Impact of source tissue and ex vivo expansion on the characterization of goat mesenchymal stem cells

Background

There is considerable interest in using goats as models for genetically engineering dairy animals and also for using stem cells as therapeutics for bone and cartilage repair. Mesenchymal stem cells (MSCs) have been isolated and characterized from various species, but are poorly characterized in goats.

Results

Goat MSCs isolated from bone marrow (BM-MSCs) and adipose tissue (ASCs) have the ability to undergo osteogenic, adipogenic and chondrogenic differentiation. Cytochemical staining and gene expression analysis show that ASCs have a greater capacity for adipogenic differentiation compared to BM-MSCs and fibroblasts. Different methods of inducing adipogenesis also affect the extent and profile of adipogenic differentiation in MSCs. Goat fibroblasts were not capable of osteogenesis, hence distinguishing them from the MSCs. Goat MSCs and fibroblasts express CD90, CD105, CD73 but not CD45, and exhibit cytoplasmic localization of OCT4 protein. Goat MSCs can be stably transfected by Nucleofection, but, as evidenced by colony-forming efficiency (CFE), yield significantly different levels of progenitor cells that are robust enough to proliferate into colonies of integrants following G418 selection. BM-MSCs expanded over increasing passages in vitro maintained karyotypic stability up to 20 passages in culture, exhibited an increase in adipogenic differentiation and CFE, but showed altered morphology and amenability to genetic modification by selection.

Conclusions

Our findings provide characterization information on goat MSCs, and show that there can be significant differences between MSCs isolated from different tissues and from within the same tissue. Fibroblasts do not exhibit trilineage differentiation potential at the same capacity as MSCs, making it a more reliable method for distinguishing MSCs from fibroblasts, compared to cell surface marker expression.

Electronic supplementary material

The online version of this article (doi:10.1186/2049-1891-6-1) contains supplementary material, which is available to authorized users.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Cell-free extract from porcine induced pluripotent stem cells can affect porcine somatic cell nuclear reprogramming

Pretreatment of somatic cells with undifferentiated cell extracts, such as embryonic stem cells and mammalian oocytes, is an attractive alternative method for reprogramming control. The properties of induced pluripotent stem cells (iPSCs) are similar to those of embryonic stem cells; however, no studies have reported somatic cell nuclear reprogramming using iPSC extracts. Therefore, this study aimed to evaluate the effects of porcine iPSC extracts treatment on porcine ear fibroblasts and early development of porcine cloned embryos produced from porcine ear skin fibroblasts pretreated with the porcine iPSC extracts. The Chariot^TM reagent system was used to deliver the iPSC extracts into cultured porcine ear skin fibroblasts. The iPSC extracts-treated cells (iPSC-treated cells) were cultured for 3 days and used for analyzing histone modification and somatic cell nuclear transfer. Compared to the results for nontreated cells, the trimethylation status of histone H3 lysine residue 9 (H3K9) in the iPSC-treated cells significantly decreased. The expression of Jmjd2b, the H3K9 trimethylation-specific demethylase gene, significantly increased in the iPSC-treated cells; conversely, the expression of the proapoptotic genes, Bax and p53, significantly decreased. When the iPSC-treated cells were transferred into enucleated porcine oocytes, no differences were observed in blastocyst development and total cell number in blastocysts compared with the results for control cells. However, H3K9 trimethylation of pronuclear-stage-cloned embryos significantly decreased in the iPSC-treated cells. Additionally, Bax and p53 gene expression in the blastocysts was significantly lower in iPSC-treated cells than in control cells. To our knowledge, this study is the first to show that an extracts of porcine iPSCs can affect histone modification and gene expression in porcine ear skin fibroblasts and cloned embryos.     

The Potential of Mesenchymal Stem Cell in Prion Research

Scrapie and bovine spongiform encephalopathy are fatal neurodegenerative diseases caused by the accumulation of a misfolded protein (PrP^res), the pathological form of the cellular prion protein (PrP^C). For the last decades, prion research has greatly progressed, but many questions need to be solved about prion replication mechanisms, cell toxicity, differences in genetic susceptibility, species barrier or the nature of prion strains. These studies can be developed in murine models of transmissible spongiform encephalopathies, although development of cell models for prion replication and sample titration could reduce economic and timing costs and also serve for basic research and treatment testing. Some murine cell lines can replicate scrapie strains previously adapted in mice and very few show the toxic effects of prion accumulation. Brain cell primary cultures can be more accurate models but are difficult to develop in naturally susceptible species like humans or domestic ruminants. Stem cells can be differentiated into neuron‐like cells and be infected by prions. However, the use of embryo stem cells causes ethical problems in humans. Mesenchymal stem cells (MSCs) can be isolated from many adult tissues, including bone marrow, adipose tissue or even peripheral blood. These cells differentiate into neuronal cells, express PrP^C and can be infected by prions in vitro. In addition, in the last years, these cells are being used to develop therapies for many diseases, including neurodegenerative diseases. We review here the use of cell models in prion research with a special interest in the potential use of MSCs.     

