经睾丸网移植小鼠精原干细胞的研究

目的研究经睾丸网移植小鼠同种精原干细胞的可行性。方法经~(60)Co-γ射线全身照射后,在基因型相似的Balb/c小鼠间经睾丸网进行精原干细胞移植,移植后3个月,观察其生精恢复情况。结果形态学证实,成功移植的小鼠精原干细胞生精过程部分恢复。结论在基因型相似的Balb/c小鼠间经睾丸网进行精原干细胞移植是可行的。     

Characteristics of spermatogonial stem cells derived from neonatal porcine testis

The aim of this study was to isolate and characterise porcine spermatogonial stem cells (PSSCs). The putative porcine germline stem cells from testis were isolated successfully by an improving way of enrichment with lymphocyte separation medium (LSM). Results from RT‐PCR analyses showed that PSSCs were positive for OCT4, SOX2, NANOG, PGP9.5, c‐MYC, KEL4 and PRDM‐14 which are multipotent stem cell markers. At the protein level, the results of immunofluorescence analyses showed that PSSCs were positive for OCT4, PGP9.5, SOX2 and CD29. We successfully differentiated these PSSCs into adipocytes and muscle cells and then defined their characteristics, including morphology, surface stem cell markers, and mechanical properties. But the experiment of teratoma formation was negative. The results indicated the PSSCs could be multipotent. Atomic force microscopy was used to characterise the morphological and mechanical properties of undifferentiated PSSCs, as well as the differentiated adipocytes and muscle cells, which could be potentially useful for distinguishing PSSCs from differentiated cells.     

胚胎干细胞源性肝细胞的获得及体内移植研究

目的 探讨体外定向诱导小鼠胚胎干细胞(ES细胞)分化为肝细胞的方法及肝损伤模型肝内移植的可行性.方法 常规培养ES细胞后,继续悬浮培养4 d以形成拟胚体(EBs),转移EBs到铺有明胶的6孔板中贴壁培养,并添加3 mmol/L丁酸钠开始诱导分化,7 d后加入淤胆血清筛选、纯化ES源性肝细胞,分化过程中用光学显微镜和电子显微镜观察细胞形态及超微结构的改变;用逆转录-聚合酶链反应(RT-PCR)方法检测肝细胞特异性标志基因:白蛋白(ALB)、甲胎蛋白(AFP)、甲状腺素运载蛋白(TTR)、α1抗胰蛋白酶(AAT)、葡萄糖6磷酸酶(G6P)、酪氨酸转氨酶(TAT)mRNA水平的表达;以荧光示踪剂CFDA-SE标记诱导获得的肝细胞,并移植到肝损伤小鼠肝内,观察移植细胞在肝内的定居、增殖情况.结果 诱导分化过程中,ES细胞形态逐渐出现肝细胞样改变,其超微结构与小鼠肝细胞超微结构十分相似;RT-PCR结果显示,随着诱导时间的推进,标志肝细胞发育过程的ALB、AFP、TTR、AAT、G6P、TAT mRNA顺序表达;肝内移植实验结果显示:ES源性肝细胞可在肝损伤小鼠肝内定居并增殖.结论 丁酸钠联合淤胆血清可以诱导ES细胞分化为肝细胞,ES源性肝细胞肝损伤模型体内移植是可行的,这有可能为细胞移植治疗难治性肝病提供一种新的细胞来源.     

