 was localized at pre-necrotic area at an early phase (5 days); it increased and extended to the area around the necrotic foci when necrosis became manifest (10 days); it decreased when macrophages and fibroblasts migrated to the necrotic foci (15 days); and then it reached a normal level and localized a little in perivascular and calcified regions (30 days). To our knowledge, this is the first study which demonstrates that TGF-β1 mRNA increases in viral-induced myocarditis at acute inflammatory phase. This result suggests that TGF-β1 promotes the migration of macrophages and fibroblasts to necrotic foci and induces fibrosis.

Summary DNA methylation regulates development and many epigenetic processes in mammals [1], and it is required for somatic cell growth and survival [2, 3]. In contrast, embryonic stem (ES) cells can self-renew without DNA methylation [4–6]. It remains unclear whether any lineage-committed cells can survive without DNA-methylation machineries. Unlike in somatic cells, DNA methylation is dispensable for imprinting and X-inactivation in the extraembryonic lineages [7–12]. In ES cells, DNA methylation prevents differentiation into the trophectodermal fate [13]. Here, we created triple-knockout (TKO) mouse embryos deficient for the active DNA methyltransferases Dnmt1, Dnmt3a, and Dnmt3b (TKO) by nuclear transfer (NT), and we examined their development. In chimeric TKO-NT and WT embryos, few TKO cells were found in the embryo proper, but they contributed to extraembryonic tissues. TKO ES cells showed increasing cell death during their differentiation into epiblast lineages, but not during differentiation into extraembryonic lineages. Furthermore, we successfully established trophoblastic stem cells (ntTS cells) from TKO-NT blastocysts. These TKO ntTS cells could self-renew, and they retained the fundamental gene expression patterns of stem cells. Our findings indicated that extraembryonic-lineage cells can survive and proliferate in the absence of DNA methyltransferases and that a cell's response to the stress of epigenomic damage is cell type dependent.

先体反応誘起後のヤギ精予の運動能,生存能および透明帯除去ハムスター卵子への侵入能保持時間を調べた.また,これらの特性に対するカフェインおよびイミダゾールの影響についても検討した.ヤギ射出精子を洗浄後,密封ガラス管内において39.5°Cで2時間加温して先体反応を誘起した.ついで,これらの精子をBO液,カブェイン添加BO液またはイミダゾール添加BO液に再浮遊して37°Cで5時間加温し,その間の運動性および生存性の変化を経時的に調べた.また,BO液またはイミダゾール添加BO液中に再浮遊直後の精子あるいは各媒液中で3時間加温した精子を透明帯除去ハムスター卵子とともに37°Cでさらに5時間培養し,その間の卵子への精子の侵入状況を経時的に調べた.得られた結果の概要は以下の通りである.1) ヤギ糖子では先体反応誘起の前と後で運動様式が異なり,誘起後の精子はムチ振り様の運動を行なった.2) 先体反応誘起後のヤギ精子の運動能および生存能は誘起前に比べて著しく低下した.3) ヤギ先体反応精子の透明帯除去ハムスター卵子への侵入能保持時間は3時間以内であった.4) 精子浮遊液へのイミダゾールの添加はヤギ先体反応精子の運動能および生存能の維持ならびに透明帯除去ハムスター卵子への侵入能保持時間の延長に有効であった.一方,カフェインの添加は運動性の一時的な付活には有効であったが,運動能および生存能の維持に対してはむしろ有害であった.

