Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Molecular Biology Interdepartmental Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
Cell Rep. 2020 Dec 29;33(13):108562. doi: 10.1016/j.celrep.2020.108562.
Generating mammalian cells with desired mitochondrial DNA (mtDNA) sequences is enabling for studies of mitochondria, disease modeling, and potential regenerative therapies. MitoPunch, a high-throughput mitochondrial transfer device, produces cells with specific mtDNA-nuclear DNA (nDNA) combinations by transferring isolated mitochondria from mouse or human cells into primary or immortal mtDNA-deficient (ρ0) cells. Stable isolated mitochondrial recipient (SIMR) cells isolated in restrictive media permanently retain donor mtDNA and reacquire respiration. However, SIMR fibroblasts maintain a ρ0-like cell metabolome and transcriptome despite growth in restrictive media. We reprogrammed non-immortal SIMR fibroblasts into induced pluripotent stem cells (iPSCs) with subsequent differentiation into diverse functional cell types, including mesenchymal stem cells (MSCs), adipocytes, osteoblasts, and chondrocytes. Remarkably, after reprogramming and differentiation, SIMR fibroblasts molecularly and phenotypically resemble unmanipulated control fibroblasts carried through the same protocol. Thus, our MitoPunch "pipeline" enables the production of SIMR cells with unique mtDNA-nDNA combinations for additional studies and applications in multiple cell types.
生成具有所需线粒体 DNA (mtDNA) 序列的哺乳动物细胞，可用于研究线粒体、疾病建模和潜在的再生疗法。MitoPunch 是一种高通量线粒体转移设备，通过将来自小鼠或人类细胞的分离线粒体转移到原代或永生 mtDNA 缺陷 (ρ0) 细胞中，产生具有特定 mtDNA-核 DNA (nDNA) 组合的细胞。在限制培养基中分离的稳定分离的线粒体受体 (SIMR) 细胞永久性保留供体 mtDNA 并重新获得呼吸作用。然而，尽管在限制培养基中生长，SIMR 成纤维细胞仍保持 ρ0 样细胞的代谢组和转录组。我们将非永生的 SIMR 成纤维细胞重编程为诱导多能干细胞 (iPSC)，随后分化为多种功能细胞类型，包括间充质干细胞 (MSC)、脂肪细胞、成骨细胞和软骨细胞。值得注意的是，在重编程和分化后，SIMR 成纤维细胞在分子和表型上都类似于通过相同方案进行的未处理的对照成纤维细胞。因此，我们的 MitoPunch“流水线”可用于生成具有独特 mtDNA-nDNA 组合的 SIMR 细胞，以用于多种细胞类型的进一步研究和应用。
