In vitro and In vivo analysis of gene expression changes in mouse neural progenitor cells derived from embryonic stem cells after exposure to specific molecules or grafting into traumatic spinal cord injury

Comparison of genomic data from neural progenitor cells derived from mouse embryonic stem cells under different experimetnal conditions in vitro and invivo. We conducted genome-wide RNA sequencing of immunoprecipitated specific ribosome-associated mRNA using RiboTag methods from: (i) mouse embryonic stem cell (ESC), (ii) derived neural progenitor cells, (iii) differentiated neural progenitor cells (in vitro), (iv) grafted neural progenitor cells (recovered from different in vivo tissue enivornments - healthy spinal cord, spinal cord injury lesions) and (v) host astrocytes using GFAp-Cre RiboTag mice. Overall design: Mouse ESC were derived from the inner cell mass of E3.5 blastocyst stage embryos generated from crosses of male homozygous B6N.129-Rpl22tm1.1 Psam/J “Ribotag” mice to females hemizygous for a dominant, maternal effect cre allele, B6.Cg-Tg(SOX2-cre)1Amc/J and heterozygous for the “RiboTag” allele. NPC were derived by neural induction and expansion protocols. Neural progenitors were differenetaitatd in vitro for 4 days by removal of progenitor maintaing mitogens (EGF/FGF) and treatment with either CNTF, IGF-1 or FBS in different concentrations. NPC mRNA was recoveved using the RiboTag protocol. NPC were grafted into young adult mice that underwent severe crush SCI at thoracic level T10. NPC grafting was performed at 2 days after injury. NPC mRNA was recoveved using the RiboTag protocol at 5 and 14 days after injury.

本数据集对比了体外（in vitro）与体内（in vivo）不同实验条件下，由小鼠胚胎干细胞（Embryonic Stem Cell, ESC）衍生的神经前体细胞（Neural Progenitor Cell, NPC）的基因组数据。我们采用RiboTag方法对免疫沉淀得到的特异性核糖体结合mRNA进行全基因组RNA测序，样本涵盖以下五类：(i) 小鼠胚胎干细胞；(ii) 原始衍生神经前体细胞；(iii) 体外分化神经前体细胞；(iv) 移植后回收的神经前体细胞（取自不同体内组织微环境：健康脊髓、脊髓损伤(Spinal Cord Injury, SCI)病灶）；以及(v) 借助GFAp-Cre RiboTag小鼠获取的宿主星形胶质细胞。
实验整体设计如下：小鼠胚胎干细胞源自纯合雄性B6N.129-Rpl22tm1.1Psam/J「RiboTag」小鼠，与携带显性母源效应cre等位基因的杂合雌性B6.Cg-Tg(SOX2-cre)1Amc/J、且为「RiboTag」等位基因杂合子的亲本交配所获得的E3.5囊胚期胚胎的内细胞团。神经前体细胞通过神经诱导与扩增方案制备获得。通过移除维持神经前体细胞增殖的有丝分裂原：表皮生长因子（Epidermal Growth Factor, EGF）与成纤维细胞生长因子（Fibroblast Growth Factor, FGF），并分别以不同浓度的睫状神经营养因子（Ciliary Neurotrophic Factor, CNTF）、胰岛素样生长因子1（Insulin-like Growth Factor 1, IGF-1）或胎牛血清（Fetal Bovine Serum, FBS）进行处理，将神经前体细胞在体外诱导分化4天。采用RiboTag方案回收该体外分化神经前体细胞的mRNA。将神经前体细胞移植至胸段T10水平遭受重度挤压性脊髓损伤的年轻成年小鼠体内，移植操作于损伤后2天进行。分别于损伤后5天和14天，采用RiboTag方案回收移植的神经前体细胞的mRNA。