Background: Eosinophils accumulated in sites of allergic inflammation are thought to play a crucial role in the pathogenesis of allergic disorders including asthma, allergic rhinitis, and atopic dermatitis, and tissue eosinophilia is attributable to increased eosinophil survival or decreased eosinophil apoptosis. Objective: Effects of the antiallergic, histamine H 1 blocker oxatomide on viability and apoptosis of eosinophils isolated from the peripheral blood of atopic subjects were studied. Methods: Eosinophil viability and apoptosis were evaluated by using a colorimetric assay and annexin V-labeling, caspase-3 activity, and DNA fragmentation assay. Results: The viability of eosinophils increased in the presence of IL-5 (10 ng/mL), confirming that IL-5 prolongs eosinophil survival in vitro. Application of oxatomide at concentrations over 20 μmol/L for 24 hours decreased the IL-5-induced enhancement of eosinophil viability. Double staining of the cells with annexin V and propidium iodide showed that deprivation of IL-5 promoted spontaneous eosinophil apoptosis and that oxatomide facilitated apoptosis and suppressed the prolongation of eosinophil survival stimulated by IL-5. In the absence of IL-5, approximately 71% and 96% of eosinophils after 24 and 48 hours, respectively, underwent spontaneous apoptosis. IL-5 decreased the rate of eosinophil apoptosis to 38% and 52% after 24 and 48 hours, respectively. Oxatomide increased eosinophil apoptosis in a concentration-dependent manner in the presence of IL-5. Furthermore, oxatomide increased caspase-3 activity and DNA fragmentation. Conclusion: We demonstrated that oxatomide possesses a novel therapeutic effect of apoptosis promotion on eosinophils and prevents the antiapoptotic effects of IL-5, suggesting that oxatomide may contribute to resolution of tissue eosinophilia in allergic inflammation. (J Allergy Clin Immunol 2003;111:567-72.)

In this study, we analyze the effect of several retinoids on the expression of nonsteroidal anti-inflammatory drug-activated gene (NAG-1) in normal human tracheobronchial epithelial (HTBE) cells and several lung carcinoma cell lines. The retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) greatly enhances the expression of NAG-1 mRNA and protein in a time- and dose-dependent manner in human lung adenocarcinoma H460 cells and several other carcinoma cell lines. This induction was specific for AHPN because retinoic acid, a retinoic acid receptor-, and a retinoid X receptor pan-agonist were unable to induce NAG-1, suggesting that this induction is not mediated through activation of retinoid receptors. Although NAG-1 is a p53-responsive gene, AHPN-induced NAG-1 expression does not require p53. The induction of NAG-1 expression by AHPN is caused at least in part by an 8-fold increase in the stability of NAG-1 mRNA. In contrast to carcinoma cells, NAG-1 expression is effectively induced by retinoic acid and the RAR-selective pan-agonist in normal HTBE cells and accompanies the inhibition of squamous differentiation and the initiation of normal differentiation. In vivo, NAG-1 expression was observed in the normal tracheobronchial epithelium, whereas no expression was found in either squamous metaplastic tracheal epithelium or in sections of human lung tumors. Our results suggest that the induction of NAG-1 expression by retinoids in normal HTBE and lung carcinoma cells is regulated by distinct mechanisms and is associated with different biological processes. The linkage between AHPN treatment and NAG-1 expression revealed in this study provides a new mechanism for the antitumorigenic activity of AHPN.

Nuclear receptors are critical regulators of many physiological processes and have been shown to be involved in a variety of disease processes, including malignant neoplasms. Our laboratory is investigating the function of the retinoid-related orphan receptor gamma (RORgamma) and its possible role in disease. Studies of mice deficient in the expression of RORgamma demonstrated that this receptor plays a crucial role in the regulation of thymopoiesis and lymph node organogenesis. In this study, we show that changes in homeostasis in the thymus of RORgamma-/- mice are associated with a high incidence of T-cell lymphomas. Over 50% of the deficient mice of mixed genetic background die within the first 4 months as a result of thymic lymphomas. A high incidence of lymphomas was also observed in RORgamma-/- 129/SvEv mice. The lymphoblastic cells metastasized frequently to spleen and liver. No other tumor types were detected in any of RORgamma-/- mice that died during the course of the experiment, and none of the heterozygous mice developed thymic lymphomas. Lymphoma formation was associated with increased cellular proliferation and an increase in the number of apoptotic cells. When placed in culture, the RORgamma-/- lymphoblastic cells underwent accelerated "spontaneous" apoptosis at a rate similar to that of RORgamma-/- thymocytes. Upon prolonged culture, several lymphoblastic cell lines could be established. Analysis of the immunophenotype of the lymphoblastic cells showed that the CD4 and CD8 subpopulations varied substantially among different lymphomas. The established cell lines consisted mostly of CD44-CD25+CD4-CD8- cells. Our studies indicate that loss of RORgamma disturbs homeostasis in the thymus by enhancing apoptosis and cellular proliferation. The latter may enhance the probability of individual cells to acquire genetic alterations that make them escape negative selection and normal differentiation programs and as a consequence lead to increased susceptibility to the development of T-cell lymphoma.

