摘要
目的胚胎干细胞(embryonic stem cells, ESCs)成为当今生命科学和生物技术研究的热点,这是因为它具有自我更新和多能分化的能力。多年来,“成体组织中是否存在胚胎干细胞”一直是一个很有争议的问题,很多实验室已报道可以从骨髓中分离出来ESCs样的多能干细胞,这些细胞都表达ESCs特异性标志Oct4基因,能像ESCs一样无限分裂繁殖,而且可以分化成几乎所有类型的细胞。但是对于这些原始细胞的来源和存在机制,人们并不清楚。因此,骨髓中是否存在ESCs就变成了一个很有争议的问题。
之前的研究都是通过不同的方法从骨髓中分离、提取并培养这些ESCs样的干细胞,而我们首次通过相反的方式直接将小鼠ESCs注入到小鼠的骨髓腔里,通过观察ESCs在体内的分布生长情况,从而思考体内是否有这样一个适宜ESCs生存的环境。
方法为了更好地追踪ESCs在体内的生长情况,我们新建两株带绿色荧光标记(green fluorescent protein, GFP)的ESCs——oGFP+ESCs(C57BL／6xOct4／EGFP转基因OG2小鼠)；和aGFP+ESCs(129／SvxCAG／EGFP转基因C57BL／6-Tg小鼠)。oGFP+ESCs中GFP的表达受Oct4启动子调控,Oct4表达时GFP才表达；aGFP+ESCs中GFP的表达受chicken-beta-actin启动子调控,无论细胞分化与否,GFP均表达。为了避免了ESCs在体内其它器官的阻滞和损失,我们直接将ESCs通过胫骨注射植入到同源的C57BL／6小鼠(分照射组和非照射组)骨链腔内。
移植后,通过双光子荧光显微镜来观察GFP+细胞在小鼠骨髓腔里存活的时间和状态。另在不同的时间点,我们从移植小鼠的骨髓腔里再次分离出这些GFP+的细胞,采用建立ES细胞系的方法使这些Oct4+-GFP+细胞建成稳定细胞系,并检测这些新建细胞系的功能。通过基因芯片检测来比较这些细胞系和母代ESCs在基因表达水平上的差异。另一方面,我们通过流式细胞学检测和体外培养的方式,分析移植的ESCs在骨髓这种造血微环境下的分化特征。
结果通过双光子荧光显微镜观察发现100天后,骨髓中仍然存在Oct4-GFP表达的类似ESCs的细胞。大多数GFP+细胞位于移植侧胫骨的注射部位,也有少部分GFP+细胞位于注射骨对侧的骨髓腔内。在不同的时间点,我们从移植小鼠的骨髓腔里分离出这些Oct4+-GFP+的细胞,建成稳定细胞系。到目前为止,我们成功建立了8株细胞系,形态同ESCs。通过对这些Oct4+细胞系的功能性检测发现它们具有与ESCs相似的特性,但这种细胞系生成嵌合鼠的嵌合率很低,且不能生殖系传递。芯片结果显示这些细胞与母代ESCs比较,部分基因表达水平发生了改变,表观遗传学也发生了逐渐的变化。
另外,移植的ESCs在骨髓腔内大多数分化成了非造血的细胞,形态多样,与骨质连接紧密,而仅有少部分分化成了造血细胞,并可进入血循环。结论1)我们的研究首次通过直接将ESCs移植入小鼠骨髓腔来证实了成体环境中长期存在ESCs的可能性,对以后干细胞的应用,在成体内的发育和分化研究具有一定的提示作用；
2)由于原始的胚胎干细胞在体内可以长期存在,因此在使用ESCs或ESCs衍生物做细胞治疗或移植过程中,安全性一定要长期关注；
3)胚胎干细胞在成体骨髓中存活一段时间后,逐渐发生了表观遗传学方面的变化,这种变化影响了ESCs的多能性改变,因此在以后细胞移植过程中,我们需要关注这些细胞表观遗传学方面的影响；
4)ESCs在骨髓腔内存活一段时间后,大多数分化成了非造血细胞。我们的研究首次说明了ESCs在骨髓微环境的分化趋势,对ESCs的分化潜能和周围环境的关系以及ESCs的应用具有一定的提示作用。
Objective Embryonic stem cells (ESCs), derived from the inner cell mass of a blastocyst, possess two important characteristics:self-renewal and pluripotency toward all three embryonic germ layers. These remarkable characteristics make ESCs an attractive cell source in regenerative medicine. The important question regarding whether ESCs can survive in adult organs/tissues for the long-term remains unanswered. Bone marrow (BM) has been identified as a major source containing hematopoietic stem cells (HSCs) as well as non-hematopoietic stem cells, which include mesenchymal stem cells, multipotent adult progenitor cells, marrow-isolated adult multilineage inducible cells, and very small embryonic-like stem cells. Some of these non-hematopoietic bone-marrow-derived stem cells have been shown to have features in common with ESCs. Some express the Oct4 gene, and multipotent adult progenitor cells have been shown to differentiate into each of the three germ layer cells. However, the claim regarding the existence of ESC-like cells in adult tissues/organs such as bone marrow remains controversial largely due to the difficulty in isolating these extremely rare cells.
In contrast to the previous studies that have examined this important yet unanswered question, we chose to approach it from the opposite direction by transplanting ESCs directly into the bone marrow to verify whether the bone marrow could provide a potential micro-environment for sustaining the pluripotent stem cells. A further rationale for carrying out our current study is the existing safety concerns regarding the potential of leftover or contaminated ESCs in therapeutic ESC products.