甘蓝型油菜DH群体抗倒伏相关性状遗传分析

为明确油菜抗倒伏性的遗传规律，本文利用抗倒伏性差异显著的2个甘蓝型油菜品系配制杂交组合，构建含280个株系的DH群体，采用作物茎秆强度抗倒测量仪等考种工具对该群体进行连续两年的抗倒伏性鉴定，并利用植物数量性状的主基因 + 多基因混合遗传模型及偏度和峰度分析对抗倒伏性进行遗传分析。结果表明，茎秆抗折力和茎秆抗折强度都受到0对主基因 + 9对微效多基因控制，茎秆抗折力和单株生物量的基因间无互作，茎秆抗折强度的基因间有互补作用，茎秆抗折力和茎秆抗折强度两年的平均遗传率在50%左右，而茎秆直径两年的平均遗传率为69.338 %，单株生物量两年的遗传率分别为52.198%和69.284%，遗传率都较低，性状受环境影响都较大，因此对于茎秆抗折力、茎秆抗折强度、茎秆直径和单株生物量，在育种选择的早期阶段都不宜太严格。同时茎秆抗折强度相比茎秆抗折力更能说明作物的抗倒伏能力，因而在抗倒伏选择育种时应更加关注。     

不同生育期水稻干物质量与产量的关系研究

为明确不同生育期的干物重对粤晶丝苗2号、粤农丝苗、合美占3个水稻(Oryza sativa L.)品种的产量及产量构成因素的影响,设置了早造和晚造两次不同氮肥梯度随机区组栽培试验。结果表明,叶片干物质的分配比例在拔节孕穗期时最高,且其随着生育进程逐渐降低,在乳熟期达到最低值;茎干物质的分配比例在乳熟期降至最低;齐穗期的穗干物质分配比例不到20%,而乳熟期的穗干物质量将近占总干物质量的43%;早造和晚造水稻的结实率差异极显著,总干重差异显著,而穗数、每穗粒数、千粒重、产量差异不显著;齐穗期以后,结实率和千粒重均与干物重呈负相关,而叶干重与穗数呈显著正相关,且相关性较大,茎干重与各产量性状密切相关,总干重与产量呈正相关。水稻干物重是决定高产的重要因素。     

基于茎秆直径微变化的拔节期玉米水分亏缺诊断指标研究

为了探索利用茎秆直径微变化诊断玉米水分亏缺状况的最优指标,采用桶栽的方法,开展了不同水分处理条件下拔节期夏玉米茎秆直径微变化中的日最大收缩量(MDS)、日增长量(DI)和当日恢复时间(RT)3个指标的变化规律及其与土壤相对含水率之间相关关系方面的试验研究。结果表明,(1)不同处理间的MDS和DI受水分亏缺影响差异较为明显,RT对土壤相对含水率的响应较为一致;(2)玉米日最大收缩量MDS和日增长量DI受到环境因素的影响较大,与土壤相对含水率θ的相关系数较小;(3)恢复时间RT与土壤相对含水率θ的相关系数最高,且受水分亏缺处理的影响较小,可以作为诊断玉米水分亏缺状况的较好指标。综上可知,与MDS和DI相比,RT更适合诊断夏玉米水分亏缺状况。     

玉米茎结构参量与产量关联及最优化分析

高效高产农业是可持续生产的重点,为研究玉米茎结构参量综合利用效率及最优利用率产量提高幅度,本研究使用2015年北京市小汤山镇获取的玉米数据,将玉米茎结构参量作为输入指标,生物量、产量和经济系数为产出指标,运用数据包络分析探讨利用效率及最优利用效率。结果表明:‘京华8号’玉米茎结构参量利用效率都保持在0.7以上,仅67%达到最优利用效率水平;小喇叭口期和抽雄期茎结构参量利用效率中上部茎粗最差,下部茎粗最优但仍有提高潜力;玉米茎结构参量综合利用效率最优效率时资源充分利用产量有不同程度的提高,其中抽雄期提高产量最大为32%。玉米茎结构参量利用效率尚未达到最优,提高其利用效率尤其抽雄期可显著提高产量。     

不同种植密度对夏玉米品种株型及产量性状的影响

为了阐述不同玉米品种株型及产量性状对密度响应，以目前生产上广泛种植的6个品种（‘郑单958’、‘先玉335’、‘伟科702’、‘浚单20’、‘金海5号’、‘利民33’）为材料，研究4个种植密度(60000、90000、112500、127500株/hm2)下不同品种株型和产量性状的变化。结果表明：6个品种棒三叶面积及茎粗系数均随着密度增加而减小，但减小幅度在不同品种或不同叶位存在差异。产量构成因素如穗长、穗粗、穗行数、行粒数、穗粒数和千粒重随着密度增加而减小。4个种植密度中，‘金海5号’、‘伟科702’、‘郑单958’在密度为90000株/hm2时产量为最高；其他3个品种随密度增加，产量降低。回归分析发现在10个已知影响玉米产量因素中，穗粒数对产量的直接影响最大，其次为行粒数。此结果为玉米品种选育和生产上高效栽培提供理论依据。