The bone morphogenic proteins (BMPs) constitute a novel subfamily of the transforming growth factor type beta (TGF-beta) supergene family and play a critical role in modulating mesenchymal differentiation and inducing the processes of cartilage and bone formation. In this study we isolated the 5'-flanking region of the human BMP-5 gene. Nucleotide sequencing, primer extension, DNase I foot printing and functional analysis by transient expression showed that the cloned 1.5 kb promoter region contains two transcription start sites, a canonical TATA box (-17 approximately -12) and a number of consensus recognition sequences including GATA-1 (-582 approximately -576) and engrailed (-549 approximately -541).

Binding proteins to the Src homology 3 (SH3) domains of Nck were screened by the use of glutathione S-transferase fusion proteins. Two proteins of 140 and 125 kDa were detected, both of which associated preferentially with the first SH3 domain of Nck. The 125-kDa protein, designated as Nap1 for Nck-associated protein 1, was purified and the corresponding rat cDNA was isolated. The predicted amino acid sequence revealed that p125 Nap1 dose not contain any known functional motif but shows sequence homology to Hem family gene. Using specific antibodies, p125 Nap1 was shown to associate with Nck both in vitro and in intact cells. Further characterization of p125 Nap1 may clarify the protein-protein interaction in the downstream signaling of Nck.

Inactivation of the "deleted in colon cancer" (DCC) gene on chromosome 18 is known to be associated with the tumorigenesis and metastasis of colorectal cancer. In the present study, we investigated the expression of DCC and the c-erbB-2 product in surgical specimens from 45 patients with breast cancer by immunohistochemical staining, and found the expression of DCC to be decreased in 23 (51%) tumors. In 8 years of follow-up, 11 of 22 (50%) patients with DCC-positive staining tumors, and 17 of 23 (74%) patients with DCC-negative tumors developed recurrence. The stratified analysis, according to the status of axillary lymph node metastasis, showed the same tendency. Overexpression of erbB-2 was detected in 13 (29%) of the 45 breast cancer specimens, but there were no differences in the relapse rate between patients with erbB-2 positive and those with erbB-2 negative tumors. Although the individual alteration of DCC or erbB-2 did not possess independent prognostic significance for the prediction of recurrence, patients with tumors having the double alteration of DCC-negative and erbB-2-positive showed adverse relapse-free survival (0.025 < P < 0.05). These findings suggest that a decrease in DCC expression and erbB-2 overexpression may influence the progression of breast carcinoma.

ヒト先体反応精子の判別法であるトリプルステイン(TS)法とその簡便法がブタ精子に応用できるか否かを検討した。これらの染色法によって,ブタ精子は正常先体を持つ生存精子と死滅精子,正常先体を持たない生存精子と死滅精子の4種類に分けられた。また,ハムスターテストの結果,"正常先体を持たない生存精子"が先体反応精子と推定された。TS法および簡便法はともにブタ先体反応精子の判別に利用可能と考えられる。しかし,簡便法では先体反応精子の割合を過大に評価する危険があるので,ブタ精子ではTS法を採用すべきと思われる。

