人胚胎干细胞定向诱导分化为造血细胞的实验研究
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摘要
血细胞输注和造血干细胞(hematopoietic stem cells,HSCs)移植是目前细胞治疗的常用手段,广泛应用于恶性血液疾病、遗传性疾病、感染性疾病、重症免疫缺陷及肿瘤放化疗治疗后的造血支持治疗等领域。然而,血细胞来源紧张及病原微生物传播等问题给其临床应用的安全性和广泛性带来了很大的挑战。因此,人们期望寻找更为安全、经济和有效的血细胞和造血干细胞的资源。
胚胎干细胞(embryonic stem cells,ES cells)在体外具有高度增殖和多向分化潜能,可定向诱导分化为神经细胞、心肌细胞、肝脏细胞、胰岛β细胞、造血细胞等多种类型的细胞。ES细胞具有比成体细胞更为广泛的分化潜能和可塑性,是最丰富的干细胞来源,备受科学家的青睐。
大量的研究表明人ES细胞在体外可定向诱导分化为造血细胞,有可能为临床输血和造血干细胞移植,及恶性血液疾病的治疗提供新途径。目前,HSCs主要来源于脐带血、骨髓和动员后的外周血。从临床应用考虑,与传统来源的HSCs相比,人ES细胞来源的HSCs具有很多优势:合适的供体骨髓的来源短缺,虽然脐带血已建库,但脐血中的HSCs数量仍相对不足,限制了其在成人病人中的广泛应用。随着干细胞及其相关技术的发展,如核移植技术以及细胞重编程技术获得诱导性多能干细胞(induced pluripotent stem cells, iPS细胞)等有可能为HSCs的获得提供患者自身遗传背景的ES细胞。此外,ES细胞在体外具有无限增殖潜能,可大量扩增培养,进而生成足够数量的HSCs,有可能成为治疗各种血液疾病的新的HSCs来源。
目前,研究者主要采用可溶性细胞因子诱导法或造血基质细胞共培养法定向诱导人ES细胞向造血细胞分化。尽管ES细胞向造血细胞分化的研究已经取得了很大进展,且其研究和应用前景非常广阔,但体外大规模地定向诱导人ES细胞分化为成熟红细胞并最终应用于临床仍有很多关键的问题需要解决。首先,造血发育的过程有着复杂的基因调控,目前造血细胞分化的分子调控机制尚不明确,探讨ES细胞向造血细胞定向诱导分化调控的机制也将会提高ES细胞体外诱导分化的效率。其次,诱导中使用的各种细胞因子多为基因工程产品,价格昂贵。此外,鼠源饲养层是目前常用的诱导方法。然而,鼠源饲养层所带来的异源污染极大地限制了该研究在临床上的广泛应用。
本研究中,我们首先探讨了前列腺素E2(prostaglandin,PGE2)在人ES细胞向造血细胞分化的作用并初步探讨了其机制,在此基础之上建立了转染促红细胞生成素(erythropoietin,EPO)基因的人胎肝基质细胞(human fetal liver stromalcells,hFLSCs),利用其条件培养基诱导人ES细胞向红系细胞分化,建立了一种新型诱导人ES细胞向造血细胞分化的方法,既降低了实验成本,又可以避免异源污染。
本研究主要包括两部分内容:
一、PGE2促进人ES细胞向造血细胞分化
人ES细胞在体外可定向诱导分化为造血细胞,尽管目前已建立了多种不同的诱导体系,仍处于起步阶段,细胞分化调控的机制尚不清楚,成为广大科研工作者研究的热点。
PGE2是一种重要的细胞生长和调节因子。研究发现PGE2在正常及缺氧、感染等异常情况下对造血发育都有重要的调节作用。近年来,研究表明PGE2能够促进造血干细胞的存活、增殖及内环境调节方面具有一定的作用。最近研究指出PGE2能够促进放射性损伤后的成年斑马鱼恢复造血重建能力,提示PGE2在胚胎的造血发育过程中发挥重要的作用。此外,PGE2也可促进小鼠ES细胞向造血干细胞分化。因此,我们推测PGE2在人ES细胞向造血分化过程中也发挥一定作用。
我们通过与OP9基质细胞共培养的方法考察PGE2对人ES细胞向造血细胞分化的影响:实验组添加PGE2,对照组不添加PGE2。结果发现,100ng/ml的PGE2可抑制集落的生长,20ng/ml的PGE2对集落生长有明显的促进作用,接种于半固体培养体系生成的造血集落最多,诱导产生的细胞具有向造血各谱系分化能力,加入PGE2抑制剂吲哚美辛后集落形成能力受到明显的抑制。为了观察PGE2对人ES细胞向造血细胞分化的影响,我们于显微镜下观察共培养细胞的形态学变化,结果发现不添加PGE2的情况下,人ES细胞接种于OP9细胞上,部分向上皮样细胞分化;部分形成血岛样的结构,随着培养时间的延长类似于血岛的结构会逐渐消失、变平。在加入PGE2的情况下,大部分会形成血岛样的结构,随着培养时间的延长血岛样的结构体积变大,但形态不会发生改变。收集共培养3～9d的细胞,用半定量PCR检测胚胎干细胞标志Oct4及造血和内皮相关基因的表达情况。