支持细胞对体外培养精原干细胞的作用途径研究

目的:探讨支持细胞对体外培养精原干细胞的作用途径。方法:选用7 d龄雄性昆明种小鼠,两步酶消化法获得睾丸组织细胞悬液,差异时间贴壁法分离精原干细胞和支持细胞,免疫荧光法和油红O染色法分别对其进行生物学鉴定,流式细胞仪对精原干细胞进行纯度分析。按培养条件的不同将实验分为3组:精原干细胞与支持细胞共培养组(A组)、条件培养基组(B组)、常规培养基组(C组)。其中条件培养基按单纯支持细胞培养清液∶双倍浓缩的DMEM/F12∶胎牛血清=4.5∶4.5∶1的比列配置;常规培养基即含体积分数10%胎牛血清的DMEM/F12。台盼蓝法测定各组贴壁率,四甲基偶氮唑盐(MTT)法测定各组精原干细胞的吸光度并绘制增殖曲线。倒置显微镜下观察各组精原干细胞增殖特点及集落形成情况。比较各组精原干细胞24 h贴壁率、增殖曲线及存活时间的不同。结果:A组精原干细胞24 h贴壁率大于B组及C组(P<0.05),而B、C组间无差异(P>0.05);A组精原干细胞接种起即稳定增殖,于7~10 d形成稳定集落并维持约30 d的特性。B组和C组均表现为精原干细胞经短暂的增殖后呈现快速减少的趋势,培养1周后,精原干细胞数目明显减少。结论:支持细胞对体外精原干细胞的作用是依靠两者之间的直接联系和支持细胞的旁分泌2种途径;而仅依靠支持细胞的旁分泌作用不能促进精原干细胞的贴壁和增殖。     

Transwell细胞龛室环境下绵羊肌源性干细胞与阴道平滑肌细胞体外共培养的研究

目的:探讨绵羊肌源性干细胞(MDSC)与阴道平滑肌细胞(VSMC)共培养分化为平滑肌的可行性。方法:利用Transwell细胞龛室建立MDSC/VSMC共培养体外模型,共培养2d、4d、6d,收集MDSC。Western blot法检测干细胞标记物Desmin及平滑肌细胞标记物a-SMA的表达情况。结果:利用Transwell细胞龛室成功构建MDSC/VSMC共培养体外模型。免疫荧光法及Western blot法检测发现,随着共培养时间延长,共培养组MDSC干细胞标记物Desmin表达逐渐下降,但培养6d仍有表达,平滑肌细胞标记物aSMA逐渐增多。结论:Transwell细胞龛室成功构建绵羊MDSC/VSMC共培养体外模型,达到了真正意义上分开两种细胞的同时,兼顾了细胞间相互影响的共培养环境,在体外证实了绵羊MDSC分化为平滑肌细胞的可行性。     

短期糖尿病大鼠睾丸的细胞学变化

目的:探讨糖尿病发病初期大鼠发生性与生殖功能障碍的细胞学机制。方法:通过给成龄雄性大鼠腹腔注射链脲菌素制成糖尿病动物模型,分别观察1d、3d、5d、7d、14d。观察期满后断头处死,取睾丸。应用常规组织学方法观察睾丸组织形态的改变;应用流式细胞分析术检测睾丸间质细胞、支持细胞及各级生精细胞的细胞周期的改变。结果:睾丸间质细胞及生精细胞的形态、数目从致病d7开始有明显变化,支持细胞的形态无明显变化;睾丸中各类细胞在高糖环境下24h即发生增殖减慢、细胞滞留在G1期、DNA合成减少等改变。结论:糖尿病大鼠生殖功能障碍与睾丸间质细胞、生精细胞的形态、数目变化及睾丸各类细胞的增殖改变有关。     

