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通讯作者:

谭韬(1981-),男,云南昆明人,教授,博士生导师,主要从事灵长类生殖与发育生物学研究。E-mail:tant@lpbr.cn。孙念琴,姚辉为共同第一作者

中图分类号:R329

文献标识码:A

文章编号:2096-8965(2022)04-0015-07

DOI:10.12287/j.issn.2096-8965.20220402

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目录contents

    摘要

    基于多能干细胞的异种嵌合是现代生物学研究的前沿,也是有望解决器官短缺这一世界性难题的主要途径之一。将具有不同多能性和发育潜能的干细胞显微注射到植入前囊胚,这些干细胞能“嵌合”到胚胎或胚胎外组织中,并产生部分或完全由其发育而来的组织、器官。异种嵌合体使产生可供移植的功能性人类器官在理论上成为可能。本文系统回顾了近年来以啮齿类和灵长类为主要研究对象的哺乳动物异种嵌合体研究进展,探讨了异种嵌合体研究面临的技术壁垒和伦理挑战,展望了异种嵌合研究的发展方向,希望为解决移植器官严重短缺问题提供新思路和新手段。

    Abstract

    Interspecies chimerism based on pluripotent stem cells is the frontier of modern biology research and one of the main techniques to solve the worldwide dilemma of organ shortage. Stem cells with different states of pluripotency and developmental potential are microinjected into preimplantation blastocysts, which can contribute to embryos or extra-embryonic tissues, and then produce tissues and organs partly or completely developed from stem cells. It makes in vitro generation of functional human organs for transplantation theoretically possible. This article systematically reviews current progress in interspecies chimeras in mammals (mainly rodents and primates), explores technological barriers and ethical challenges, and envisions the future directions in interspecies chimeras research, and hopes to provide new ideas and means to solve the serious shortage organs for transplantation.

  • 0 前言

  • 器官移植是终末期器官衰竭病人的唯一选择,全球每年大概有 200 万人亟待器官移植拯救生命,但平均器官供需比仅为 1∶20~1∶30。可供移植的捐献器官严重短缺,很多患者在等待中去世。据《中国器官移植发展报告 (2020)》中数据显示,我国目前器官移植供需比不足 1∶8,捐献器官数量远不能满足临床与患者的需求。将多能干细胞 (Pluripotent Stem Cells,PSCs) 显微注射到宿主胚胎中,异种供体细胞能够嵌合至空缺的器官发育生态位并生成由供体细胞发育形成的组织、器官[12]。然而,迄今为止,仅有大小鼠间胰腺互补获得成功,物种进化距离限制了人类细胞在动物体内的嵌合贡献[3]。啮齿类等小型哺乳动物与人类在器官大小和构造等方面差异显著,难以在其体内生成人类器官。猴、猪等大型哺乳动物是理想的嵌合器官生成宿主。最近的研究发现,人多能干细胞 (human Pluripotent Stem Cells,hPSCs) 能够嵌合到猪、猴胚胎中,有望破解鼠、兔等小型哺乳动物来源器官小、与人类器官构造差异大等问题,转而在猪、猴等大型哺乳动物体内生成符合人体器官移植要求的低免疫原性器官[45]。研究异种嵌合能为生成人造器官提供理论支撑和可行路径,有助于解析发育及疾病发生机理并为临床前药物筛选和毒理检测提供平台。开展异种嵌合研究对保障人口健康和抢占国际科学研究前沿高地意义重大且深远。