结果表明,在不添加PGE2的情况下,诱导的细胞持续表达Oct-4,从培养的第3d开始表达中胚层标志Brachyury,早期造血标志CD34和内皮细胞标志VE-cad,从第5d开始表达造血调控相关基因Runx1,GATA1,SCL和内皮标志vWF。在添加PGE2的情况下Oct-4的表达从培养第3d开始降低,共培养的细胞从培养第3d开始表达Brachyury,CD34,Runx1,GATA1,SCL和VE-cad,从培养第5d开始表达vWF。这提示外源性添加PGE2可明显促进造血和内皮相关基因的表达。我们进一步考察了PGE2处理后Smad信号通路的变化。Western-blot结果显示,与对照组相比,PGE2处理能够上调p-Smad1/5的表达,同时Smad4的表达也上调。而加入PGE2的抑制剂后p-Smad1/5和Smad4的表达受到抑制,提示Smad信号通路可能参与了PGE2介导的人ES细胞向造血细胞分化。
二、EPO基因修饰的hFLSCs条件培养基诱导人ES细胞向红系细胞分化
在ES细胞体外诱导分化为造血细胞的过程中,研究造血微环境的调控机制是其发生和分化的重要环节。在胚胎发育的过程中,不同的造血微环境对造血的发生发挥着重要的作用。造血发生经历了卵黄囊造血、胎肝造血和骨髓造血三个阶段。人卵黄囊造血时期发生在胚胎的4～6周;胎肝造血时期发生在胚胎的6～22周;骨髓造血时期从22周至出生以后。胎肝是造血早期发育的主要位点,是造血细胞发育和分化的重要微环境,构成其微环境的胎肝基质细胞很有可能在造血细胞的发育和分化过程中发挥重要的作用。此外,血细胞的产生还受到造血干细胞内在基因和造血因子的共同调控,EPO是其产生的最重要的因子。EPO是一种糖基化的蛋白质激素,主要来源于肾脏和肝脏,而脑、肺、脾也能产生少量。EPO可促进造血干细胞向原始红细胞分化,加速幼红细胞的分裂、增殖,促进血红蛋白的合成,在红细胞诱导分化研究中发挥重要作用。而造血因子提供的方式对其作用的发挥也有重要的影响。研究表明以造血生长因子基因修饰的基质细胞比相同条件下的造血生长因子更能有效地调控造血。
因此,本部分研究中我们采用慢病毒系统建立了稳定高表达EPO基因的hFLSCs,能够高效分泌具有生物学活性的EPO蛋白,从而避免了添加外源性细胞因子,极大地降低了实验成本。随后,将人ES细胞培养于低黏附的细胞培养皿中诱导生成拟胚体(embryoid bodies,EBs),用过表达EPO基因的FLSCs条件培养基(EPO/hFLSCs-CM)诱导EBs细胞向造血细胞分化,未转染EPO基因的FLSCs条件培养基(hFLSCs-CM)作为阴性对照,未转染EPO基因的FLSCs条件培养基外源性添加重组的EPO(hFLSCs-CM+EPO)作为阳性对照。收集诱导不同天数的细胞,通过流式细胞术分析诱导过程中CD34阳性的表达,以确定不同培养条件下EBs向造血细胞分化的情况。结果表明三种不同的培养体系均能诱导人ES细胞向造血细胞分化。然而,hFLSCs-CM组的诱导效率较低,在诱导第12d中CD34阳性细胞低于11%。添加外源性EPO之后,人ES细胞向造血细胞定向分化的能力增强,其中CD34+细胞最高可达13.1%。EPO/hFLSCs-CM组能更为有效地促进人ES细胞向造血细胞分化,其中CD34+细胞可高达22.74%。此外,我们还应用实时定量PCR检测不同组细胞造血相关基因的表达情况;应用克隆形成实验检测了诱导细胞的造血集落形成能力;通过瑞氏-吉姆萨染色观查了诱导细胞的形态;应用联苯胺染色和RT-PCR方法检测了诱导造血集落珠蛋白的表达情况。结果表明,诱导生成的细胞具有造血细胞的一般特性,在半固体培养基中可分化为不同种类的造血集落,不同培养条件下产生的造血细胞的集落形成能力与珠蛋白的表达量不同,EPO/hFLSCs-CM诱导产生细胞的造血基因的表达量及所形成的造血克隆最多,且以红系克隆为主。此外,我们还检测了诱导产生的红系细胞的携氧能力。结果显示,EPO/hFLSCs-CM诱导的红系细胞具有和脐带血相似的“S”形氧离曲线,这表明体外诱导的红系细胞具有和脐带血相似的携氧能力。
综上所述,在本研究中我们初步考察了PGE2在人ES细胞向造血细胞分化过程中的作用并初步探讨了其作用机制;并利用稳定表达EPO基因的FLSCs条件培养基诱导人ES细胞向红系细胞分化,该方法可高效诱导人ES细胞定向诱导分化为造血细胞,同时降低了研究成本,且有效避免了鼠源饲养层带来的异源基因污染。上述研究为深入探讨造血细胞的发育调控机制及以胚胎干细胞或造血干细胞为启动细胞大规模地诱导产生红细胞奠定了一定的实验基础。
