利用化學藥物有效率地將皮膚纖維母細胞轉換成寡突細胞的譜系

Abstract

寡突細胞是僅存在於中央神經系統中的膠質細胞，其主要的功能是提供神經細胞養分和結構上的支持及隔離 (髓鞘)，保護並促進神經的信號傳遞。寡突細胞的受損或功能失調會導致不同的脫髓鞘病變。而其中以多發性硬化為最為常見的脫髓鞘病變之一。然而雖然抗發炎藥物的治療在疾病初期能有效地減緩多發性硬化的病情，可是越後期成效越不佳，且無法治癒由免疫細胞攻擊myelin basic protein (MBP)所造成的脫髓鞘病變。所以現今人們開始嘗試以細胞治療來解決脫髓鞘病變的問題。此係利用具有正常功能的寡突前驅細胞去取代或協助產生能正常進行髓鞘再生的寡突細胞，以重新進行髓鞘包覆來治療或減緩病情。 在過去的文獻中，人類寡突譜系的細胞主要從胚胎幹細胞或誘導性多功能幹細胞的分化來取得。然而胚胎幹細胞和誘導性多功能幹細胞分化產生寡突譜系細胞分化需耗時45天、為高成本、有致癌或病毒基因載體的殘留的可能等風險。 為了降低這些風險及降低細胞療法的成本，我們嘗試建立以化學藥物來誘導轉化人類纖維母細胞成寡突譜系細胞的方法。在呂仁老師實驗室中，已篩選出Y27632 (Rho-associated, coiled-coil containing protein kinase抑制劑)、basic fibroblast growth factor (bFGF)、SU9516 (cyclin-dependent kinase抑制劑) 是初步誘導皮膚纖維母細胞產生類似寡突譜系細胞型態上的變化的複合配方 (含九至十種成分)所必須的。本論文中，不使用複合配方時，我們進一步發現單一加入Y27632 或SU9516就足以產生細胞型態上的變化。且僅同時加入Y27632、bFGF及SU9516， 就足以產生表現寡突譜系細胞標誌O4的細胞。進一步以G06983 (protein kinase C抑制劑) 取代bFGF， 或前二天加入Valproic acid (VPA)，就足以產生更多更分化的寡突譜系細胞，並表現O4表現標誌。 我們接著尋找在常用的23個藥物中，是否有藥物可進一步促進寡突譜系細胞的生成。我們發現Forskolin (Fsk)可促進化學藥物在三天內誘導轉化成寡突譜系細胞的能力。而其轉化效率超過八成。轉化而成的細胞能表現多個寡突細胞譜系的標記所染上，例如O4、Olig2、Sox10以及 Myelin basic protein (MBP)等。 依此研究結果，我們有望在三天內高效率取得病人自體皮膚細胞所轉分化的寡突譜系的細胞，並使之應用到脫髓鞘疾病的細胞治療或藥物篩選中。

Oligodendrocytes are the glial cells reside in the central nervous system, which play critical roles in the transmission of neural signals and neural protection by myelinated the neurons. It supports the nutrients of neuron and provides physical barrier between different neurons. Dysfunction or loss of oligodendrocytes leads to many different demyelination diseases. Multiple sclerosis (MS) is one of the most common demyelination disease which is caused by autoimmunity or genetic mutation that the immune system target at myelin basic protein (MBP). Anti-inflammatory drugs can ameliorate the symptoms of MS at the early stage. However, anti-inflammatory drug doesn’t work effectively in later stage and cannot reverse the symptom that caused by demyelination. Thus, cell therapy with oligodendrocyte progenitor cells (OPCs) to regenerate the functional oligodendrocytes and then restore the myelination shed lights on the next generation treatment of MS. In previous studies, human oligodendrocyte lineage cells are derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). However, the differentiation protocol required more than 45 days which is labor consuming and high cost. Besides, the ESC/iPSCs derived cells may have the biosafety risks of tumor formation and virus insertion mutagenesis. To reduce the cost and biosafety risk, we try to establish a method to convert human dermal fibroblasts into oligodendrocyte-lineage cells with defined chemicals. Previously, in Dr. Jean Lu’s laboratory, Y27632 (Rho-associated, coiled-coil containing protein kinase inhibitor), basic fibroblast growth factor (bFGF), and SU9516 (cyclin-dependent kinase 2) was suggested to be essential for a cocktail with nine to ten factors to induce morphological change of skin fibroblasts. Here, we further demonstrated that in the absence of 9-10 factor cocktail, only one chemical, Y27632 or SU9516, is sufficient to induce different morphological changes. Of note, combination of three factors, Y27632, bFGF, and SU9516 is sufficient to transdifferentiate fibroblasts into oligodendrocyte lineage cells which expressed O4 marker. To obtain O4 positive oligodendrocyte lineage cells with more differentiated morphology, we substitute bFGF with G06983 (protein kinase C inhibitor), and add Valproic acid [VPA, histone deacetylase (HDAC) inhibitor] in the first two days. Next, to further improve the protocol, we screen 23 common inhibitors or activators. We found the addition of Forskolin (Fsk) can reprogram the fibroblasts into oligodendrocyte lineage cells within 3 days with the efficacy more than 80%. The transdiffeneitated cells expressed multiple oligodendrocyte lineage markers, such as O4, Olig2, Sox10 and MBP. By this study, we can efficiently obtain the autologous oligodendrocyte-linage cells by transdifferentiating skin fibroblasts into oligodendrocyte lineage cells within 3 days. There cells can be applied in the cell therapy or drug screening of demyelination diseases.