Investigation of cholestasis-related changes in characteristics of spermatogonial stem cells in testis tissue of male Wistar rats

Paternal metabolic status is an important factor in the health status of offspring. Cholestasis, as a metabolic disorder, significantly disrupts spermatogenesis. Spermatogonial stem cells (SSCs) are considered the dividing germ cells, which maintain spermatogenesis throughout the lifespan. Here, we investigated the in vivo and in vitro effect(s) of cholestasis on SSCs. Cholestasis was induced in rats by bile duct ligation. Four weeks after the cholestasis induction, testicular tissues were analysed using histopathological examinations. The expression of SSC markers, including Plzf and Thy-1, was determined using the immunofluorescent technique. Also, SSCs were isolated from animals, and their proliferation was examined in vitro. The histological examinations revealed that cholestasis caused irregularities in the structure of seminal tubes. Immunostaining showed that the total number of Thy-1- and Plzf-expressing cells was declined in the cholestasis group compared with the control group. In vitro culture of SSCs indicated that the number of SSC colonies and those expressing Plzf were significantly reduced in the culture medium of the cholestasis group. Our results indicated that cholestasis affects the functionality of SSCs and reduces the number and proliferation of them. This finding may be of interest to the effect of metabolic diseases such as cholestasis on spermatogenesis.     

半成熟树突状细胞在胚胎干细胞源性肝细胞移植中诱导耐受的研究

目的 在小鼠胚胎干细胞(ESC)源性肝细胞移植中,应用半成熟树突状细胞(smDC)诱导免疫耐受.方法 绿色荧光蛋白(GFP)标记的129小鼠ESC按前期方法向肝细胞诱导分化,129小鼠smDC亦按前期方法由骨髓单核细胞诱导获得.smDC注入BALB/C小鼠3d后,再将ESC分化的肝细胞移植入BALB/C小鼠肝被膜下.检测肝转氨酶、外周血白细胞介素-2(IL)-2和IL-10水平变化,观察移植细胞生长及淋巴细胞浸润情况.结果 移植后7d,smDC组肝功能指标丙氨酸转氨酶(ALT)为212.58 U/L,较对照组512.27 U/L明显改善(P＜0.05),7d和14 d外周血IL-10水平较对照组明显升高,而IL-2水平较对照组明显降低(P＜0.05).移植细胞存活率在14、21 d smDC组较对照组明显增高,CD4 +T细胞浸润较对照组明显减轻.结论 在ESC源性肝细胞异基因移植中,smDC预输注可诱导部分免疫耐受,有望作为一种良好的移植耐受原应用于ESC分化细胞移植研究中.     

Isolation and enrichment of type A spermatogonia from pre‐pubertal buffalo ( Bubalus bubalis) testis

The aim of this study was to isolate and subsequently enrich type A spermatogonia from pre‐pubertal buffalo testis. Two‐step enzymatic digestion was used to isolate spermatogonia from 10 to 14 months pre‐pubertal buffalo calves, resulting in maximal release of spermatogonia from the seminiferous tubules. After enzymatic digestion, the type A spermatogonia were subsequently enriched by differential plating and Percoll gradient centrifugation. The identity of type A spermatogonia was determined by light microscopy and further characterised by Dolichos biflorus agglutinin, a specific marker for bovine type A spermatogonia by fluorescence‐activated cell sorting analysis. After enzymatic isolation, the cell suspension contained about 27% of type A spermatogonia, which was enriched up to 71% with >70% cell viability. Further flow cytometric analysis showed the presence of THY1+ cells (cells expressing thymocyte differentiation antigen 1), suggesting that THY1 is a conserved marker of the undifferentiated spermatogonial cells in buffalo. The isolation of the enriched type A spermatogonia from buffalo testis opens ways to study the further biochemical characteristics of this important class of germ cells in this species.     

Improved transplantation outcome through delivery of DNA encoding secretion signal peptide‐linked glucagon‐like peptide‐1 into mouse islets