Currently , blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important means for cellular therapy. These methods are widely used in the treatment of incurable hematological disorder, genetic diseases, infectious diseases and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. So people try hard to obtain more safe, effective and economic resource of blood cells. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of preimplantation embryos possess indefinite proliferative capacity in vitro, and maintain pluripotency as well. They also have the capacity to differentiate into all cell types.
Recently, the potential of hESCs to differentiate into hematopoietic cells has been reported in several studies, suggesting clinical applications in cellular therapy, such as blood transfusion or hematopoietic stem cell transplantation, and treatment for hematological disorder. Currently, the major sources of transplantable HSCs for clinical therapy are human bone marrow (BM), mobilized peripheral blood and umbilical cord blood (CB), but their availability for clinical use is limited by both quantity and compatibility. Pluripotent hESCs may provide an alternative.
With the repid development of biology research, pluripotent human embryonic stem cells (hESCs) may provide a unique tool for hematopoietic transplantation and the study of human embryonic hematopoiesis.
Currently, various methods including cytokinses administration and co-culture with stromal cells have been used to promote the hematopoietic differentiation of embryonic stem cells (ESCs). Although hESCs are competent for development into hematopoietic cell fate, the cellular and molecular mechanisms that control this process are very poorly understood and yield heterogeneous outcomes. Furthermore, the risk of mouse-related disease, high cost of cytokines and low differentiation efficiency greatly limit the clinical applications.
In the present study, we first demonstrate the role for PGE2 in regulating hematopoietic differentiation from hESCs, then we establish a novel method which can induce hESCs differentiated into hematopoietic lineages safely, efficiently, and economicly.