体外着床模型中人孵化后早胚细胞角蛋白和肌动蛋白的表达

目的:建立理想的体外胚胎着床模型,并检测模型中人孵化后早胚细胞角蛋白、肌动蛋白和hCG。方法:孵化后早胚与人子宫内膜蜕膜化的基质细胞共培养,观察胚泡在基质细胞层上的定位、黏附、铺展和侵入过程;用免疫荧光染色技术,测定共培养系统中的细胞角蛋白和肌动蛋白;用免疫荧光分析技术,测定培养液中的hCG水平。结果:胚泡和基质细胞共培养5h起,胚泡开黏附在基质细胞层上,最终侵入蜕膜化的基质细胞间。共培养48h后,细胞角蛋白仅仅在滋养层细胞中表达;肌动蛋白在人蜕膜化的基质细胞和滋养层细胞中均有表达。囊胚与子宫内膜基质细胞共培养的培养液中的hCG水平明显高于囊胚单独培养的(P<0.01)。结论:成功建立了一个能反映人胚泡黏附、铺展及侵入到人子宫基质细胞的体外着床模型,细胞角蛋白、肌动蛋白和hCG在着床早胚细胞中起相应变化。     

胶质细胞源性神经营养因子在小鼠生精恢复过程中的表达和意义

目的:分析并探讨胶质细胞源性神经营养因子(GDNF)在小鼠恢复生精过程中的表达变化,及其与精原干细胞增殖与分化的关系。方法:间隔24 d 2次腹腔注射白消安建立小鼠生精恢复过程的动物模型。根据第二次给药后的不同时间随机分为1、2、3、4、6、8、10周共7组(8只/组),8只正常小鼠作对照组,分别于相应时点取材,进行光镜和电镜的研究。采用半定量逆转录聚合酶链反应(RT-PCR)检测不同时间GDNF mRNA的表达变化;并采用原位杂交技术检测GDNFmRNA表达定位。结果:2次给药后第1-2周,GDNF mRNA的表达明显增强,在第2周达到高峰;在给药后第3-4周明显下降,并在第4周达到低谷;随后几周内表达又逐渐增强,于第10周恢复到正常水平。原位杂交显示,GDNF由sertoli细胞表达。结论:在小鼠生精恢复过程中,GDNF的高水平表达促进精原干细胞自我更新,对于维持生精上皮干细胞数量的稳定具有十分重要的意义。     

精原干细胞及其研究进展

精原干细胞既具有自我更新能力又能分化成精子,是雄性动物中唯一能将遗传信息传递给后代的成体干细胞。它起源于早期胚胎的原始生殖细胞,表达一些相对特异的分子标记,其增殖和分化过程受到许多基因(GDNF、Plzf和c-Kit等)的精确调控。而且最近发现精原干细胞具有很强的可塑性,能在体外诱导为胚胎干细胞样的多能干细胞。精原干细胞研究不仅能促进男性不育治疗的发展,而且在人类再生医学领域具有巨大的应用价值。     

姜黄素、佛波酯对人卵巢癌细胞株SKOV3细胞生长的影响

目的:通过研究抑癌剂姜黄素(CCM)、促癌剂佛波酯(TPA)对体外培养的人卵巢癌细胞株SKOV3细胞增殖、细胞形态及细胞周期的影响,探讨肿瘤细胞SKOV3增殖抑制的效应机制.方法:采用MTT法测定CCM、TPA不同浓度及不同时间对人卵巢癌细胞株SKOV3增殖的抑制作用,计算细胞生长抑制率.光镜和电镜下观测细胞形态学及超微结构的改变.流式细胞仪检测细胞周期.结果:TPA具有抑制人卵巢癌细胞株SKOV3细胞增殖的作用,呈剂量和时间依赖性,细胞生长停滞于G1/S期.部分细胞出现凋亡形态学改变.TPA抑制人卵巢癌细胞株SKOV3细胞的增殖,呈剂量和时间依赖性.细胞周期发生G1→S期阻滞.结论:CCM、TPA通过抑制细胞增殖,细胞周期发生停滞,显著地抑制卵巢癌细胞SKOV3的体外生长.     