Glucagon‐like peptide‐1 (GLP‐1) stimulates cell proliferation and has anti‐apoptotic effects on pancreatic islet β cells. In our previous study, the transduction of mouse islets with a recombinant adenovirus containing GLP‐1 cDNA enhanced islet graft survival. In this study, we sought to deliver the GLP‐1 gene using a nonviral vector, which raises fewer safety issues in clinical application. We constructed a plasmid, pβ‐SP‐GLP‐1, in which a secretion signal peptide (SP) was inserted to increase GLP‐1 secretion, and transfected mouse islets using the nonviral carrier Effectene. Transfection of pβ‐SP‐GLP‐1 induced a significant increase in bioactive GLP‐1 levels in islet cultures. Islets transfected with pβ‐SP‐GLP‐1 were protected from H₂O₂‐induced cell damage in vitro. In addition, glucose‐stimulated insulin secretion was significantly increased in pβ‐SP‐GLP‐1‐transfected islets. Diabetic syngeneic mice transplanted under the kidney capsule with a marginal mass of pβ‐SP‐GLP‐1‐transfected islets rapidly became normoglycemic, with 88% of recipients being normoglycemic at 30 days post‐transplantation compared with 52% of mice that received pβ‐transfected islet grafts (P < 0.05). Islet grafts retrieved 7 days after transplantation revealed that the pβ‐SP‐GLP‐1‐transfected group had significantly more Ki67‐positive cells as compared with the pβ‐transfected group. In conclusion, delivery of a plasmid containing a secretion SP and GLP‐1 cDNA using a nonviral carrier leads to efficient secretion of GLP‐1 in mouse islet cells, enhances islet cell survival during the early post‐transplant period, and improves islet transplantation outcome.     

Phosphorylated mixed lineage kinase domain‐like protein in human seminal plasma: A potential novel biomarker of spermatogenic function

Keeping the self‐renewal and differentiation of spermatogonial stem cell (SSC) in balance is essential for maintaining spermatogenesis. However, whether the cell death of SSC also plays a vital role in the human being remains unknown. To explore the necroptosis of SSC, the activation marker of necroptosis, phosphorylated mixed lineage kinase domain‐like protein (pMLKL) in testes was evaluated by immunofluorescence staining and Western blot. Meanwhile, a total of 81 semen samples were divided based on the sperm concentration (>15, 10–15, 5–10 and 0–5 million/ml) to study the relationships between pMLKL levels and sperm counts. We found that the pMLKL was increased in the ageing human testes (p < 0.05). Moreover, the seminal pMLKL expression was decreased in groups with sperm concentration 0–5 and 5–10 million/ml when compared with normal sperm concentration in young men (p < 0.05). Further analysis revealed that pMLKL showed an age‐related increased expression in men aged 22–60 years with normal sperm concentration. These data demonstrated that the necroptosis of SSC was important for the spermatogenic function and would raise in advance on the point of testicular hypofunction. In conclusion, the pMLKL may serve as a potential biologically seminal indicator for the spermatogenic function in men.     

非接触状态下小鼠胚胎干细胞在小鼠肝癌细胞H22诱导下分化的动态观察

目的 观察小鼠源性肿瘤细胞分泌的细胞因子对小鼠胚胎干细胞(ES cells)的诱导分化.方法 将 ES细胞形成的拟胚体分为诱导组和对照组.诱导组在加入小鼠肝癌细胞H22上清液制成的条件培养基中生长,对照组在去除白血病抑制因子(LIF)的ES细胞培养液中生长.于诱导分化的第7、9、12、15天在倒置显微镜下动态观察两组分化情况,并分别测量上清液中AFP的含量.结果 诱导分化的第7天观察到诱导组拟胚体细胞群落的中心和周边有较为典型的小鼠肝癌样细胞形成,第9、12、15天肝癌样细胞数量逐渐增加.对照组拟胚体向三胚层细胞分化,未见肝癌细胞形成.第9,12,15天诱导组和对照组AFP含量经方差分析,差异有统计学意义(P<0.05).结论 小鼠胚胎干细胞在小鼠肝癌细胞H22分泌的细胞因子的诱导下可以分化为肝癌细胞.     