  • 1 哺乳动物异种嵌合研究进展

  • 嵌合体是指来自于不同基因型的细胞组成的生物体,即“Chimera”。Chimera 原指希腊神话中狮首羊身蛇尾的神兽,在 1907 年被德国科学家 H.温克勒引入学界,特指“嵌合体”。嵌合现象最早于 1646 年由费拉里乌斯在柑橘中观测到,而随着奥伊格斯特发现蜜蜂嵌合体,自然嵌合现象也被扩展到动物界。早期的嵌合研究集中于同一物种内,即同种嵌合。不同物种间的嵌合,即异种嵌合,直到 20 世纪 70 年代才被首次报道用于研究嵌合细胞在胚胎发育期间的谱系分化和命运决定。PSCs 能否形成嵌合体胚胎是评估干细胞分化潜能的黄金标准。随着本世纪初多能干细胞制备技术日益发展成熟[6-8],异种嵌合研究也相继取得突破进展。2008 年,Rossont等[9] 成功获得木鼠-小家鼠(Mus MusculusMus Caroli)异种嵌合体,发现木鼠胚胎干细胞对小家鼠包括生殖细胞在内的所有主要器官的发育有广泛贡献 (某些组织高达 40%),并能够分化成各种组织的细胞类型。之后,Kobayashi团队[2] 指出敲除了小鼠的 Pdx1 基因,制备了胰腺发生功能丧失小鼠囊胚,通过向 Pdx1-/-小鼠囊胚显微注射大鼠 PSCs,开创性的在小鼠体内生成了大鼠胰腺。这些工作无一不表明体外培养的具有不同发育潜能的干细胞显微注射到植入前囊胚能“嵌合”到胚胎或胚胎外组织中产生部分或完全由干细胞发育而来的组织、器官。

  • 啮齿类是嵌合研究最常用也是最经济的动物模型,但啮齿类与人类在器官大小和构造等方面差异显著,不能在其体内产生大小合适的人类器官。体型更大、与人类器官构造更相近的猪、猴等大型哺乳动物比啮齿类更适合作为异种嵌合器官生成宿主。已有研究成功获得了世界首例人-猪、人-猴嵌合胚胎[410],初步探索造成人类细胞难以在其他动物体内、胚胎中发生嵌合的原因,为解决异种嵌合效率低下等问题提供了新思路。因此,本文分别以啮齿类和灵长类为例综述异种嵌合研究进展。

  • 1.1 啮齿类动物异种嵌合体

  • 利用种间囊胚互补 (Interspecies Blastocyst Complementation) 技术产生异种嵌合体,在动物体内生成人类器官是当前有望解决世界范围内移植器官严重短缺的主要途径之一。1993 年,Chen 等[11] 首次提出囊胚互补的概念并证明小鼠种内嵌合的可行性,但通过囊胚互补成功的种间器官发生也只在小鼠和大鼠之间实现过。2010年,Kobayashi团队[2] 首先在小鼠体内生成了大鼠胰腺,但大鼠胰腺尺寸只有小鼠器官大小,不足以分离出治疗大鼠糖尿病所需的胰岛数量。此后,通过反向实验,他们将小鼠多能干细胞 (mouse Pluripotent Stem Cells, mPSCs) 注射到 Pdx1-/-缺陷大鼠囊胚中,生成了 mPSCs来源、大鼠器官大小的小鼠胰腺。在没有进行免疫抑制干预的情况下,形成的小鼠胰腺成功地使患有糖尿病的小鼠血糖水平正常维持了 370 天以上[12]

  • 尽管世界各地的实验室做了许多尝试,但截至目前,仍然没有在异种动物体内生成除胰腺外的功能性器官。Usui等[13] 曾试图利用种间囊胚互补技术生成大鼠肾脏,但敲除 Sall1 基因的小鼠胚胎并未能为大鼠肾脏“腾出”一个完整的生态位,由 mPSC 形成的肾脏包含不受 Sall1 表达影响的结构,如集合管、微血管。显微注射的大鼠多能干细胞 (rat Pluripotent Stem Cells,rPSCs) 也被发现能够挽救小鼠肺发育不全表型[14]。但无一例外,这些尝试都未能形成完整器官。虽然啮齿类与人在发育 (即胚胎大小和妊娠期) 方面存在显著差异,不适合作为嵌合研究转化宿主模型,但对啮齿类的研究为异种嵌合完成了大量的理论基础和技术储备。

  • 1.2 人-动物异种嵌合体

  • 人-动物异种嵌合研究由来已久,早期的研究聚焦生物医学领域,用于研究癌症和人类免疫紊乱等疾病,并不涉及体外生成异种嵌合器官。由人成纤维细胞 (Human Fibroblast) 得到的诱导多能干细胞 (induced Pluripotent Stem Cells,iPSCs) 具备分化为体内任意细胞的潜力,研究人员希望 iPSC 技术可以用于制造患者特异性器官,以解决移植器官短缺难题。据报道,naïve 状态的 hPSCs 在小鼠、猪、甚至兔和猴胚胎中几乎没有嵌合贡献,这可能源于多能干细胞状态、进化距离和/或基因组进化导致的发育不兼容或种间障碍[515-18]。但最近的研究发现,人扩展多能干细胞(human Extended Pluripotent Stem Cells,hEPSCs)在猴子胚胎中的嵌合效率能达到 4%,远远超过之前人-猪嵌合体胚胎的 1/100 000[4]。因此,下文主要以人-鼠、人-猪和人-猴嵌合体为例介绍异种嵌合研究进展 (见表1)。

