第一个造血干细胞分化道路上的分岔

2020/7/27 16:31:48 本站原创 佚名 【字体:

廖联明   编译

 

哺乳动物胚胎中第一个具有长期再增殖潜能的造血干细胞(HSCs)在妊娠中期从主要位于背主动脉的造血内皮细胞(HECs)通过内皮-造血转化(EHT)而分化出来。尽管人们普遍认为HECs起源于动脉ECs,但尚不完全了解HECs的内皮细胞(ECs)前体的确切分子身份和来源。

Hou等人,通过单细胞RNA测序检测造血内皮细胞(hemogenic endothelial cells, HECs)的起源。他们表明,动脉内皮细胞和HECs来源于一种共同的早期动脉内皮细胞前体,且某些HECs及其近几代保留产生内皮细胞和造血细胞的能力的时间比以前认为的更长。

单细胞技术的进步使得罕见的异种细胞群可以在功能和分子上被分开。单个HECs进行EHT的活体影像学研究表明,它们经历动态形态学变化,从内皮上脱落,并且在哺乳动物胚胎中积聚在动脉内造血细胞簇(IAHCs)。一些研究小组已经利用单细胞qRT-PCR或单细胞RNA测序(scRNA-seq)来鉴定HECs的新的细胞表面标记物,如CD44,并对位于HECHSC之间过渡细胞pre-HSC进行分子鉴定,该细胞表达内皮细胞标记物、c-Kit和高水平CD201

为了揭示HECs的早期特征和相关的前体细胞信息,Hou等人利用scRNA-seq对小鼠发育的E8-E11连续阶段的所有内皮细胞群进行非常均匀的检测。这一时间框架包括最初的背主动脉形成、HECsEHT、前HSCs和最初的HSCs的出现。这一方法能够发现产生HECsECs的早期细胞分化的机制,以及HECs自身的详细情况。通过对不同发育阶段的内皮细胞进行取样,他们能够构建从原始ECs到前HSCs的连续轨迹。该轨迹揭示了从原始ECsHECs的两个主要分支:原始ECs必须先选择在动脉生长,而不是静脉,然后早期动脉ECs必须采用造血途径成为HECs并与晚期动脉ECs分离。

 

晚期动脉ECsHECs有共同的早期动脉前体的发现与两个谱系起源于不同前体细胞的观点相矛盾。CD44是动脉内皮标记物,而E9.5E10.5之间背主动脉中向HSC分化的HECs也表达CD44,这个数据进一步证实了HECs的动脉内皮起源。HSC诱导的HECs的动脉起源也已在人类中得到证实;该群细胞显示出明确的动脉细胞特征,IAHCs也表达动脉基因。此外,NOTCH介导的HECs动脉化是从人多能干细胞中产生多系造血祖细胞所必需的。综上所述,这些发现有力地支持了HECs直接来源于早期动脉内皮前体的理论模型。

Hou等人,同时利用他们的scRNA-seq数据信息,发现想HSC分化的HECs的新标记。通过比较不同的内皮细胞群与其假定的HEC群体,研究者用CD31CD44CD201c-KIT作为标记物,从E9.5-E10.0小鼠胚胎中分离出一个精纯的HEC群。这一细胞群在培养过程中分化为内皮管或造血细胞,一些单细胞能同时产生这两种细胞。

Hou等人,能够通过从生物信息学筛选鉴定出的新的HEC特征基因Neurl3生成报告基因来进一步完善其HEC种群。他们建立了一个Neurl3:EGFP报告基因小鼠,观察到EGFP的表达仅限于IAHCs和一部分主动脉内皮细胞,其中大多数还表达HECs的一个已知标记物转录因子RUNX1。在功能测定中,neur3:EGFP+HECs可同时产生造血细胞和内皮细胞,且所有长期增殖的HSC均由neur3:EGFP+ECs体外培养产生,表明neur3HSC诱导的HECs的可信标记物。

