以Osr1基因剔除之小鼠胚及腎臟囊胚代償技術產生外源性腎臟之可行性

Abstract

因為現代的生活習慣的演進，腎臟衰竭愈發常見，相較於其他方式，目前最理想的治療方式為器官移植，而目前器官移植來源短缺的問題非常嚴重，如何另闢器官移植來源的途徑，儼然是再生醫學的重要目標之一。而利用多分化潛能幹細胞 (pluripotent stem cells, PSCs) 或許是可行的方法之一，但器官生成還需組織和細胞內部之間的複雜的相互溝通，難以僅用體外培養環境達到生成如腎臟等組成複雜的器官。而作為替代方案，科學家們開發了囊胚代償 (blastocyst complementation) 的技術，能夠經過基因編輯後的胚剔除掉生成特定器官的基因，以PSCs進行囊胚代償，期望能彌補該缺陷並生成缺少的器官，達到在體內環境誘導分化成目標器官的目標。 由Kobayashi等人於2012年以Sall1基因剔除進行囊胚代償的實驗中，小鼠的腎臟雖然完全由外源性的PSCs細胞所構成，但出生後的仔鼠卻因為此基因會影響到腦部發育，PSCs無法代償至腦部缺損的部位，導致仔鼠不具備尋找乳頭吸取乳汁的能力而早夭。因此本試驗挑選另一與腎臟發育有關，但不影響腦部發育的相關基因Osr1進行剔除並進行囊胚代償。首先確認Osr1的基因編輯效率，參考Wang於2005年發表的Osr1文章中提到，大部分的純合子剔除胎兒會在胚胎發育時間12.5-13.5天死亡，本試驗共移置了66個基因編輯後的囊胚至受胚母鼠，並於12.5或13.5天時取出分析生長狀況，而其中有34個著床點，仍有胎鼠形狀的有13個，正在被母鼠吸收者有21個。後續進行囊胚代償試驗，原預期囊胚代償能夠救援Osr1基因編輯的胚移置於受胚母鼠，並使其產下仔鼠，預計產下的組別中有三隻受胚母鼠都無產下子代，因此在12.5、13.5、15.5天提前進行取樣，以H E及免疫染色觀察代償後的胚胎之發育。在免疫染色的切片顯示有極低的機率成功代償出大部分由外源性細胞所組成的腎臟，但目前的試驗結果顯示以Osr1基因剔除進行囊胚代償的策略尚未成功產下具有外源性腎臟的胎兒。 綜觀上述，本試驗之結果將有助於了解Osr1進行囊胚代償之可行性，並提供再生醫學工程產製外源性腎臟相關研究之參考。顯示Osr1雖不影響到腦部發育，但剔除後造成其他器官的缺陷如心臟等，是否可能經由囊胚代償補足胎兒的發育所需仍有待進一步釐清。

Due to the evolution of morden lifestyle, kidney failure is becoming more common over time. Compared with other solution, the most ideal treatment of the kidney failure patients is organ transplantation. But the shortage for organ transplantation is a severe problem. The use of pluripotent stem cells (PSCs) may be one of the feasible methods, but organogenesis requires complex communication between tissues and cells. It is difficult to generate complex organs such as kidneys by using in vitro culture system. As an alternative, scientists have developed blastocyst complementation technology, first, to knockout the embryo’s ability to generate specific organs through gene editing tools, and via blastocyst compensation, it is expected that PSCs will rescue the defects and generate the deficient organs, in order to achieve the expectation of differentiation into target organs through the in vivo environment. In Kobayashi et al.'s experiment of blastocyst compensation with the Sall1 gene knockout in 2012, although the kidney of the mouse was completely composed of exogenous PSCs cells, the neonate mice dead soon after birth because of this gene will affect the development of brain, PSCs cannot be compensated to the location of the brain defect, leading to the premature death of the pups without the ability to find nipples suckle. In this experiment, another gene Osr1 related to kidney development but not affecting brain development will be selected for blastocyst complementation. First experiment is to confirm the gene editing efficiency of Osr1, referring to the Osr1 article published by Wang in 2005, most of the homozygous knouckout fetuses died within E12.5-13.5 days. To confirm the efficiency of gene editing, 66 gene edited embryo were transferred in our experiment, and were harvested the fetus on E12.5 or 13.5 to analyze the growth status, and 34 of them were implanted, 13 were normal, and 21 were being resorbing by the mother. And then blastocyst complementaion experiment was expected to birth the Osr1 gene-edited embryos transferred to the pseudopregnancy mice and would allow them to give birth to pups. It is expected that three pseudopregnancy mice in the group but none of them gave birth to pups. For the next experiment, harvest the fetus in on E12.5, 13.5 and 15.5 days, and observe the development of the compensated embryos with H E and immunostaining. Immunostained sections show a very low probability of successfully complement to kidneys and mostly composed of exogenous cells, but the current test results show that the Osr1 gene knockout strategy for blastocyst complementation haven’t success so far of give birth to complement pups. To sum up, the results of our experiment would like to help to understand the feasibility of Osr1 for blastocyst complementation, and provide a reference for the research on the production of exogenous kidneys by regenerative medicine engineering. Further data shows that Osr1 does not affect brain development, but the complement of defects in other organs such as the heart, and may still need more experiment to confirm.