  • 表1 人-动物异种嵌合体研究进展

  • 注:N-hPSC,naive human PSC; P-hPSC,primed human PSC; rs-hPSC,region-specific human PSC; hEPSC,human extended PSC; NHP-PSC,nonhuman primate PSC。

  • 1.2.1 人-鼠异种嵌合体

  • 异种嵌合体产生的前提是供体 PSCs 在宿主胚胎内具备嵌合能力。对啮齿类的研究发现能够形成嵌合体的PSCs仅限于naïve PSCs,人“naïve”状态的 hPSCs 在动物体内几乎不会发生嵌合[18]。 2013 年,Gafni等[19] 制备了一种新型“naïve”hPSCs,这种细胞具备与小鼠 naïve ES细胞高度相似的分子特征和功能特性,并与传统的 primed hPSCs 截然不同。将这种“naïve” hPSCs 注射到小鼠桑葚胚后,研究人员发现其能嵌合到小鼠胚胎的不同器官中,提示供体细胞的发育阶段可能是嵌合体形成的关键。随后,Wu 等[20] 从植入前和植入后胚胎的外胚层 (Epiblast, EPI) 细胞中分离出一种稳定的 primed多能细胞类型,它在克隆效率、细胞生长动力学、转录组、表观基因组和代谢特征方面与外胚层干细胞 (Epiblast Stem Cells,EpiSCs) 不同,是一种介于 naïve 和 primed 状态之间的多能干细胞。这种新发现的 PSCs 选择性地嵌合至着床后胚胎的后部区域,并在体外产生种内和种间嵌合胚胎[20]。邓宏魁团队[21] 也获得了类似状态的人类干细胞。最近报道的几种 naïve-like hPSCs 能够形成人鼠嵌合胚胎[222526],转录组分析发现这些 hPSCs 更接近胚龄第8-10天,而不是第6天的人外胚层细胞,表明它们处于中间状态而不是naïve状态[1727]。总之,这些研究表明,处于naïve和primed中间状态的hPSCs 更具备形成种间嵌合体的能力。

  • 1.2.2 人-猪异种嵌合体

  • 猪和人的器官在大小、解剖结构和生理学上都很相似,是理想的人类器官培育动物。2017 年, Wu等[5] 报道了世界上第一例人-猪嵌合体胚胎。他们将几种类型的hiPSCs显微注射到第7天的猪囊胚中,培养两天后,naïve 和中间态的 hiPSCs 都能形成人-猪嵌合囊胚,但 hiPSCs的嵌入可能干扰了猪的正常发育。在植入后的人猪嵌合胚胎中,中间态的 hiPSCs 表现出更高的嵌合贡献,并能分化为多种细胞类型。但是,猪胚胎中所有 hiPSCs 的嵌合水平比大鼠-小鼠嵌合体要低得多,只有大约万分之一的细胞是人类细胞[5]。异种嵌合器官携带宿主内皮细胞,会导致病人机体产生急性免疫排斥。 2020年,Das 等[25] 将 hiPSCs (BCL2+/+) 注射到 ETV2 (缺失致死) 缺失猪囊胚中,获得内皮细胞全部来自人类的人-猪嵌合胚胎,表明ETV2突变可以与其他基因突变联合产生免疫原性降低的外源性器官,表明理论上有望在猪体内产生低免疫原性嵌合器官。但就目前的研究来看,人-猪嵌合体仍然存在供体细胞嵌合效率低、发育不兼容和难以形成完整器官等难题。

