Spontaneous Efficient Differentiation of Human Pluripotent Stem Cells (hPSC) Upon Co-culture of hPSCs with Human Neonatal Foreskin Fibroblasts in 3D.

Q4 Biochemistry, Genetics and Molecular Biology
Muhammad Nihad, Sudheer Shenoy P, Bipasha Bose
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Abstract

Pluripotent stem cells (PSCs) form well-formed embryoid bodies (EBs) in 3D culture. These EBs are formed in culture media lacking leukemia inhibitory factor (LIF) or basic fibroblast growth factor (bFGF) in mouse and human PSCs, respectively. EBs are excellent technical tools for understanding developmental biology and inducing controlled differentiation in succeeding experimental steps. Technically speaking, EBs are spontaneously differentiated PSCs in 3D and exhibit all three lineages in a time-point/sequential manner. For example, ectoderm will form first, followed by mesoderm and endoderm. We have attempted to co-culture human neonatal foreskin-derived fibroblast cells in our laboratory with the PSCs first in 2D conditions followed by the induction of EBs (PSC+fibroblasts co-cultured) in low attachment dishes. We also performed spontaneous differentiation of such EBs (co-cultured with fibroblasts). We checked the presence of markers of various lineages, namely, ectoderm, mesoderm, and endoderm in days 6, 10, and 12 day EBs. We have also compared the fibroblast co-cultured EBs, along with control EBs (derived from only PSCs). This co-culture system mimics the natural conditions of uterine implantation and the role of the endometrial fibroblasts in the induction of further embryonic development. The fibroblast co-cultured iPSC EBs had better roundness scores than the normal iPSC EBs and had a higher expression of lineage-specific markers.

人多能干细胞(hPSC)与人新生儿表皮成纤维细胞三维共培养后的自发高效分化。
多能干细胞(PSCs)在三维培养中会形成形态良好的类胚体(EBs)。小鼠和人类多能干细胞分别在缺乏白血病抑制因子(LIF)或碱性成纤维细胞生长因子(bFGF)的培养基中形成这些EB。EB 是了解发育生物学和在后续实验步骤中诱导受控分化的绝佳技术工具。从技术上讲,EB 是三维空间中自发分化的造血干细胞,以时间点/顺序的方式表现出所有三个系。例如,外胚层首先形成,然后是中胚层和内胚层。我们实验室曾尝试在二维条件下先将人新生儿包皮源性成纤维细胞与间充质干细胞共培养,然后在低附着力培养皿中诱导 EB(间充质干细胞+成纤维细胞共培养)。我们还对这种 EB(与成纤维细胞共培养)进行了自发分化。我们检查了第 6 天、第 10 天和第 12 天 EB 中各系(即外胚层、中胚层和内胚层)标记的存在情况。我们还比较了成纤维细胞共培养 EB 和对照 EB(仅来源于造血干细胞)。这种共培养系统模拟了子宫植入的自然条件以及子宫内膜成纤维细胞在诱导胚胎进一步发育中的作用。与正常的iPSC EB相比,成纤维细胞共培养的iPSC EB具有更好的圆度评分,并且有更高的系特异性标记表达。
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来源期刊
Methods in molecular biology
Methods in molecular biology Biochemistry, Genetics and Molecular Biology-Genetics
CiteScore
2.00
自引率
0.00%
发文量
3536
期刊介绍: For over 20 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice.
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