Swiers等人在此前报道,从E8.5E9.5E10.5胚胎中分离的单个HECs未能产生内皮细胞和造血细胞,这与Hou等人报道的结果相反。这些结果的差异可能是由于两组用于分离HECs的标记物;Swiers等人使用转基因报告小鼠,其中GFP的表达是从+23runx1增强子驱动表达,以分离Runx1:GFP+HECs进行分析。表达Runx1基因的HECs失去了自身的内皮和造血分化的双向潜能,Hou等分离出一种稍早的HEC前体,该前体处于分化为造血细胞之前,并且尚未失去内皮细胞的潜力。

Hou等人描述的双潜能HEC不同于血管母细胞,血管母细胞是指位于动静脉EC分开之前的造血细胞和内皮细胞的胚胎前体。相反,侯和他的同事的双潜能细胞可能成为一种正在想造血细胞分化的动脉HEC,并没有分化为内皮细胞或造血细胞。

为了研究EHTHECs细胞的命运,研究者分离了HSC前体,即产生HSCHECs的后代和HSCs的前体。值得注意的是,研究者发现前HSC还含有少量具有内皮和造血分化双潜能的细胞,这表明至少有一部分前HSC尚未参与造血命运。这是一个令人惊讶的结果,因为前HSC被认为已经完全经历了EHT,并丧失了向内皮细胞分化的命运。那么,一个有趣的问题是,一个细胞在整个发育过程中的哪一点最终只具备了成为一个血细胞的分化潜力?

scRNA-seq是一种非常有价值的工具,可用于识别和表征引起HECs的罕见短暂亚群。在本研究中,Hou等人能够证明从scRNA-seq数据进行生物信息分析的能力,以确定HECs的直接前体,并发现HEC形成的新标记和调控因子。

 

原文:

Forks in the road to the first hematopoietic stem cells

The first hematopoietic stem cells (HSCs) with long-term repopulating potential in mammalian embryos differentiate at mid-gestation from hemogenic endothelial cells (HECs) located primarily in the dorsal aorta through an endothelial-to-hematopoietic transition (EHT). The precise molecular identity and source of the endothelial cell (EC) precursors of HECs is not fully understood, although it is widely believed that HECs originate from arterial ECs

Hou et al. examine the origin of hemogenic endothelial cells (HECs) by single-cell RNA sequencing. They show that arterial endothelial cells and HECs are derived from a common early arterial endothelial cell precursor, and that some HECs and their immediate progeny retain the capacity to generate both endothelial and hematopoietic cells longer than previously thought.

Advances in single cell technologies have allowed for rare heterogenous cell populations to be dissected functionally and molecularly. Live imaging studies of single HECs undergoing EHT showed that they undergo dynamic morphological changes, detach from the endothelium, and in mammalian embryos accumulate in intra-arterial clusters of hematopoietic cells (IAHCs). Several groups have exploited single-cell qRT-PCR or single-cell RNA sequencing (scRNA-seq) to identify novel cell surface markers of HECs such as CD44 and to molecularly characterize a transitional cell situated between HEC and HSC called a pre-HSC, which expresses endothelial markers, the cell surface marker c-Kit, and high levels of CD201

 