  • 1.2.3 人-猴异种嵌合体

  • 进化距离和/或基因组进化导致的发育不兼容或种间障碍会限制嵌合体的形成。非人灵长类与人在基因、发育、生理和行为特征等方面都非常相似,胚胎发育差异小,种间障碍低。深入研究人-灵长类嵌合体有望破译物种隔离产生的人类-动物嵌合阻碍。与其他物种相比,使用非人灵长类作为嵌合宿主可减少生长因子配体、受体、细胞粘附分子和其他促进细胞整合和分化的调节因子的物种特异性差异[28]。据报道,人和非人类灵长类的 naïve PSCs 在注入宿主胚胎后的定植过程中难以保持有丝分裂活性,G1 期停滞并过早分化,不能有效地产生嵌合体,这与 Wu 等之前的发现一致[15]。2021 年,季维智等[4] 将hEPSCs显微注射到食蟹猴早期囊胚中,首次获得了人-猴嵌合胚胎,其能在食蟹猴植入后体外培养体系中培养至原肠胚阶段。分析发现 hEPSCs 能够嵌合至食蟹猴早期胚胎的几个主要谱系,同时指出 hEPSCs 对 EPI 的贡献较高 (受精后第 15 天的贡献最高,为 7.08%),对内胚层 (Hypoblast,HYP) 的贡献相对较低 (受精后第 19 天的贡献最高,为 4.96%)。据统计,人类细胞在食蟹猴胚胎中的嵌合效率在 4% 左右,远远超过之前人-猪嵌合体胚胎的1/100 000[5]

  • 深入研究嵌合胚胎中人、猴的细胞互作,研究人员发现人猴外胚层细胞之间存在特异的互作关系,在猴胚胎的微环境中,hEPSCs 与受体食蟹猴胚胎细胞的转录组特征互相变得相似。总的来说,人-猴嵌合体研究回答了异种嵌合细胞如何互作及发育程序差异如何调节等科学问题,为克服人-动物异种嵌合效率低下提供了新思路,为理解人类发展和进化、创建人性化动物疾病模型以及在动物身上生成可移植的人体器官奠定了基础[29]

  • 2 异种嵌合体研究面临的技术挑战

  • PSCs 状态是形成嵌合体的关键因素,能够形成种间嵌合体的供体细胞需具备能高效整合、与正常细胞周期速率相匹配的广泛增殖、归位于发育生态位、按照宿主细胞命运轨迹分化、以及抵御宿主细胞竞争的能力。传统的人和猴的 PSCs 保持在类似于 EPI 细胞的状态,这与内细胞团 (Inner Cell Mass,ICM) 具有的生物学特性完全不同[30]。通过 RNA 转染瞬时传递重编程因子,可以有效地将人体细胞重编程到 naïve 状态的 PSCs[31],但“naïve” hPSCs印记基因容易丢失,基因组很不稳定。限于技术水平,目前很难捕捉到稳定态的鼠、兔、猪、猴和人等的 naïve PSCs,也未在任何物种中报道过真正能够自我更新的全能干细胞在培养中表现出内在稳定性[32]

  • 供体细胞和宿主细胞之间的竞争是难以产生嵌合器官的另一个主要原因。与宿主细胞相比,异种供体细胞常作为相对较弱的竞争对手而被清除,导致没有足够的细胞补充缺失器官的发育生态位[33]。 Qin等[34] 开发了一种克服人和小鼠 PSCs细胞竞争的策略,从而提高了早期小鼠胚胎中人类细胞的存活率和嵌合效率。但不同 PSCs 环境下运行的细胞竞争机制也存在多样性[35],这为克服细胞竞争增加了难度。破译细胞适应性是如何在群体中被感知,以及不适合的细胞是如何被清除,将对控制干细胞命运能力产生重大影响。

  • 如何确保嵌合胚胎正常发育也是种间嵌合体产生的固有技术挑战。种间器官发生的先决条件是嵌合体能正常形成。高嵌合贡献对器官形成有利,但却容易导致受体胚胎死亡,尤其是亲缘关系较远的异种嵌合往往会由于高嵌合贡献导致发育不协调而致使胚胎死亡。许多生物学因素也会影响供体细胞的贡献程度,包括细胞分裂率,人与宿主细胞之间发育进展的动力学差异,供体细胞嵌合位置与效率,物种间细胞-细胞相互作用差异等[28]

  • 3 人-动物嵌合体研究的伦理挑战

  • 人-低等动物嵌合研究由来已久,主要的伦理问题为:动物福利,供体细胞来源。虽然,所有的实验动物管理与使用委员会 (Institutional Animal Care and Use Committee,IACUC) 及其同等的国内外评审委员会都对动物实验研究开展标准的伦理审批,但嵌合体因其特殊性,仍然可能存在一些特殊问题,例如改变动物的感觉或行为。对此,可以借鉴转基因动物相关规定进行审查。

