不同培养体系对鼠-猪异种嵌合胚胎发育和嵌合能力的比较

In vitro development and chimeric efficiency of mouse-porcine interspecies chimeric embryos in different culture systems

摘要:

近年来，随着干细胞分化与再生医学研究的不断深入，异种嵌合已成为当前干细胞和再生医学领域的热点问题，并有望为未来解决器官移植供体来源严重短缺等再生医学难题开辟新的方向。异种嵌合以及异种器官再造过程中面临众多科学问题和技术难题，而异种嵌合过程中嵌合胚胎时期的选择，后续培养液的选择以及这些环节所造成的供体细胞与受体胚胎之间的发育平衡成为建立异种器官再造的第一个科学问题。猪由于具有与人类器官大小相似、繁殖快等特点，成为异种嵌合最适合的潜在研究对象。为了提高鼠-猪异种嵌合胚胎中小鼠供体细胞——诱导多潜能干细胞 (Induced pluripotent stem cells, iPSCs) 的存活率和增殖率，我们尝试以iPSCs培养液 (N2B27) 以及N2B27→PZM-3梯度更换的培养液 (N2B27(3.5 h)) 作为研究异种嵌合胚胎体外发育培养的对象，并与猪胚胎培养液 (PZM-3，Porcine zygotic medium)体系下发育进行比较，从而评价了这3种培养液在8-细胞和囊胚期注射后，对嵌合胚胎后续发育的影响及嵌合情况。结果显示，8-细胞期注射后，PZM-3不仅对嵌合胚胎的后续发育较为有利，更有利于小鼠iPS嵌合到猪胚胎中；囊胚期注射后3种培养体系下GFP阳性嵌合率差异不显著，但其嵌合率显著低于8-细胞期嵌合率。结果表明，PZM-3培养体系更有利于鼠-猪异种嵌合胚胎的体外发育，对8-细胞期胚胎进行嵌合操作有益于提高鼠-猪异种嵌合后胚胎的嵌合率。

Abstract:

With the advancements of stem cells and regenerative medicine, interspecies chimera has become a hot topic and will pave a new way of providing donor sources in organ transplantation. However, the interspecies chimera is confronted with a number of scientific questions and technical obstacles, including selections of appropriate embryonic stage and appropriate culture medium; those factors will deeply influence the developmental balance between donor cells and receptor embryos. Due to its relatively rapid reproductive cycle and similar organ size to human’s, porcine is a very potential donor candidate to study these questions. To compare the development and chimeric efficiency of interspecies embryos, we tested and evaluated three different culture systems, PZM-3 (Porcine zygotic medium), culture medium for iPSCs (N2B27) and 3.5 h of N2B27 before PZM-3 (N2B27(3.5 h)), and two different embryonic stages, 8-cell and blastocyst in mouse-porcine chimeric embryos using parthenogenetically activated porcine embryos and mouse induced pluripotent stem cells (miPS). The results showed that, PZM-3 was beneficial for both development of chimeric embryos and miPSCs proliferation in porcine embryos in the 8-cell injection group. After early blastocyst injection, the chimeric efficiency did not appear significantly different among the three culture systems but was lower than 8-cell injection. In summary, the results suggest that 8-cell injection and PZM-3 culture medium are more beneficial to the in vitro development and chimeric efficiency of mouse-porcine chimeric embryos.