Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin
Derivation of new human embryonic stem cell lines reveals rapid epigenetic progression in vitro that can be prevented by chemical modification of chromatin
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DOI：
10.1093/hmg/ddr506
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发表时间：
2012-02-15
影响因子：
3.5
通讯作者：
Clark, Amander T.
中科院分区：
文献类型：
--
作者：
Perez, Silvia V. Diaz;Kim, Rachel;Clark, Amander T.
人胚胎干细胞（human embryonic stem cells，hESC）是来源于人胚泡内细胞团的多能细胞类型。最近的数据表明，大多数已建立的雌性XX hESC系在分化前已经经历了X染色体失活（XCI），并且hESC的XCI可以是XIST依赖性的（II类）或XIST不依赖性的（III类）。女性hESC的XCI排除了XX hESC作为检查XCI机制的基于细胞的模型的使用，并且对于研究X连锁疾病将是一个挑战，除非开发策略来重新激活失活的X。为了恢复具有两个活性X染色体（I类）的细胞核，我们开发了一种重编程策略，通过向hESC培养基中补充小分子丁酸钠和3-去氮Planocin A（DZNep）。我们的数据表明，成功的重编程可以发生在XIST依赖的II类核状态，但不是III类核状态。为了确定这些小分子是否预防XCI，我们在常氧条件下衍生了六个新的hESC系（UCLA 1-UCLA 6）。我们表明，I类细胞核存在于前20代人胚胎干细胞衍生冷冻保存前，补充丁酸钠或DZNep保存I类细胞核的自我更新状态。总之，我们的数据表明，自我更新和生存的I类细胞核是兼容的含氧量正常的人胚胎干细胞衍生，和衍生后的化学补充提供了一种策略，以防止表观遗传进展和保留细胞核与两个活跃的X染色体在自我更新状态。
Human embryonic stem cells (hESCs) are pluripotent cell types derived from the inner cell mass of human blastocysts. Recent data indicate that the majority of established female XX hESC lines have undergone X chromosome inactivation (XCI) prior to differentiation, and XCI of hESCs can be either XIST-dependent (class II) or XIST-independent (class III). XCI of female hESCs precludes the use of XX hESCs as a cell-based model for examining mechanisms of XCI, and will be a challenge for studying X-linked diseases unless strategies are developed to reactivate the inactive X. In order to recover nuclei with two active X chromosomes (class I), we developed a reprogramming strategy by supplementing hESC media with the small molecules sodium butyrate and 3-deazaneplanocin A (DZNep). Our data demonstrate that successful reprogramming can occur from the XIST-dependent class II nuclear state but not class III nuclear state. To determine whether these small molecules prevent XCI, we derived six new hESC lines under normoxic conditions (UCLA1-UCLA6). We show that class I nuclei are present within the first 20 passages of hESC derivation prior to cryopreservation, and that supplementation with either sodium butyrate or DZNep preserve class I nuclei in the self-renewing state. Together, our data demonstrate that self-renewal and survival of class I nuclei are compatible with normoxic hESC derivation, and that chemical supplementation after derivation provides a strategy to prevent epigenetic progression and retain nuclei with two active X chromosomes in the self-renewing state.