The aim of the present study was to examine the effects of the elimination of energy substrate from the medium and the effects of the preincubation vessel, temperature, and time on the induction of the acrosome reaction in ejaculated goat spermatozoa in chemically defined medium. Washed spermatozoa were resuspended in Brackett-Oliphant (BO) medium or substrate-free BO medium to give a high concentration and preincubated in open test tubes or sealed glass tubes at 37.0 or 39.5 degrees C for 1, 2, or 3 h. Sperm acrosome reaction was evaluated using a simplified triple-stain technique and a hamster test. It was found that the goat sperm acrosome reaction occurred more readily in the sealed glass tubes than in open test tubes, in substrate-free BO medium than in BO medium, and at 39.5 than at 37.0 degrees C. During preincubation with substrate-free BO medium in sealed glass tubes at 39.5 degrees C, the optimum time necessary to induce the acrosome reaction in goat spermatozoa was 2-3 h.

Synovium-derived mesenchymal stem cells (SDMSCs) are one of the most suitable sources for cartilage repair because of their chondrogenic and proliferative capacity. However, the isolation methods for SDMSCs have not been extensively characterized. Thus, our aim in this study was to optimize the processes of enzymatic isolation followed by culture expansion in order to increase the number of SDMSCs obtained from the original tissue. Human synovium obtained from 18 donors (1.5 g/donor) was divided into three aliquots. The samples were minced and subjected to collagenase digestion, followed by different procedures: Group 1, Tissue fragments were removed by filtering followed by removing floating tissue; Group 2, No filtering. Only floating fragments were removed; Group 3, No fragments were removed. Subsequently, each aliquot was sub-divided into two density subgroups with half. In Group 1, the cell-containing media was plated either at high (5000 cells/cm2) or low density (1000 cells/cm2). In Groups 2 and 3, the media containing cells and tissue was plated onto the same number of culture dishes as used in Group 1, either at high or low density. At every passage, the cells plated at high density were consistently re-plated at high and those plated at low density were likewise. The expanded cell yields at day 21 following cell isolation were calculated. These cell populations were then evaluated for their osteogenic, adipogenic, and chondrogenic differentiation capabilities. The final cell yields per 0.25 g tissue in Group 1 were similar at high and low density, while those in Groups 2 and 3 exhibited higher when cultured at low density. The cell yields at low density were 0.7 ± 1.2 × 107 in Group 1, 5.7 ± 1.1 × 107 in Group 2, 4.3 ± 1.2 × 107 in Group 3 (Group 1 vs Groups 2 and 3, p, Highlights • The processes of enzymatic isolation of MSCs from synovium have been optimized. • Exclusion of filtering the undigested synovial debris provides higher number of SDMSCs. • Exclusion of filtering the undigested synovial debris provides higher number of MSCs.

To identify the physiological functions of the retinoid-related orphan receptor γ (RORγ), a member of the nuclear receptor superfamily, mice deficient in RORγ function were generated by targeted disruption. RORγ−/−mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that RORγ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3+and CD4+lymphocytes is reduced 6- and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of RORγ−/−mice contains 74.4% ± 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4+CD8+subpopulation. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining demonstrated a 4-fold increase in apoptotic cells in the cortex of the thymus of RORγ−/−mice. The latter was supported by the observed increase in annexin V-positive cells. RORγ−/−thymocytes placed in culture exhibit a dramatic increase in the rate of “spontaneous” apoptosis. This increase is largely associated with CD4+CD8+thymocytes and may, at least in part, be related to the greatly reduced level of expression of the anti-apoptotic gene Bcl-XL. Flow cytometric analysis demonstrated a 6-fold rise in the percentage of cells in the S phase of the cell cycle among thymocytes from RORγ−/−mice. Our observations indicate that RORγ is essential for lymphoid organogenesis and plays an important regulatory role in thymopoiesis. Our findings support a model in which RORγ negatively controls apoptosis in thymocytes.