Epigenetic reprogramming to generate novel chondro-osseous stem cells for bone tissue engineering

最近的研究利用BMP 2诱导组织和/或骨形成的能力， 肢体再生导致了一种新的软骨-骨祖细胞的鉴定， 进入活跃骨形成的部位，即使是全身递送。这些细胞是用谱系 追踪胶质细胞高亲和力谷氨酸转运蛋白GLAST，它已被证明仅限于神经 干细胞、星形胶质细胞和软骨细胞。使用单细胞RNAseq的转录组分析导致了 一种高度复制的干细胞的鉴定，该干细胞似乎经历了上皮细胞向间充质细胞的转化 (EMT)成为表达大量软骨细胞和成骨细胞相关转录物的细胞 类似于最近报道的骨膜干细胞。令人惊讶的是，当复制干细胞表达 GLAST以及另一种突触蛋白神经颗粒蛋白和β-微管蛋白3/TUJ 1;而成熟的软骨细胞 并不表达这种支持其神经联系的蛋白质。在进一步支持几个或所有集群中，还 表达几种雪旺细胞标记物，包括N-钙粘蛋白，Krox 20和髓鞘碱性蛋白。 转录组分析还显示该簇高表达H2A.Z和SET甲基转移酶 与假时间或细胞成熟相关的其它簇/细胞中的合成减少。数据 共同导致我们假设雪旺细胞在暴露于BMP 2后经历了表观遗传， 导致神经嵴的引发的组织特异性发育增强子的重编程和再活化 祖细胞/干细胞表型。此外，这些干细胞/祖细胞然后能够经历再分化为 软骨细胞和成骨细胞。为了验证这一假设，我们建议利用磷脂蛋白1（Plp 1） 启动子驱动Cre重组酶和番茄红细胞STOPflox小鼠能够初始标记雪旺细胞 并允许报告基因在下游重编程细胞中表达。48小时获得的GLAST-TR+细胞 在交付BMP 2后，也将包括以获得潜在的早期群集。GLAST-TR+和/或Plp 1-TR+ - 来自BMP 2递送后24小时从头骨形成区域周围的软组织的细胞， 进行单细胞RNAseq和单细胞ATACseq，这将鉴定细胞内染色质的变化。 这些细胞群可能反映了细胞向高度复制性干细胞的重编程。所得 数据将与scRNAseq数据相关联，以提供这些基因的产生的分子机制。 细胞从这些数据中，人们可以开始设想细胞的体外重编程以形成这些软骨， 骨祖细胞，它们将回到活跃的骨形成部位。

Recent studies to harness the capacity of BMP2 to induce de novo bone formation for tissue and/or limb regeneration resulted in the identification of a novel chondro-osseous progenitor that is primed to engraft into sites of active bone formation, even when delivered systemically. These cells were identified using lineage tracing for glial high affinity glutamate transporter GLAST, which has been shown to be restricted to neural stem cells, astrocytes, and chondrocytes. Transcriptome analysis, using single cell RNAseq, led to the identification of a highly replicating stem cell that appears to undergo an epithelial to mesenchymal transition (EMT) to become a cell that expresses a large number of chondrocyte and osteoblast associated transcripts similar to a recently reported periosteal stem cell. Surprisingly, while the replicating stem cell expressed GLAST as well as neurogranin, another synaptic protein, and β-tubulin 3/TUJ1; whereas mature chondrocytes did not express this protein supporting its neural association. In further support several or all clusters also expressed several Schwann cell markers including N-cadherin, Krox20, and myelin basic protein. Transcriptome analysis also revealed that this cluster is highly expressing H2A.Z and SET methyltransferase with decreasing synthesis in other clusters/cells correlating with pseudotime or cell maturation. The data collectively has led us to hypothesize that Schwann cells upon exposure to BMP2 undergo epigenetic reprogramming and reactivation of primed tissue specific developmental enhancers leading to a neural crest progenitor/stem cell phenotype. Further, these stem/progenitors are then able to undergo re-differentiation into chondrocytes and osteoblasts. To test this hypothesis, we propose to utilize the phospholipid protein 1 (Plp1) promoter driving Cre recombinase and tomato redfloxSTOPflox mouse to be able to initially label Schwann cells and allow for reporter expression in downstream reprogrammed cells. GLAST-TR+ cells obtained 48 hours after delivery of BMP2, will also be included to obtain potential earlier clusters. GLAST-TR+ and/or Plp1-TR+ cells from soft tissues surrounding the region of de novo bone formation 24 hours after delivery of BMP2 and subjected to single cell RNAseq and single cell ATACseq, which will identify changes in chromatin within the cell populations that might reflect reprogramming of the cells to the highly replicative stem cell. The resultant data will be correlated with the scRNAseq data to provide a molecular mechanism for the generation of these cells. From this data one can then start to envision ex vivo reprogramming of cells to form these chondro- osseous progenitors that will home to sites of active bone formation.