造血幹細胞多樣性應用之研發：血小板前驅細胞-巨核細胞的體外誘導增殖技術

Ex vivo induction and expansion of megakaryocyte

陳德偉(Te-Wei Chen)

指導教授 ： 朱一民

國立清華大學/工學院/化學工程學系/博士(2009年)

https://doi.org/10.6843/NTHU.2009.00018

摘要

巨核細胞(Megakaryocytes，Mks或是CD41a+CD61+細胞)是由造血幹細胞(Hematopoietic stem cells，HSCs)分化而來，在骨髓中成熟並且生成以及釋放血小板(platelets)，因此被稱為血小板的前驅細胞(progenitor)。病患在接受高劑量化療後的幹細胞移植過程中，首先藉由體外誘導培養提升巨核細胞數量，進而與幹細胞共同移植後能夠加速血小板的恢復時間。在本研究中，使用無血清增殖過後的造血幹細胞為細胞來源，藉由二階部分因子實驗設計結合陡升路徑法針對巨核細胞來開發一個無血清、無滋養層以及具有最適細胞激素添加的體外誘導培養系統(SF-Mk medium)：Iscove’s modified Dulbecco’s medium (IMDM)中含有HIT (8 g/L HSA，1.8 μg/ml Insulin，50.5 μg/ml Transferrin) + CC-Mk (3.0 ng/ml TPO，2.9 ng/ml IL-3，12.5 ng/ml SCF，0.5 ng/ml IL-6，1.4 ng/ml FL，1.7 ng/ml IL-9以及7.3 ng/ml GM-CSF)。經過無血清培養基(SF-HSC medium)增殖過後的造血幹細胞，將其培養在SF-Mk培養基中一個星期後，CD41a+CD61+細胞累積數量之增殖倍率可達4000倍以上。
SF-Mk培養基在誘導與增殖CD41a+CD61+細胞的能力上，是優於Panserin 401TM，X-VIVO 10TM，X-VIVO 15TM，X-VIVO 20TM，PRO 293TM等商業無血清培養基。此外，經過無血清培養基誘導出之巨核細胞皆能保有其功能性(包含：CD41a以及CD61專有表面抗原表現，NF-E2與GATA-1基因表現，多核性分佈表現，以及體外活化血小板能力)。在動物實驗的結果上顯示，經過放射線照射後之NOD/SCID小鼠移植人類巨核細胞之後，在短時間內可以觀察到人類血小板出現於小鼠週邊血液。
因此，我們所開發之SF-Mk培養基具有低細胞激素添加濃度、高經濟價值、短誘導時間以及高巨核細胞誘導能力等優勢；誘導後之巨核細胞皆有表現出正常功能且能在NOD/SCID小鼠體內加速人類血小板的生成。結合SF-HSC與SF-Mk培養基的全程無血清環境培養下生成巨核細胞，可以提供一個適合的巨核細胞與血小板細胞來源，未來在解決臨床上病患血小板恢復時間過長的問題，是具有應用淺力的。

關鍵字

造血幹細胞 ； 巨核細胞 ； 體外誘導 ； 無血清

並列摘要

Megakaryocytes (Mks, CD41a+CD61+ cells) are the progenitor cells of platelets, and they differentiate from CD34+ hematopoietic stem cells (HSCs) and progenitor cells through complex development processes in the bone marrow (BM). Increasing the number of Mk by ex vivo induction and expansion culture for stem cells transplant may provide an approach to accelerate platelet reconstruction in patient after high-dose chemotherapy. In this study, a serum-free, stromal-free and optimal cytokine cocktail containing medium (SF-Mk medium: HIT (8 g/L HAS, 1.8 μg/ml Insulin, 50.5 μg/ml transferrin) + CC-Mk (3.0 ng/ml TPO，2.9 ng/ml IL-3，12.5 ng/ml SCF，0.5 ng/ml IL-6，1.4 ng/ml FL，1.7 ng/ml IL-9以及7.3 ng/ml GM-CSF) in Iscove’s modified Dulbecco’s medium (IMDM)) was established by 2-level factorial design and steepest ascent path methods for Mk generation from serum-free expanded CD34+ cells. After serum-free induction, the maximum expansion for the accumulated Mks increased over 4000-fold.
Furthermore, SF-Mk medium was superior and comparable with Panserin 401TM, X-VIVO 10TM, X-VIVO 15TM, X-VIVO 20TM and PRO 293TM commercial serum-free medium in induction of Mks. The serum-free induced Mks were characterized by surface marker expression of CD41a and CD61, gene expression of NF-E2 and GATA-1, polyploidy distribution, and platelet activation ability. Importantly, transplantation of serum-free induced Mks could accelerate human platelet recovery in NOD/SCID mice. In conclusion, we have developed a serum-free Mk induction (SF-Mk) medium, and the combination of SF-Mk and SF-HSC media can generate a large amount of functional Mk efficiently. Our method may represent a promising source of Mk and platelets for future cell therapy.

並列關鍵字

Hematopoietic stem cells ； Megakaryocytes ； Ex vivo induction ； Serum-free

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