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

表观遗传重编程产生用于骨组织工程的新型软骨骨干细胞

基本信息

批准号：
10215392
负责人：
Alan ROBERT Davis
金额：
$ 17.07万
依托单位：
BAYLOR COLLEGE OF MEDICINE
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-15 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10215392
关键词：
ATAC-seq Active Sites Adult Affinity Algorithms Astrocytes Automobile Driving BMP2 gene Bone Diseases Bone Regeneration Bone Tissue Cartilage Cell Maturation Cell physiology Cells Cellular Assay Chondrocytes Chromatin Cluster Analysis CpG dinucleotide DNA Data Data Set Development EGR2 gene Embryo Engineering Enhancers Enterobacteria phage P1 Cre recombinase Epigenetic Process Erythrocytes Exposure to GLAST Protein Generations Genes Genetic Transcription Genomics Glutamate Transporter Grant Heterotopic Ossification Histones Home Hour Label Lead Link Literature Malignant Neoplasms Methyltransferase Modeling Molecular Mus Myelin Basic Proteins N-Cadherin Natural regeneration Nerve Neural Crest Nucleosomes Ontology Osteoblasts Osteogenesis PMP22 gene Pathway interactions Peripheral Nerves Phenotype Phospholipids Physiologic pulse Polycomb Population Process Proteins Red nucleus structure Reporter Reporting Repression Repressor Proteins Role Schwann Cells Site Synapses Tamoxifen Technology Testing Tissue Engineering Tissues Tomatoes Transcript Transposase Variant aortic valve disorder beta Tubulin blastomere structure bone calcification cancer stem cell cell type chromatin remodeling embryo tissue epigenetic memory epigenetic regulation epithelial to mesenchymal transition experimental study fetal reactivity genomic platform histone methylation in vivo in vivo evaluation limb regeneration nerve stem cell neurogranin next generation sequencing novel progenitor promoter relating to nervous system repair model repaired single-cell RNA sequencing soft tissue stem stem cells stem-like cell tissue regeneration transcription factor transcriptome

项目摘要

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.

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甲基转移酶在与假时间或细胞成熟相关的其它簇/细胞中合成减少。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。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

MAGI1 inhibits interferon signaling to promote influenza A infection.

DOI：
10.3389/fcvm.2022.791143
发表时间：
2022
期刊：
Frontiers in cardiovascular medicine
影响因子：
3.6
作者：
通讯作者：

A Population of M2 Macrophages Associated With Bone Formation.

DOI：
10.3389/fimmu.2021.686769
发表时间：
2021
期刊：
Frontiers in immunology
影响因子：
7.3
作者：
Olmsted-Davis E;Mejia J;Salisbury E;Gugala Z;Davis AR
通讯作者：
Davis AR

Free Cholesterol Bioavailability and Atherosclerosis.

DOI：
10.1007/s11883-022-01011-z
发表时间：
2022-05
期刊：
CURRENT ATHEROSCLEROSIS REPORTS
影响因子：
5.8
作者：
Abe, Rei J.;Abe, Jun-ichi;Nguyen, Minh T. H.;Olmsted-Davis, Elizabeth A.;Mamun, Abrar;Banerjee, Priyanka;Cooke, John P.;Fang, Longhou;Pownall, Henry;Le, Nhat-Tu
通讯作者：
Le, Nhat-Tu

A replicating stem-like cell that contributes to bone morphogenetic protein 2-induced heterotopic bone formation.