  • 长期以来,人-非人灵长类嵌合体的创造一直是公众批判的对象[36],指责嵌合体模糊了两个物种之间的界限[37]。对于反对人-动物嵌合体研究的理由主要集中于嵌合体的道德地位及潜在的社会问题[38]。尤其是人-非人灵长类嵌合胚胎的伦理是复杂且关键的[39]。人们担忧人类细胞会在嵌合胚胎内出现神经和生殖系统嵌合[40],造成人-动物嵌合体 “人化”,包括在动物身上表现出独特的人类心理特征以及动物大脑中出现人类细胞,并产生类人的认知能力和意识以及类人的外观[41]。因此,研究人员应该仔细分析配子和中枢神经系统中人类细胞的嵌合程度,并事先确定人类细胞贡献的最大阈值,同时采取规避手段。鉴于体外人-猴嵌合体研究在推进再生医学方面的前景,相关研究应在符合当地法规、社区参与,并受适当伦理监督的情况下,以谨慎的分步方式进行研究。

  • 4 异种嵌合体潜在转化应用与前景

  • 当胚胎嵌合体与分子工具结合使用时,嵌合体为精确、深入和大规模分析基因功能提供了无限的选择。嵌合体分析有望成为对人和其他哺乳动物胚胎和成体组织干细胞特征进行体内评估最全面和严格的必要工具[42]。已经报道的包含人类组织细胞的种间嵌合体,包括肝脏[43]、神经干细胞[44] 和造血免疫系统[45] 等,表明在人-小鼠和人-猪嵌合体中异种器官重塑具有可行性 (见图1)。

  • 图1 人-动物嵌合体产生异种器官[46]

  • 过表达抗凋亡基因和诱导增殖可能促进嵌合体形成,但抑制 iPSCs凋亡会有致瘤风险。对注入的供体干细胞进行基因编辑能触发其定向分化,并对特定组织或器官产生贡献,这使得器官衰竭终末期患者接受自体器官移植治疗成为一种可能。人-动物嵌合体不仅在缓解器官短缺方面具有巨大前景,还是研究器官发育、发病机制、免疫防御和药物筛选的可靠模型。借助人-动物种间嵌合体可为个性化药物的药效、毒性试验提供更好的体内药物筛选平台,以便更准确地开展临床前药物毒理学研究和预测临床结果。通过对宿主动物和供体细胞进行基因编辑能够获得低免疫原性异种供体器官[47]。2022 年 1 月 7 日,David Bennett 接受了猪 (10 处基因修饰) 心脏移植,并存活2个月,这例突破性的手术表明异种器官移植存在可行性[48]。未来,利用基因工程猪生产出适合移植的人性化器官并延长患者整体存活时间是嵌合器官研究的理想方向。

  • 5 展望

  • 异种嵌合体在器官移植、开发新药和促进干细胞研究以及研究人类疾病进程等方面具有巨大潜力。未来,人-动物嵌合体研究应该逐步推动实现 hPSCs在嵌合模型中的全部潜力,包括探索更广泛的人类供体细胞;优化供体和宿主细胞间的竞争平衡,以获得更高的嵌合水平;了解胚胎微环境所起的作用;扩大细胞在嵌合环境中如何适应和变化的研究;探索细胞-细胞双向交流[49]

  • 2021 年,国际干细胞研究学会 (International Society for Stem Cell Research,ISSCR) 更新了其指导方针[50],将涉及动物的干细胞研究形式划分为两种,并呼吁进行不同程度的监督。因此,人-动物,尤其是人-非人灵长类种间嵌合须在严格的伦理审查和监督中进行,需要平衡促进涉及部分人类嵌合体的伦理研究,并防止进行具有不确定性和潜在实质性道德地位的不道德的嵌合体实验。另外,有必要重新设计对部分人类嵌合体研究的监管[51]

  • 在伦理层面之外,人-动物异种嵌合体研究对于理解人干细胞多能性和人类早期发育具有重大意义,特别是在制造可供移植的异种嵌合器官方面,很可能会超越目前设想的转化益处。但在临床应用之前,异种嵌合还有很长的路要走。

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