精子发生的转录组学和蛋白组学

<正>1精原干细胞研究的新技术成人睾丸中的精原干细胞(spermatogonial stemcells,SSCs)来源于原始生殖细胞,SSCs是典型的多能干细胞,具有经过一系列有丝分裂分化成精原细胞的能力,并且可通过细胞的自我更新和凋亡来维持细胞数量的稳定[1,2]。随着分子生物学的发展,这些     

小鼠囊胚在子宫内膜体外共培养模型上着床的超微结构观察

目的:进一步证明已建立的体外着床模型的可行性。方法:将妊娠d4的小鼠囊胚培养在已建立的体外子宫内膜共培养模型上,电镜观察胚胎植入时囊胚与子宫内膜上皮细胞相互关系的超微结构。结果:小鼠囊胚能正常脱透明带、黏附和扩展。扫描电镜见黏附在子宫内膜上的囊胚呈椭圆形或扁平形;胚胎表面具微绒毛;在胚胎与子宫内膜细胞接触点可见胚胎滋养细胞黏附于胞饮突上。透射电镜观察显示,胚胎滋养细胞通过微绒毛黏附于上皮细胞上,有些滋养细胞通过微绒毛开始植入,局部滋养细胞与上皮细胞形成紧密连接。胚胎滋养细胞与上皮细胞间形成许多镶嵌连接。超微结构显示粗面内质网、线粒体、溶酶体和核糖体非常丰富。结论:小鼠囊胚在子宫内膜体外模型上生长发育良好,该胚胎与子宫内膜共培养系统是研究胚胎着床机理的理想体外模型。     

体外培养方法诱导大鼠精母细胞减数分裂

目的:探讨大鼠睾丸精母细胞在体外减数分裂、分化为精子细胞或精子的可能性。方法:取出生后20-22dWistar大鼠睾丸组织,经胶原酶消化分离细胞,分别用含2%胎牛血清(FBS)的DMEM/F12培养基(对照组)和含2%FBS的DMEM/F12培养基+多种性激素+多种细胞生长因子等(实验组)作体外培养,于培养后d3、d7、d14收集细胞,采用流式细胞技术,检测培养前后生精细胞染色体倍体的变化;RT-PCR法检测精子细胞特异性基因过渡蛋白1(TP1)mRNA的表达。结果:培养d2,可见支持细胞贴壁生长,生精细胞粘附于支持细胞表面,实验组生精细胞存活率>90%,对照组<5%;培养14d,实验组可见长有鞭毛的长形精子细胞。流式细胞检测结果显示,培养前生精细胞为二倍体和四倍体细胞群,培养d14实验组单倍体细胞占总细胞数的6.2%,对照组未见单倍体峰。RT-PCR结果显示与流式细胞技术检测结果一致:培养前生精细胞未检测到TP1mRNA的表达,培养7d后实验组可检测到该基因的表达,培养14d,其表达量显著增加。结论:采用生精细胞与支持细胞混合培养并在培养液中添加性激素、细胞生长因子等的方法,大鼠精母细胞可以在体外进行减数分裂,分化成长形精子细胞。     

In vitro culture and characterization of spermatogonial stem cells on Sertoli cell feeder layer in goat (Capra hircus)

Purpose

To develop an efficient protocol for isolation, purification and long-term culture of spermatogonial stem cell (SSC) in goat.

Methods

The isolation of SSC was performed by testicular disaggregation by enzymatic digestion using collagenase IV, trypsin and DNase I. Further SSCs were enriched using Percoll density gradient centrifugation. The purity of SSCs was assessed by immunocytochemistry (ICC) using α6 integrin. The SSCs were co-cultured on Sertoli cell feeder layer. The SSC colonies were characterized by studying the expression of SSC specific markers (viz., α6 integrin and PLZF) using ICC. The abundance of mRNAs encoding the markers of SSC (viz., β1 integrin and Oct-4) and Sertoli cells (viz., vimentin) was also assayed using quantitative real-time PCR (qPCR).