1. Prostaglandin E2 promotes hematopoietic development from hESCs
Recent studies have suggested that PGE2 and the prostaglandin pathway are essential for hematopoietic stem cell growth, survival and development. These findings have led to a greater interest in the possible role of the prostaglandin pathway in human hematopoietic development. Here we evaluated the effects of PGE2 on hematopoietic differentiation of hESCs. The induced cells from hESCs/OP9 co-culture and in the presence of PGE2 were characterized by RT-PCR, flow cytometry, colony-forming, assays and Wright-Giemsa staining. Our results demonstrated that the hematopoietic colonies increased in a dose-depentent manner after exposure to PGE2. The expression of Oct-4 and hematopoiesis-inductive transcription factors and hematoendothelial specific genes, including Brachyury, CD34, SCL, GATA-1, Runx1, vWF and VE-cad were examined with RT-PCR. PGE2 exposure also altered the morphology of co-cultured hESCs and resulted in a robust hematopoietic differentiation with the higher frequencies of CD34+ and CD45+ cells. Furthermore, PGE2 supplement increased the activation of p-Smad1/5 and enhanced the expression of Smad4 compared to the non-treated counterpart. When the effect of PGE2 was blocked by its specific inhibitor indomethacin, p-Smad1/5 and Smad4 expression declined correlatively. This research may improve our knowledge of stem cell regulation, which hopefully will lead to improved stem cell-based therapeutic options.
2. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells
Development of hematopoietic system in human embryo involves several anatomical sites including the yolk sac hematopoiesis occurring from 4 to 6 weeks, the fetal liver hematopoiesis occurring from 6 to 22 weeks, and the bone marrow hematopoiesis occurring during the remainder of life. Different hemopoietic microenvironment plays important roles in the different stages. In the mode of hematopoietic differentiation from hESCs, the selection of microenvironment is an important part. The fetal liver is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated. So we suppose that the fetal liver microenvironment could promote the hematopoietic differentiation from hESCs. On the other hand, the most important cytokine regulator of erythropoiesis is erythropoietin (EPO). It acts on erythroid progenitor cells, stimulates proliferation, promotes differentiation, and prevents apoptosis. The main EPO production site is the liver in the fetus and the kidney in the adult. As a conclusion, the combination of fetal liver cells and EPO must improve the erythropoietic differentiation. It was also shown that direct secreted cytokines from stromal cells exert their function better than medium supplement. Based on these understanding, a transgenic stromal line was built.
We have developed a novel method to promote the differentiation of hESCs toward hematopoietic lineages. The human fetal liver stromal cell (hFLSCs) expressing EPO were established using lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce hESCs differentiate into hematopoietic cells without exogenous cytokines.
In this experiment, hESCs were cultured in low cell-attachment dishes to obtain EBs. While the EBs were induced into hematopoietic cells by different inducing systems including hFLSCs-CM, hFLSCs-CM+EPO, and EPO/hFLSCs-CM after treated by BMP4. Then the expression of CD34 was analyzed by flow cytometry. For EBs treated with hFLSCs-CM CD34+ cells peaked to 10.57% on day 12. For the EBs treated with hFLSCs-CM+ EPO, the number of CD34+ cells reached the maximum of 13.1% at day 12. For the EBs treated with EPO/hFLSCs-CM, CD34+ cells peaked to 22.74% at 12 days of culture. These results demonstrated that EPO/hFLSCs-CM treated EBs could yield more hematopoietic cells than other groups. In addition, the genotypic expression was characterized by realtime RT-PCR. Among these different inducing systems, EPO/hFLSCs-CM had the highest hematopoitic gene expression level. The features of hESCs-derived erythroid cells were characterized by morphology, flow cytometry, wright-Giemsa staining, 3’3-diaminobenzidine staining, and RT-PCR. The results showed that the induced cells showed the feature of hematopoietic cells. Among the inducing systems, the EPO/hFLSCs-CM could promote hematopoietic differentiation of hESCs, especially into erythrocytes. Furthermore, the hESC-drived erythroid cells were also able to function as oxygen carriers, and exhibited an oxygen dissociation pattern similar to human CB.
In summary, our studies support an expected role for PGE2 in regulating hematopoietic differentiation from hESCs. We demonstrate that PGE2 treatment, together with OP9 stromal cell co-culture, promotes hematopoietic development from hESCs. Furthermore, we established the transgenic human fetal liver stromal cells (EPO/hFLSCs) that stably express EPO gene and used the conditioned medium to induce erythropoietic differentiation of hESCs. Furthermore, this method can avoid the mouse-related disease and cut down the cost of experiment. The system we developed will provide a reliable alternative for the research in the future therapeutic applications of human embryonic stem cells.