To uncover information about early specification of HECs and the relevant precursors, Hou et al. utilized scRNA-seq to unbiasedly examine all endothelial populations that spanned continuous stages from E8 to E11 of mouse development. This time frame encompasses initial dorsal aorta formation, the appearance of HECs, EHT, pre-HSCs and the first HSCs. This strategy allowed for examination of the early cell fate decisions of ECs that give rise to HECs, as well as detailed examination of the HECs themselves. By sampling endothelial populations from several developmental stages, they were able to construct a continual trajectory from primitive ECs to pre-HSCs. The trajectory revealed two major bifurcations along the path from primitive ECs to HECs; primitive ECs must first choose an arterial, but not venous fate, and then early arterial ECs must adopt a hematopoietic fate to become HECs and segregate away from late arterial ECs. The identification of an early arterial precursor of both late arterial ECs and HECs argue against the notion that the two lineages arise from distinct precursors. The arterial endothelial origin of HECs is further supported by data showing that CD44, an arterial endothelial marker, reliably marks HSC-primed HECs in the dorsal aorta from E9.5 to E10.5. The arterial origin of HSC-primedHSC分化的 HECs has also been shown in humans; this population displayed an unambiguous arterial signature, and IAHCs also expressed arterial genes. Additionally, NOTCH-mediated arterilization of HECs is required for generating multi-lineage hematopoietic progenitor cells from human pluripotent stem cells. Taken together, these findings strongly support a model wherein HECs are derived directly from an early arterial endothelial precursor.

Hou et al. also leveraged information from their scRNA-seq data to uncover novel markers of HSC-primed HECs. By comparing different endothelial populations with their putative HEC population, the authors isolated a refined HEC population from E9.5–E10.0 mouse embryos using CD31, CD44, CD201, and c-KIT as markers. This population differentiated into either endothelial tubes or hematopoietic cells in culture, and some single cells produced both.

Hou et al. were able to further refine their HEC population by generating a reporter gene from a novel HEC signature gene, Neurl3, that was identified from a bioinformatics screen. They created a Neurl3:EGFP reporter mouse, and observed that EGFP expression was restricted to IAHCs and a subset of aortic ECs, most of which also expressed the transcription factor RUNX1, a known marker of HECs. Neurl3:EGFP+ HECs could produce both hematopoietic cells and endothelial tubes in functional assays, and all long-term repopulating HSCs were generated exclusively from Neurl3:EGFP+ ECs in ex vivo cultures, indicating that Neurl3 is a faithful marker of HSC-primed HECs.

Swiers et al. previously reported that single HECs isolated from E8.5, E9.5, and E10.5 embryos never gave rise to both endothelial and hematopoietic cells, in contrast to the results reported by Hou et al.. The difference in these results could be due to the markers that the two groups used to isolate HECs; Swiers et al. used a transgenic reporter mouse in which GFP expression is driven from the +23 Runx1 enhancer to isolate Runx1:GFP+ HECs for their analyses. It is possible that HECs expressing the Runx1 transgene had lost their dual endothelial-hematopoietic potential, and that Hou et al. have isolated a slightly earlier HEC precursor that is in the midst of hemogenic specification and has not yet extinguished endothelial potential.

The dual-potential HEC described by Hou et al. is distinct from a hemangioblast, a term used to describe an embryonic precursor of both hematopoietic and endothelial cells situated prior to the branch point of arterial and venous ECs. Rather, Hou and colleagues’ dual-potential cell appears to be an arterial HEC that is undergoing hemogenic specification, and has not been committed to either the endothelial or hematopoietic cell fates.

To examine the fate of the HECs cells after EHT, the authors isolated pre-HSCs, the progeny of HSC-producing HECs and a precursor to HSCs. Strikingly, the authors found that pre-HSCs also contained a low frequency of cells with dual endothelial-hematopoietic potential, suggesting that at least a subset of pre-HSCs are not yet committed to the hematopoietic fate. This is a surprising result, as pre-HSCs are thought to have fully undergone EHT, and extinguished their endothelial fate. An interesting question, then, is at what point in the entire process a cell is finally fully committed to becoming a blood cell?

scRNA-seq has been an invaluable tool for identifying and characterizing rare transient subpopulations that give rise to HSCs. In this study, Hou et al. were able to demonstrate the power of bioinformatic analysis from scRNA-seq data to define the immediate precursors of HECs, and to uncover novel markers and regulators of HEC formation.

 

References

Hou, S. et al. Cell Res. https://doi.org/10.1038/s41422-020-0300-2 (2020).

 

 

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