Results

The viability of isolated testicular cells was > 90 % and the Percoll density gradient method resulted in 3.65 folds enrichment with a purity of 82.5 %. Co-culturing of SSCs with Sertoli cell feeder layer allowed the maintenance of stable SSC colonies even after one and half months of culture. The results of ICC analysis showed the expression of α6 integrin and PLZF in almost all the SSC colonies. qPCR analysis revealed a differential expression of mRNAs encoding β1 integrin, Oct-4 and vimentin markers.

Conclusion

Results of this study demonstrate a simple enzymatic digestion and Percoll density gradient method for isolation and enrichment of SSCs, and suitability of Sertoli cell feeder layer for long term in vitro culture of SSC in goats. Results also suggest the possible application of non-caprine antibodies against SSC specific markers for the identification and subsequent assessment of SSCs in goats.     

Recent advances of in vitro culture systems for spermatogonial stem cells in mammals

Background

Spermatogonial stem cells (SSCs) in the mammalian testis are unipotent stem cells for spermatozoa. They show unique cell characteristics as stem cells and germ cells after being isolated from the testis and cultured in vitro. This review introduces recent progress in the development of culture systems for the establishment of SSC lines in mammalian species, including humans.

Methods

Based on the published reports, the isolation and purification of SSCs, identification and characteristics of SSCs, and culture system for mice, humans, and domestic animals have been summarized.

Results

In mice, cell lines from SSCs are established and can be reprogrammed to show pluripotent stem cell potency that is similar to embryonic stem cells. However, it is difficult to establish cell lines for animals other than mice because of the dearth of understanding about species‐specific requirements for growth factors and mechanisms supporting the self‐renewal of cultured SSCs. Among the factors that are associated with the development of culture systems, the enrichment of SSCs that are isolated from the testis and the combination of growth factors are essential.

Conclusion

Providing an example of SSC culture in cattle, a rational consideration was made about how it can be possible to establish cell lines from neonatal and immature testes.     

Development of a xeno-free non-contact co- culture system for derivation and maintenance of embryonic stem cells using a novel human endometrial cell line

Purpose

Mouse embryonic fibroblast feeder layers (MEF) have conventionally been used to culture and maintain the pluripotency of embryonic stem cells (ESC). This study explores the potential of using a novel human endometrial cell line to develop a non-xeno, non-contact co-culture system for ESC propagation and derivation. Such xeno-free systems may prove essential for the establishment of clinical grade human ESC lines suitable for therapeutic application.

Methods

A novel line of human endometrial cells were seeded in a 6-well dish. Filter inserts containing mouse ESCs were placed on these wells and passaged 2–3 times per week. Inner cell masses derived from mouse blastocysts were also cultured on transwells in the presence of the feeder layer. In both cases, staining for SSEA-1, SOX-2, OCT-4 and alkaline phosphatase were used to monitor the retention of stem cells.

Results

ESC colonies retained their stem cell morphology and attributes for over 120 days in culture and 44 passages to date. Inner cell mass derived ESC cultures were maintained in a pluripotent state for 45 days, through 6 passages with retention of all stem cell characteristics. The stem cell colonies expressed stem cell specific markers SSEA-1, Sox 2, Oct-4 and alkaline phosphatase. Upon removal of the human feeder layer, there was a distinct change in cell morphology within the colonies and evidence of ESC differentiation.

Conclusions

Human feeder layers offer a simple path away from the use of MEF feeder cells or MEF conditioned medium for ESC culture. Furthermore, indirect co-culture using porous membranes to separate the two cell types can prevent contamination of stem cell preparations with feeder cells during passaging.     