Methods We approached this question by directly injecting mouse ESCs into syngeneic BM and then monitoring the survival and differentiation potential of the injected ESCs in BM based on the expression of GFP protein of the injected cells. To this end, an ESC line was derived from the Oct4-GFP transgenic mice in which GFP protein expression is driven by the Oct4 promoter, thereby indicating the status of pluripotency. As a control, an ESC line was also derived from the mice that constitutively express GFP that is driven by the chicken-β-Actin. The cells were injected via intra-tibia and then monitored at different time points after injection. The transplanted cells were visualized at several time points using two-photon microscopy to determine whether the bone marrow could sustain the pluripotent stem cells. Moreover, we further characterized the properties of the re-established cell lines recovered from the transplanted cells. To explore why the pluripotency of the re-derived cells are decreased, we compared the global gene-expression profiles of ESCs and the re-derived cell lines. To analyze the differentiation potential of ESCs after bone marrow transplantation, we examined the engrafted cells by flow cytometric analysis and the colony-forming unit-fibroblast assay.
Results Despite the fact that some of the animals (28-29%in the irradiated hosts) developed tumors as expected, we intended to focus on the engrafted ESCs in the animals in which no tumor had occurred. We found that the Oct4+ cells could exist in recipient's BM for more than 100 days after transplantation as examined under the two-photon microscopy. Notably, most injected ESCs in BM differentiated into non-hematopoietic cells whereas few cells acquired hematopoietic cell surface markers. Interestingly, the engrafted cells were found in the contrary BM of the injected side as well as the blood circulation. We then harvested the Oct4+ cells in the BM at various time points after transplantation and attempted to re-establish the ESC lines. Eight ESC lines were recovered from a total of 141 irradiated recipients that were transplanted with the Oct4-driven GFP+ESCs. Those successfully recovered ESC lines from BM expressed known markers for embryonic pluripotency and were able to differentiate into three germ layers although germ line transmission is yet to be further defined. Gene expression profiling suggests time-dependent epigenetic alterations in the re-derived ESC lines as compared to their parental ESC line.
Conclusions This study provides definitive evidence for long-term survival and differentiation of exogenous ESCs in adults. It has important implications for the understanding of stem cell development and maintenance, as well as for the safety evaluation of therapeutic ESC derivatives especially given the fact that rare un-differentiated ESCs can be contained in the final cellular products.
引文
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