今年4月14日发表于Cell Stem Cell的一项研究表明，人类线粒体DNA突变会随着年龄增加而不断累积。该研究对于应用患者皮肤细胞来源的诱导多能干细胞（iPS）进行损伤组织或器官修复治疗具有重要启示。年老患者细胞来源的iPS细胞可能包含缺陷的线粒体DNA，从而削弱iPS细胞的治疗价值。

论文共同通讯作者Shoukhrat Mitalipov（俄勒冈健康与科学大学胚胎干细胞及基因治疗中心主任）和黄涛生教授（辛辛那提儿童医院线粒体医学中心主任及医学遗传学专家）提到，“因此，拟用于治疗用途的iPS细胞株，应进行线粒体DNA突变筛查与检测。”

黄涛生教授说，“人们往往只关注核基因组，但如果你想将iPS细胞应用于人类治疗，那么必须对线粒体DNA进行检测。”

线粒体DNA包含支持机体基本需求的关键基因，例如为细胞提供能量等。人类每个细胞具有两个拷贝的核DNA，但可能存在数千个拷贝的线粒体DNA，并且可能同时包含突变和正常的线粒体DNA。

黄涛生教授说，“我们称之为斑点效应，每个细胞都可以是不同的。两个相邻的细胞之间可能携带不同的突变或不同的突变比例（异质性水平）。”

这些突变在iPS细胞中可以被复制。在构建治疗用的iPS细胞株之前，一些患者细胞会被收集起来，并进行线粒体DNA突变检测。与新的下一代测序技术相比，传统的Sanger测序技术由于其灵敏度较差、不能检测出样本中比例低于20%的突变。但实际上，细胞中的线粒体DNA突变可以仅发生在少于20%的线粒体中。因此，以往对于iPS细胞中线粒体DNA突变率的问题并未被阐明。“这些线粒体DNA突变实际是被隐藏了” Mitalipov教授说。

不过，构建的iPS细胞株其实是来源于患者皮肤活检或血液样本的单个细胞。iPS细胞株的每个细胞均携带与初始成年患者细胞相同的线粒体DNA突变。因此，Mitalipov和黄教授从每个患者组织中获得了10个而仅非一个iPS细胞株克隆进行测序，以更好的探讨线粒体DNA突变率的情况。

他们从24岁到72岁的个体中分别采集了血液和皮肤标本进行检测。当直接检测血液或皮肤标本时，线粒体DNA的突变比例较低。但在iPS细胞株中，线粒体DNA突变的数目增加，特别是那些来源于60岁以上患者的细胞中。此外，细胞中线粒体突变的比例也更高。当突变的线粒体DNA比例越高，那么对细胞功能的损害也就越大。

由于每个iPS细胞株均来源于不同的细胞，每个细胞株可能包含不同类型的线粒体DNA突变以及突变负荷。作者建议，要对每个患者的多个细胞株进行筛选，并选择损伤最少的细胞株。“对iPS细胞克隆进行线粒体DNA突变检测是一个非常好的主意，可以确保你挑选了一个好的细胞株”，黄教授说。

“线粒体基因组相对较小，只有16.5 kb，包含37个基因，因此使用下一代测序进行筛查是可行的，这种方法相对便宜且易于实施“，Mitalipov教授最后说到。

黄涛生教授研究小组中，包括来自中国的王新建和罗仕玉两名学者。

Unchecked mitochondrial DNA mutations could be a problem for stem cell therapies

《Cell Press》

Mutations accumulate in human mitochondrial DNA with age, reports a study published April 14 in Cell Stem Cell. The finding has implications for potential therapies using induced pluripotent stem（iPS）cells, which are generated from patient skin cells and may be used to repair damaged tissue or organs. IPS cells derived from an elderly patient's cells could contain faulty mitochondrial DNA that could undermine the iPS cells' therapeutic value.

As a result, iPS cell lines intended for therapeutic use should be screened and checked for mitochondrial DNA mutations, say co-senior authors Shoukhrat Mitalipov, director of the Center for Embryonic Cell and Gene Therapy at Oregon Health & Science University, and Taosheng Huang, a medical geneticist and director of the Mitochondrial Medicine Program at Cincinnati Children's Hospital.

"People tend to look just at the nuclear genome," says Huang. "But if you want to use iPS cells in a human, you must check for mutations in the mitochondrial genome."

Mitochondrial DNA contains genes that support basic needs, such as energy production for the cell. While each cell stores two copies of nuclear DNA, there may be a thousand copies of the mitochondrial genome in a single cell. Those copies can contain both mutated and healthy mitochondrial DNA.

"We call it the freckled effect," says Huang. "Every single cell can be different. Two cells next to each other could have different mutations or different percentages of mutations."

These mutations can be replicated in iPS cells. Prior to the creation of a therapeutic iPS cell line, a collection of cells is taken from the patient. These cells will be tested for mutations. If the tester uses Sanger sequencing, older technology that is not as sensitive as newer next generation sequencing, any mutation that occurs in less than twenty percent of the sample will go undetected. But mitochondrial DNA mutations might occur in less than twenty percent of mitochondria in the pooled cells. As a result, mutation rates have not been well understood. "These mitochondrial mutations are actually hidden," says Mitalipov.

But in the process of making iPS cell lines, researchers expand clones of individual cells from a patient's biopsy. Every cell in the iPS cell line will contain the same mitochondrial DNA mutations as that initial adult cell. So rather than studying one iPS cell line, Mitalipov and Huang derived and sequenced ten iPS cell clones from each patient tissue sample to get a better understanding of mitochondrial DNA mutation rates.

They took samples of blood and skin from people ranging in age from 24 to 72. When they tested the samples for mitochondrial DNA mutations, the levels of mutations appeared low. But when they sequenced the iPS cell lines, they found higher numbers of mitochondrial DNA mutations, particularly in cells from patients over 60. They also found higher percentages of mitochondria containing mutations within a cell. The higher the load of mutated mitochondrial DNA in a cell, the more compromised the cell's function.

Since each iPS cell line is created from a different cell, each line may contain different types of mitochondrial DNA mutations and mutation loads. To choose the least damaged line, the authors recommend screening multiple lines per patient. "It's a good idea to check the iPS clones for mitochondrial DNA mutations and make sure you pick a good cell line," says Huang.

The mitochondrial genome is relatively small, containing just 37 genes, so screening should be feasible using next generation sequencing, says Mitalipov. "It should be relatively cheap and do-able."

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This work was supported by the Leducq Foundation, Oregon Health & Science University institutional funds and Mayo Clinic Center for Regenerative Medicine and the Cincinnati Children's Hospital Research Foundation.

Cell Stem Cell, Kang and Wang et al.："Age-related accumulation of somatic mitochondrial DNA mutations in adult-derived human iPSCs" http://dx.doi.org/10.1016/j.stem.2016.02.005