Sox‐9 facilitates differentiation of adipose tissue‐derived stem cells into a chondrocyte‐like phenotype in vitro

The purpose of this study is to test whether ectopic expression of Sox‐9 can induce adipose tissue‐derived stem cells (ASCs) to function as real nucleus pulposus (NP) cells in vitro. Adenoviral vectors expressing Sox‐9 were reported to infect the chondroblastic and human disc cells, which resulted in increased Sox‐9 and type II collagen production. ASCs were isolated from rat inguinal adipose pad, characterized, and transduced in vitro with a retroviral vector encoding the Sox‐9 gene. Sox‐9‐engineered ASCs (ASCs/Sox‐9) were induced for the chondrocyte‐like cell differentiation by 3D cultured in alginate beads and TGF‐β3 for 2 weeks. Expression of exogenous Sox‐9 protein was detected. Type II collagen and Aggrecan gene expressions of induced ASCs/Sox‐9 were measured using real‐time PCR; proteoglycans expressions were measured by checking the glycosaminoglycan content and type II collagen production by enzyme‐linked immunosorbent assay. Isolated ASCs were CD 29⁺/CD44⁺/C‐Kit^?/Lin^?/CD34^?/CD45^?. ASCs/Sox‐9 expressed marked increase in exogenous Sox‐9 protein. After induction, type II collagen gene expression was sevenfold higher in mRNA levels, with an approximately twofold increase in protein levels of ASCs/Sox‐9 compared to ASCs. Type II collagen and proteoglycan productions were significantly increased in the ASCs/Sox‐9 compared to the ASCs. In addition, co‐culture of induced ASCs/Sox‐9 with matured NP cells resulted in enhanced increase in proteoglycan and type II collagen production. Constitutive retroviral expression of Sox‐9 could efficiently induce ASCs differentiation into chondrocyte‐like cells. This novel approach may provide a practicable system for a simple and rapid differentiation of ASCs into chondrocyte‐like cells which may be potentially used as a stem cell‐based therapeutic tool for the treatment of degenerative disc diseases. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1291–1297, 2011     

Culturing with modified EGM2 medium enhances porcine neonatal islet‐like cell clusters resistance to apoptosis in islet xenotransplantation

Background

Neonatal pig islet‐like cell clusters (NICC) are an attractive source of insulin‐producing tissue for potential transplantation treatment of type 1 diabetic patients. However, a considerable loss of NICC after their transplantation due to apoptosis resulted from islet isolation and instant blood‐mediated inflammatory reaction remains to be overcome.

Methods

EGM2 medium depleted with hydrocortisone and supplemented with 50 mmol/L isobutylmethylxanthine, 10 mmol/L nicotinamide, and 10 mmol/L glucose was used to culture NICC at day 1, the day after isolation and changed every other day. NICC cultured with EGM2 or control Ham's F‐10 medium were collected at day 7 of culture for the following assays. The viability of NICC was evaluated by AO/EB staining and FACS. Static assay and oxygen consumption rate analysis were performed to assess the function of NICC. Insulin and glucagon gene expression were measured by real‐time PCR. Tubing loops model and TUNEL assay were performed to confirm the apoptosis‐resistant ability of NICC cultured with modified EGM2 medium. Serum starvation and hypoxia treatment were used to test the tolerant capability of NICC in the microenvironment of hypoxia/nutrient deficiency in vitro. The molecules involved in apoptosis pathways in NICC were analyzed by Western blotting.

Results

Compared with Ham's F‐10 medium, culturing NICC with EGM2 medium led to increased number and viability of NICC with higher stimulation index, upregulated gene expression of both insulin and glucagon, and enhanced mitochondria function. Furthermore, fewer modified EGM2 medium cultured NICC were found under apoptosis when evaluated in an in vitro tubing loop model of IBMIR. Moreover, EGM2 medium cultured NICC demonstrated much less apoptotic cells under either serum starvation or hypoxia condition than their Ham's F‐10 medium cultured counterparts. The enhanced capability of EGM2 cultured NICC to resist apoptosis was associated with their elevated protein levels of anti‐apoptotic Bcl‐2 family member Mcl‐1.