In vitro culture and morphological characterization of prepubertal buffalo (Bubalus bubalis) putative spermatogonial stem cell

Purpose

Spermatogonial stem cells (SSCs) have the unique ability both to self-renew and to produce progeny that undergo differentiation to spermatozoa. The present study has been carried out to develop a method to purify and enrich the pure populations of spermatogonial stem cell like cells in buffalo.

Methods

The spermatogonial cells were isolated from testes of 3–7 month old buffalo calves and disaggregated by double enzymatic digestion. Mixed population of isolated cells were then plated on Datura stramonium agglutinin (DSA) lectin coated dishes for attachment of Sertoli cells. The desired cells were obtained from suspension medium after 18 h of incubation and then loaded on discontinuous density gradient using percoll (20–65 %) and different types of spermatogonia cells were obtained at interface of each layer. These cells were cultured in vitro.

Results

Spermatogonial cells isolated have spherical outline and two or three eccentrically placed nucleoli, created a colony after proliferation during first week or immediately after passage. After 7–10 days of culture, the resulted developed colonies of spermatogonial cells expressed the spermatogonial specific genes like Plzf and VASA; and other pluripotency related markers viz. alkaline phosphtase, DBA, CD9, CD90, SSEA-1, OCT-4, NANOG and REX-1.

Conclusion

Our results show that the isolated putative spermatogonial stem cells exhibit the expression of pluripotency related and spermatogonial specific genes. This study may help to establish a long term culture system for buffalo spermatogonia.     

Co-culture of mouse spermatogonial stem cells with sertoli cell as a feeder layer,stimulates the proliferation and spermatogonial stemness profile

Objectives

Sertoli cells effect the fate map of spermatogonial stem cells (SSCs) to self-renew via providing the special microenvironments. Maintenance of proliferation and self-renewal activity of SSCs may be usable as a therapeutic strategy, leads to increase the recovery of male fertility. This research was aimed to evaluate the effect of mouse sertoli cells on spermatogonia stem cells proliferation and the expression pattern of stemness markers.

β-estradiol promotes the growth of primary human fetal spermatogonial stem cells via the induction of stem cell factor in Sertoli cells

BackgroundMammalian spermatogenesis is responsible for male fertility and is supported by the self-renewal and differentiation of spermatogonial stem cells (SSCs). Sertoli cells provide a supportive microenvironment for SSCs, in part by the production of stem cell factor (SCF), which is a potent regulator of spermatogonia proliferation and survival.MethodsWe investigated the novel role of β-estradiol in modulating the proliferation and apoptosis of fetal SSCs via the regulation of SCF secretion in Sertoli cells isolated from human fetal testes. The proliferation of SSCs in the co-culture system was determined by colony formation and BrdU incorporation assays. TUNEL assay was used to measure SSC apoptosis in co-culture in response to treatment with control, β-estradiol, or the combination of β-estradiol and the estrogen receptor inhibitor ICI 182780.ResultsIn the system with purified human fetal Sertoli cells (MIS+/c-Kit−/AP−), β-estradiol upregulated the production of SCF in a dose- and time-dependent manner. In the co-culture system of primary human fetal SSCs (c-Kit+/SSEA-4+/Oct-4+/AP+) and Sertoli cells (MIS+), β-estradiol markedly increased the proliferation of SSCs. Moreover, SSC apoptosis was significantly inhibited by β-estradiol and was completely reversed by the combination of β-estradiol and ICI 182780.ConclusionHere we report, for the first time, that β-estradiol can induce the increase of SCF expression in human fetal Sertoli cells and regulates the growth and survival of human fetal SSCs. These novel findings provide new perspectives on the current understanding of the role of estrogen in human spermatogenesis.Supplementary InformationThe online version contains supplementary material available at 10.1007/s10815-021-02240-y.     

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers

Introduction

Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting.

Purpose

The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture.

Methods

One month old goats’ testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated.

Results

The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively.

Conclusions

The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.