胚胎干细胞(embryonic stem cells,ES cells)在体外具有高度增殖和多向分化潜能,可定向诱导分化为神经细胞、心肌细胞、肝脏细胞、胰岛β细胞、造血细胞等多种类型的细胞。ES细胞具有比成体细胞更为广泛的分化潜能和可塑性,是最丰富的干细胞来源,备受科学家的青睐。
大量的研究表明人ES细胞在体外可定向诱导分化为造血细胞,有可能为临床输血和造血干细胞移植,及恶性血液疾病的治疗提供新途径。目前,HSCs主要来源于脐带血、骨髓和动员后的外周血。从临床应用考虑,与传统来源的HSCs相比,人ES细胞来源的HSCs具有很多优势:合适的供体骨髓的来源短缺,虽然脐带血已建库,但脐血中的HSCs数量仍相对不足,限制了其在成人病人中的广泛应用。随着干细胞及其相关技术的发展,如核移植技术以及细胞重编程技术获得诱导性多能干细胞(induced pluripotent stem cells, iPS细胞)等有可能为HSCs的获得提供患者自身遗传背景的ES细胞。此外,ES细胞在体外具有无限增殖潜能,可大量扩增培养,进而生成足够数量的HSCs,有可能成为治疗各种血液疾病的新的HSCs来源。
目前,研究者主要采用可溶性细胞因子诱导法或造血基质细胞共培养法定向诱导人ES细胞向造血细胞分化。尽管ES细胞向造血细胞分化的研究已经取得了很大进展,且其研究和应用前景非常广阔,但体外大规模地定向诱导人ES细胞分化为成熟红细胞并最终应用于临床仍有很多关键的问题需要解决。首先,造血发育的过程有着复杂的基因调控,目前造血细胞分化的分子调控机制尚不明确,探讨ES细胞向造血细胞定向诱导分化调控的机制也将会提高ES细胞体外诱导分化的效率。其次,诱导中使用的各种细胞因子多为基因工程产品,价格昂贵。此外,鼠源饲养层是目前常用的诱导方法。然而,鼠源饲养层所带来的异源污染极大地限制了该研究在临床上的广泛应用。
本研究中,我们首先探讨了前列腺素E2(prostaglandin,PGE2)在人ES细胞向造血细胞分化的作用并初步探讨了其机制,在此基础之上建立了转染促红细胞生成素(erythropoietin,EPO)基因的人胎肝基质细胞(human fetal liver stromalcells,hFLSCs),利用其条件培养基诱导人ES细胞向红系细胞分化,建立了一种新型诱导人ES细胞向造血细胞分化的方法,既降低了实验成本,又可以避免异源污染。
本研究主要包括两部分内容:
一、PGE2促进人ES细胞向造血细胞分化
人ES细胞在体外可定向诱导分化为造血细胞,尽管目前已建立了多种不同的诱导体系,仍处于起步阶段,细胞分化调控的机制尚不清楚,成为广大科研工作者研究的热点。
PGE2是一种重要的细胞生长和调节因子。研究发现PGE2在正常及缺氧、感染等异常情况下对造血发育都有重要的调节作用。近年来,研究表明PGE2能够促进造血干细胞的存活、增殖及内环境调节方面具有一定的作用。最近研究指出PGE2能够促进放射性损伤后的成年斑马鱼恢复造血重建能力,提示PGE2在胚胎的造血发育过程中发挥重要的作用。此外,PGE2也可促进小鼠ES细胞向造血干细胞分化。因此,我们推测PGE2在人ES细胞向造血分化过程中也发挥一定作用。
我们通过与OP9基质细胞共培养的方法考察PGE2对人ES细胞向造血细胞分化的影响:实验组添加PGE2,对照组不添加PGE2。结果发现,100ng/ml的PGE2可抑制集落的生长,20ng/ml的PGE2对集落生长有明显的促进作用,接种于半固体培养体系生成的造血集落最多,诱导产生的细胞具有向造血各谱系分化能力,加入PGE2抑制剂吲哚美辛后集落形成能力受到明显的抑制。为了观察PGE2对人ES细胞向造血细胞分化的影响,我们于显微镜下观察共培养细胞的形态学变化,结果发现不添加PGE2的情况下,人ES细胞接种于OP9细胞上,部分向上皮样细胞分化;部分形成血岛样的结构,随着培养时间的延长类似于血岛的结构会逐渐消失、变平。在加入PGE2的情况下,大部分会形成血岛样的结构,随着培养时间的延长血岛样的结构体积变大,但形态不会发生改变。收集共培养3～9d的细胞,用半定量PCR检测胚胎干细胞标志Oct4及造血和内皮相关基因的表达情况。结果表明,在不添加PGE2的情况下,诱导的细胞持续表达Oct-4,从培养的第3d开始表达中胚层标志Brachyury,早期造血标志CD34和内皮细胞标志VE-cad,从第5d开始表达造血调控相关基因Runx1,GATA1,SCL和内皮标志vWF。在添加PGE2的情况下Oct-4的表达从培养第3d开始降低,共培养的细胞从培养第3d开始表达Brachyury,CD34,Runx1,GATA1,SCL和VE-cad,从培养第5d开始表达vWF。这提示外源性添加PGE2可明显促进造血和内皮相关基因的表达。我们进一步考察了PGE2处理后Smad信号通路的变化。Western-blot结果显示,与对照组相比,PGE2处理能够上调p-Smad1/5的表达,同时Smad4的表达也上调。而加入PGE2的抑制剂后p-Smad1/5和Smad4的表达受到抑制,提示Smad信号通路可能参与了PGE2介导的人ES细胞向造血细胞分化。
二、EPO基因修饰的hFLSCs条件培养基诱导人ES细胞向红系细胞分化
在ES细胞体外诱导分化为造血细胞的过程中,研究造血微环境的调控机制是其发生和分化的重要环节。在胚胎发育的过程中,不同的造血微环境对造血的发生发挥着重要的作用。造血发生经历了卵黄囊造血、胎肝造血和骨髓造血三个阶段。人卵黄囊造血时期发生在胚胎的4～6周;胎肝造血时期发生在胚胎的6～22周;骨髓造血时期从22周至出生以后。胎肝是造血早期发育的主要位点,是造血细胞发育和分化的重要微环境,构成其微环境的胎肝基质细胞很有可能在造血细胞的发育和分化过程中发挥重要的作用。此外,血细胞的产生还受到造血干细胞内在基因和造血因子的共同调控,EPO是其产生的最重要的因子。EPO是一种糖基化的蛋白质激素,主要来源于肾脏和肝脏,而脑、肺、脾也能产生少量。EPO可促进造血干细胞向原始红细胞分化,加速幼红细胞的分裂、增殖,促进血红蛋白的合成,在红细胞诱导分化研究中发挥重要作用。而造血因子提供的方式对其作用的发挥也有重要的影响。研究表明以造血生长因子基因修饰的基质细胞比相同条件下的造血生长因子更能有效地调控造血。