Conclusion

Culturing NICC with EGM2 provides a simple and effective approach not only to increase NICC yield, viability, and maturation but also to enhance their resistance to apoptosis to preserve the initial graft mass for successful islet xenotransplantation.     

Development and characterization of novel clinical grade neonatal porcine bone marrow‐derived mesenchymal stem cells

Due to recent advances in research on mesenchymal stem cells (MSCs), MSCs are expected to be used in various clinical applications. However, securing adequate cadaveric donors and safety of living donors are major issues. To solve such issues, we have examined to develop clinical grade neonatal porcine bone marrow‐derived MSCs (npBM‐MSCs). Clinical grade neonatal porcine bone marrow cells were collected, frozen, and sent to our laboratory by air. The npBM‐MSCs were isolated from thawed bone marrow cells, then frozen. The thawed npBM‐MSCs were examined for CD markers and differentiated into chondrocytes, osteocytes, and adipocytes. They were compared with human bone marrow‐derived MSCs (hBM‐MSCs) for growth rate and size. To assess the robustness of proliferation, we compared culture medium with or without gelatin. The npBM‐MSCs expressed positive MSC markers CD29, CD44, and CD90 and were differentiated into chondrocytes, osteocytes, and adipocytes. The doubling time of npBM‐MSCs was significantly shorter than that of hBM‐MSCs (17.3 ± 0.8 vs 62.0 ± 19.6 hours, P < 0.01). The size of npBM‐MSCs was also significantly smaller than that of hBM‐MSCs (13.1 ± 0.3 vs 17.5 ± 0.4 μm, P < 0.001). The npBM‐MSCs showed similar proliferation characters irrespective of with or without gelatin coating. The npBM‐MSCs secreted VEGF‐A, VEGF‐C, and TGF‐β1. We have established npBM‐MSCs which show super‐rapid growth, small size, and robust proliferation profile. The np‐MSCs might be able to solve the donor issues for MSC therapy.     

Three‐dimensional high‐density co‐culture with primary tenocytes induces tenogenic differentiation in mesenchymal stem cells

Mesenchymal stem cells (MSCs) have potential applications in regenerative medicine and tissue engineering and may represent an attractive option for tendon repair and regeneration. Thus far the ability of MSCs to differentiate into tenocytes in vitro has not been investigated. Experiments were performed with and without growth factors (IGF‐1, TGF‐β1, IGF‐1/TGF‐β1, PDGF‐BB, and BMP‐12), in co‐cultures of tenocytes and MSCs mixed in different ratios and by culturing MSCs with spent media obtained from primary tenocytes. Tenogenesis was induced in MSCs through a combination of treatment with IGF‐1 and TGF‐β1, in high‐density co‐cultures and through cultivation with the spent media from primary tenocytes. Electron microscopy and immunoblotting were used to demonstrate up‐regulation of collagen I/III, decorin, tenomodulin, β1‐Integrin, MAPKinase pathway (Shc, Erk1/2), and scleraxis in the co‐cultures and provide simultaneous evidence for the inhibition of apoptosis. In monolayer co‐cultures extensive intercellular contacts between MSCs and tenocytes were observed. Cells actively exchanged vesicles, which were labeled by using immunofluorescence and immunogold techniques, suggesting the uptake and interchange of soluble factors produced by the MSCs and/or tenocytes. We conclude that MSCs possess tenogenic differentiation potential when provided with relevant stimuli and a suitable microenvironment. This approach may prove to be of practical benefit in future tissue engineering and tendon regenerative medicine research. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 1351–1360, 2011     

Differentiation of mesenchymal stem cells to germ‐like cells under induction of Sertoli cell‐conditioned medium and retinoic acid

