綠色螢光小鼠間葉幹細胞於早期胚胎發育之分化潛能

The Differentiation Potential of Mesenchymal Stem Cells Isolated from EGFP Transgenic Mice in Early Embryogenesis

顏雅萍(Ya-Ping Yen)

指導教授 ： 吳信志

國立臺灣大學/生物資源暨農學院/動物科學技術學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.02930

摘要

近年來幹細胞及再生醫學相關研究蓬勃發展，尤其是「胚幹細胞」(embryonic stem cells, ESCs) 及諸多不同組織來源之「成體幹細胞」(adult stem cells, ASCs)已被成功分離、純化與定性，且大多已證明具備多分化潛能之特性。目前並有研究指稱將成體幹細胞藉由顯微操作方式送入囊胚腔後，確可成功分化為各胚層細胞。有鑑於此，本研究室選用源自小鼠骨髓之成體幹細胞作為試驗處理組，並以源自小鼠胚幹細胞作為本研究之對照組，觀察比較源自成體幹細胞與胚幹細胞二者分別於早期胚中嵌入及分化之潛能。
研究中所使用之綠色螢光小鼠之骨髓間葉幹細胞 (enhanced green fluorescent protein-mesenchymal stem cells, EGFP-mMSCs; type I and II) 乃分離自攜帶β-actin啟動子之綠色螢光蛋白 (enhanced green fluorescent protein, EGFP) 之轉基因小鼠骨髓液或骨頭，並分別以兩種純化方式，分離獲得之小鼠骨髓及骨間葉幹細胞。在完成初代培養並經純化獲得之EGFP-mMSCs type I及II，以不同細胞數為組別，分別送入受精後第3.5日齡小鼠胚之囊胚腔中，再經胚移置於代理孕母子宮中，俟胚在體內發育達第6.5及9.5日之際，分別進行觀察各胎小鼠對EGFP之進一步表現情形，藉由螢光顯微鏡觀察外並透過聚合酶連鎖反應 (polymerase chain reaction, PCR) 分析，試驗結果確認EGFP於胚胎6.5及9.5日之表現，顯示EGFP-mMSCs以囊胚注射方式於早期胚胎不具有分化能力。試驗二中，乃以8細胞至桑椹胚 (morula) 時期胚細胞與EGFP-mMSCs type I及II進行細胞聚合方式，並於體外培養至3.5天囊胚時期及4.5天孵化 (hatch) 時期，再透過免疫螢光染色(immuno-fluorescence stain) 進行偵測EGFP-mMSCs type I及II於胚胎內之表現情形。試驗結果亦顯示，前述方法聚合之成體幹細胞，亦未達順利於早期胚胎發育中進行分化及增生。
由以上結果顯示，小鼠胚幹細胞以囊胚注射方式將細胞送入囊胚後可與囊胚共同增生分化至成體，然而骨髓及骨間葉幹細胞卻無此能力，與原先假設骨髓及骨間葉幹細胞具有多分化潛能有所出入，同時為探究為何EGFP-mMSCs type I及II於胚胎發育過程中即不再具有表現，初步懷疑是否此種細胞於胚胎發育環境下會進行細胞凋亡作用 (apoptosis)，因此試驗過程中以半胱天冬酶-3 (caspase-3) 與EGFP進行免疫螢光複染，然而目前尚無任何證據可以證明EGFP-mMSCs於胚胎體內消失的原因為何。
骨髓間葉幹細胞雖於體外不同條件培養後可分化為各個胚層的細胞，堪稱為具有多能性之幹細胞。但在體內試驗中，因其生長環境不符合其分化條件，且此種細胞乃為成體分離之幹細胞，與早期囊胚內之未分化微環境 (niche) 相異，且內細胞群細胞 (inner cell mass) 與骨髓間葉幹細胞之膜蛋白質組成或許有極大差別，以至於它們在細胞之間的聚合作用亦有很大的阻礙。綜合以上所述，在本試驗研究條件下，骨髓間葉幹細胞之多能性表現只受限於適當的體外分化條件下始具有此特性。

關鍵字

成體幹細胞 ； 胚幹細胞 ； 骨髓間葉幹細胞 ； 多能性 ； 囊胚

並列摘要

In recent years, many tissue specific stem cells had been successfully isolated and transdifferentiated into lineages other than the tissue of origin. Several studies have reported that bone marrow derived mesenchymal stem cells (MSCs) possess the potency to differentiate into various lineages. Recent study also indicated that a tissue specific stem cell, neural stem cells could contribute to many tissues of the chimera mouse when injected into a blastocyst. Therefore, the object of this study was to investigate the differentiation potential of MSCs in vivo. The ability to contribute to the embryogenesis was determined by introducing them into the early embryonic environment and observing the fate of their progeny.
In this study, type I and type II EGFP-mMSCs were isolated from the femur of transgenic mice carrying s-actin promoter constructed with enhanced green fluorescent protein (EGFP) cDNA serving as a tracing marker. Grouping by cell numbers, EGFP-mMSCs were injected to C57BL/6JNarl blastocysts and injected embryos were transferred to the uterus of the pseudo-pregnant foster mothers to assess the potency of EGFP-mMSCs to integrate into inner cell mass (ICM) during early embryonic development. The results of fluorescence microscopy and polymerase chain reaction analysis showed that EGFP-mMSCs injected embryos failed to give rise to chimeras and EGFP signal could not be observed in conceptuses day 9.5 and day 6.5. In contrast, EGFP embryonic stem (ESCs) could proliferate spontaneously with the ICM and contribute to all cell lineages in vivo. Compared with ESCs, mMSCs don’t have the ability to differentiate in vivo. To test whether mMSCs have the stem cell plasticity via cell aggregation, type I and II EGFP-mMSCs were aggregated with eight-cell or morula stage embryos and further cultured to blastocyst and hatching stages. Analysis on preimplantation embryos by immunofluorescence stain showed that type I and II EGFP-mMSCs could not aggregate with early embryos, suggesting that these somatic stem cells are unable to participate in early embryogenesis.
Results in this thesis revealed that the hypothesis may be false that mMSCs could not integrate into the host embryos during early embryogenesis. The reason injected mMSCs disappear in day 6.5 embryos is required to be clarified, but cell death is a likely answer. Putatively, mMSCs could not aggregate with eight-cell or morula stage embryos which may due to the incompatible cell niche or microenvironment. In addition, the composition of membranous protein of mMSCs and inner cell mass may be enormously different which could be the reason why these cells would not aggregate with each other. Taken together, although mMSCs can differentiate to tri-lineage tissues via in vitro induction, the microenvironment in early embryos might not support for mMSCs survival and differentiation.

並列關鍵字

adult stem cells ； embryonic stem cells ； mesenchymal stem cells ； pluripotent ； blastocys

參考文獻

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