因此,本部分研究中我们采用慢病毒系统建立了稳定高表达EPO基因的hFLSCs,能够高效分泌具有生物学活性的EPO蛋白,从而避免了添加外源性细胞因子,极大地降低了实验成本。随后,将人ES细胞培养于低黏附的细胞培养皿中诱导生成拟胚体(embryoid bodies,EBs),用过表达EPO基因的FLSCs条件培养基(EPO/hFLSCs-CM)诱导EBs细胞向造血细胞分化,未转染EPO基因的FLSCs条件培养基(hFLSCs-CM)作为阴性对照,未转染EPO基因的FLSCs条件培养基外源性添加重组的EPO(hFLSCs-CM+EPO)作为阳性对照。收集诱导不同天数的细胞,通过流式细胞术分析诱导过程中CD34阳性的表达,以确定不同培养条件下EBs向造血细胞分化的情况。结果表明三种不同的培养体系均能诱导人ES细胞向造血细胞分化。然而,hFLSCs-CM组的诱导效率较低,在诱导第12d中CD34阳性细胞低于11%。添加外源性EPO之后,人ES细胞向造血细胞定向分化的能力增强,其中CD34+细胞最高可达13.1%。EPO/hFLSCs-CM组能更为有效地促进人ES细胞向造血细胞分化,其中CD34+细胞可高达22.74%。此外,我们还应用实时定量PCR检测不同组细胞造血相关基因的表达情况;应用克隆形成实验检测了诱导细胞的造血集落形成能力;通过瑞氏-吉姆萨染色观查了诱导细胞的形态;应用联苯胺染色和RT-PCR方法检测了诱导造血集落珠蛋白的表达情况。结果表明,诱导生成的细胞具有造血细胞的一般特性,在半固体培养基中可分化为不同种类的造血集落,不同培养条件下产生的造血细胞的集落形成能力与珠蛋白的表达量不同,EPO/hFLSCs-CM诱导产生细胞的造血基因的表达量及所形成的造血克隆最多,且以红系克隆为主。此外,我们还检测了诱导产生的红系细胞的携氧能力。结果显示,EPO/hFLSCs-CM诱导的红系细胞具有和脐带血相似的“S”形氧离曲线,这表明体外诱导的红系细胞具有和脐带血相似的携氧能力。
综上所述,在本研究中我们初步考察了PGE2在人ES细胞向造血细胞分化过程中的作用并初步探讨了其作用机制;并利用稳定表达EPO基因的FLSCs条件培养基诱导人ES细胞向红系细胞分化,该方法可高效诱导人ES细胞定向诱导分化为造血细胞,同时降低了研究成本,且有效避免了鼠源饲养层带来的异源基因污染。上述研究为深入探讨造血细胞的发育调控机制及以胚胎干细胞或造血干细胞为启动细胞大规模地诱导产生红细胞奠定了一定的实验基础。
Currently , blood cells transfusion and hematopoietic stem cells (HSCs) transplantation are important means for cellular therapy. These methods are widely used in the treatment of incurable hematological disorder, genetic diseases, infectious diseases and immunologic deficiency. However, their availability is limited by quantity, capacity of proliferation and the risk of blood transfusion complications. So people try hard to obtain more safe, effective and economic resource of blood cells. Human embryonic stem cells (hESCs) derived from the inner cell mass (ICM) of preimplantation embryos possess indefinite proliferative capacity in vitro, and maintain pluripotency as well. They also have the capacity to differentiate into all cell types.
Recently, the potential of hESCs to differentiate into hematopoietic cells has been reported in several studies, suggesting clinical applications in cellular therapy, such as blood transfusion or hematopoietic stem cell transplantation, and treatment for hematological disorder. Currently, the major sources of transplantable HSCs for clinical therapy are human bone marrow (BM), mobilized peripheral blood and umbilical cord blood (CB), but their availability for clinical use is limited by both quantity and compatibility. Pluripotent hESCs may provide an alternative.