The aim of this research was to find a way to differentiate germ cells from umbilical cord Wharton's jelly mesenchymal stem cells (MSCs) to support in vitro spermatogenesis. A small piece of Wharton's jelly was cultured in high‐glucose Dulbecco's modified Eagle's medium in present of 10% foetal calf serum. After the fourth passage, the cells were isolated and cultured in Sertoli cell‐conditioned medium under induction of two different doses of retinoic acid (10^?5, 10^?6 m ). The differentiation of MSC to germ‐like cells was evaluated by expression of Oct4, Nanog, Plzf, Stra8 and Prm1 genes during different days of culture through qPCR. The results showed that there were downregulation of Oct4 and Nanog and upregulation of pre‐meiotic germ cell marker (stra8) and haploid cell marker (Prm1) when MSCs are differentiated over time. The expression of Bax gene (an apoptotic marker) was significantly observed in high dosage of retinoic acid (RA). As a result, RA has positive effects on proliferation and differentiation of MSCs, but its effects are related to dosage. The success of this method can introduce umbilical cord MSC as a source of germ cells for treatment of infertility in future.     

Safety and efficacy of intramuscular human placenta‐derived mesenchymal stromal‐like cells (cenplacel [PDA‐002]) in patients who have a diabetic foot ulcer with peripheral arterial disease

The objective of this study was to examine the safety of cenplacel (PDA‐002) in patients with peripheral arterial disease (PAD) and a diabetic foot ulcer (DFU). Cenplacel is a mesenchymal‐like cell population derived from full‐term human placenta. This phase 1, dose‐escalation study investigated cenplacel in diabetic patients with chronic DFUs (Wagner grade 1 or grade 2) and PAD [ankle‐brachial index (ABI) >0·5 and ≤0·9], enrolled sequentially into each of four dose cohorts (3 × 10⁶, 10 × 10⁶, 30 × 10⁶ and 100 × 10⁶ cells; administered intramuscularly on study days 1 and 8 in combination with standard of care). Overall, cenplacel was well tolerated in all 15 patients in the study. Before enrollment, nine patients had an ulcer for ≥6 months and 11 had an ABI of 0·7–0·85. No patient met dose‐limiting toxicity criteria and no treatment‐related serious adverse events were reported. There was preliminary evidence of ulcer healing in seven patients (five complete; two partial) within 3 months of cenplacel treatment, and circulating endothelial cell levels (a biomarker of vascular injury in PAD) were decreased within 1 month. Cenplacel was generally safe and well tolerated in patients with chronic DFUs and PAD. Outcomes from this study informed the doses, endpoints, biomarkers and patient population for an ongoing phase 2 trial.     

In vitro culture of spermatogonial stem cells isolated from adult alpaca (Vicugna pacos) testes analysed with Dolichos biflorus by flow cytometry

Spermatogonial stem cell (SSC) is known for its self‐renewal capacity. We have studied the in vitro proliferation of isolated SSC from adult alpaca (Vicugna pacos) testes. A total of 107 samples were evaluated of which 31 were evaluated at baseline, 36 were cultivated in DMEM and 40 in STEMPRO media. Half of the cultivated samples was analysed after 14 days, and the rest after 21 days. Round cell subpopulations were identified with FITC‐DBA by flow cytometry: strongly positive DBA (sDBA+) as SSC, weakly positive DBA (wDBA+) as in early differentiation and negative DBA as differentiated. At the beginning, 4.16% of the cells were SSC, 37.61% wDBA+ while 54.12% were DBA?. After 14 days, 42.28% of SSC, 44.68% wDBA+ and 11.07% DBA? were found in DMEM while 47.09% of SSC, 32.57% wDBA+ and 18.48% DBA? in STEMPRO. After 21 days 38.66% were SSC, 52.78% wDBA and 7.65% DBA? in DMEM and on STEMPRO media 47.92% SSC, 44.20% wDBA+, 4.93% DBA?. There is a significant difference between the number of initial and SSC cultivated, as well as between DBA? (p < 0.05), while there is no significant difference between the wDBA+ (p > 0.05). Our results suggest that both culture media are appropriate for the in vitro proliferation of alpacas SSC.