With the repid development of biology research, pluripotent human embryonic stem cells (hESCs) may provide a unique tool for hematopoietic transplantation and the study of human embryonic hematopoiesis.
Currently, various methods including cytokinses administration and co-culture with stromal cells have been used to promote the hematopoietic differentiation of embryonic stem cells (ESCs). Although hESCs are competent for development into hematopoietic cell fate, the cellular and molecular mechanisms that control this process are very poorly understood and yield heterogeneous outcomes. Furthermore, the risk of mouse-related disease, high cost of cytokines and low differentiation efficiency greatly limit the clinical applications.
In the present study, we first demonstrate the role for PGE2 in regulating hematopoietic differentiation from hESCs, then we establish a novel method which can induce hESCs differentiated into hematopoietic lineages safely, efficiently, and economicly.
1. Prostaglandin E2 promotes hematopoietic development from hESCs
Recent studies have suggested that PGE2 and the prostaglandin pathway are essential for hematopoietic stem cell growth, survival and development. These findings have led to a greater interest in the possible role of the prostaglandin pathway in human hematopoietic development. Here we evaluated the effects of PGE2 on hematopoietic differentiation of hESCs. The induced cells from hESCs/OP9 co-culture and in the presence of PGE2 were characterized by RT-PCR, flow cytometry, colony-forming, assays and Wright-Giemsa staining. Our results demonstrated that the hematopoietic colonies increased in a dose-depentent manner after exposure to PGE2. The expression of Oct-4 and hematopoiesis-inductive transcription factors and hematoendothelial specific genes, including Brachyury, CD34, SCL, GATA-1, Runx1, vWF and VE-cad were examined with RT-PCR. PGE2 exposure also altered the morphology of co-cultured hESCs and resulted in a robust hematopoietic differentiation with the higher frequencies of CD34+ and CD45+ cells. Furthermore, PGE2 supplement increased the activation of p-Smad1/5 and enhanced the expression of Smad4 compared to the non-treated counterpart. When the effect of PGE2 was blocked by its specific inhibitor indomethacin, p-Smad1/5 and Smad4 expression declined correlatively. This research may improve our knowledge of stem cell regulation, which hopefully will lead to improved stem cell-based therapeutic options.
2. Human fetal liver stromal cells expressing erythropoietin promote hematopoietic development from human embryonic stem cells
Development of hematopoietic system in human embryo involves several anatomical sites including the yolk sac hematopoiesis occurring from 4 to 6 weeks, the fetal liver hematopoiesis occurring from 6 to 22 weeks, and the bone marrow hematopoiesis occurring during the remainder of life. Different hemopoietic microenvironment plays important roles in the different stages. In the mode of hematopoietic differentiation from hESCs, the selection of microenvironment is an important part. The fetal liver is a unique hematopoietic organ where both HSCs and mature blood cells are actively generated. So we suppose that the fetal liver microenvironment could promote the hematopoietic differentiation from hESCs. On the other hand, the most important cytokine regulator of erythropoiesis is erythropoietin (EPO). It acts on erythroid progenitor cells, stimulates proliferation, promotes differentiation, and prevents apoptosis. The main EPO production site is the liver in the fetus and the kidney in the adult. As a conclusion, the combination of fetal liver cells and EPO must improve the erythropoietic differentiation. It was also shown that direct secreted cytokines from stromal cells exert their function better than medium supplement. Based on these understanding, a transgenic stromal line was built.
We have developed a novel method to promote the differentiation of hESCs toward hematopoietic lineages. The human fetal liver stromal cell (hFLSCs) expressing EPO were established using lentiviral system. We observed that the supernatant from the EPO transfected hFLSCs could induce hESCs differentiate into hematopoietic cells without exogenous cytokines.
In this experiment, hESCs were cultured in low cell-attachment dishes to obtain EBs. While the EBs were induced into hematopoietic cells by different inducing systems including hFLSCs-CM, hFLSCs-CM+EPO, and EPO/hFLSCs-CM after treated by BMP4. Then the expression of CD34 was analyzed by flow cytometry. For EBs treated with hFLSCs-CM CD34+ cells peaked to 10.57% on day 12. For the EBs treated with hFLSCs-CM+ EPO, the number of CD34+ cells reached the maximum of 13.1% at day 12. For the EBs treated with EPO/hFLSCs-CM, CD34+ cells peaked to 22.74% at 12 days of culture. These results demonstrated that EPO/hFLSCs-CM treated EBs could yield more hematopoietic cells than other groups. In addition, the genotypic expression was characterized by realtime RT-PCR. Among these different inducing systems, EPO/hFLSCs-CM had the highest hematopoitic gene expression level. The features of hESCs-derived erythroid cells were characterized by morphology, flow cytometry, wright-Giemsa staining, 3’3-diaminobenzidine staining, and RT-PCR. The results showed that the induced cells showed the feature of hematopoietic cells. Among the inducing systems, the EPO/hFLSCs-CM could promote hematopoietic differentiation of hESCs, especially into erythrocytes. Furthermore, the hESC-drived erythroid cells were also able to function as oxygen carriers, and exhibited an oxygen dissociation pattern similar to human CB.
In summary, our studies support an expected role for PGE2 in regulating hematopoietic differentiation from hESCs. We demonstrate that PGE2 treatment, together with OP9 stromal cell co-culture, promotes hematopoietic development from hESCs. Furthermore, we established the transgenic human fetal liver stromal cells (EPO/hFLSCs) that stably express EPO gene and used the conditioned medium to induce erythropoietic differentiation of hESCs. Furthermore, this method can avoid the mouse-related disease and cut down the cost of experiment. The system we developed will provide a reliable alternative for the research in the future therapeutic applications of human embryonic stem cells.
引文
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