TWI487788B - 無致瘤性多能性幹細胞之增殖 - Google Patents
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- TWI487788B TWI487788B TW101113147A TW101113147A TWI487788B TW I487788 B TWI487788 B TW I487788B TW 101113147 A TW101113147 A TW 101113147A TW 101113147 A TW101113147 A TW 101113147A TW I487788 B TWI487788 B TW I487788B
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Description
本發明係關於人類多能性幹細胞之增殖，且特別是關於無致瘤性之人類胚胎幹(hES)細胞之增殖。
人類胚胎幹(hES)細胞為多能性的，且其具有分化為成人大多數細胞種類的強大能力。因此，該等細胞是再生醫學的強大保證。該等hES細胞令人嚮往的特殊性質包含不死性、多能性及無限制的未分化生長。傳統上，多能性胚胎幹細胞係培養於滋養層(feeder layer)細胞(如小鼠胚胎纖維母細胞(MEFs))上，以維持該等細胞於未分化狀態。該滋養層對於維持該等細胞是必要的；且該等hES細胞通常培養於該滋養層之層上直到需要分化時。不幸地，該等小鼠滋養層維持細胞經常與hES細胞培養而受到汙染，雖對於小鼠滋養層維持細胞不會造成多大功能上的影響，仍因此使培養於小鼠滋養層細胞之該等hES細胞不適用於臨床上。已有報告指出無滋養層培養之人類多能性幹(hPS)細胞會很快死亡，或者分化為定型細胞(committed cells)之異源性群體(heterogeneous population)。
許多報告已證實嘗試使用無細胞成分或是至少避免非人類成分或細胞，來取代該滋養層或維持細胞。該等取代物無法展現長期保證的結果，且這樣的嘗試已證實不足以維持細胞穩固(robust)且繼續的增殖(propagation)。再者，該等取代方式亦展現，於無滋養層細胞之培養中，生長於培養基取代物中之hES細胞的確在該等hES細胞群落周圍形成已分化細胞，係沒有達成最理想情況之指標。
故，需要開發使用不造成污染及形成畸胎瘤(teratomas)之另一種滋養層細胞來源以培養hES細胞之替代方案。
在一態樣中，本發明係提供一種增殖人類多能性幹細胞之方法，係共同培養人類多能性幹細胞與臍帶衍生幹細胞之。該臍帶衍生幹細胞於培養基中形成滋養層以用於使該人類多能性幹細胞增殖，且維持該人類多能性幹細胞於未分化狀態。
在一態樣中，該人類多能性幹細胞之增殖為無致瘤性增殖，使得該人類多能性幹細胞不會形成畸胎瘤。
在另一態樣中，該人類臍帶衍生幹細胞對於CD10、CD13、CD29、CD44、CD73、CD90、CD166及HLA-ABC為陽性，但對於CD1q、CD3、CD34、CD45、CD56、CD117及HLA-DR為陰性。該人類臍帶衍生幹細胞具有成骨(osteogenic)或成脂(adipogenic)細胞分化能力。
在另一態樣中，該臍帶衍生幹細胞為人類臍帶間質幹細胞(HUCMSCs)，且衍生自人類臍帶之華特氏膠(Wharton’s jelly)。
在另一態樣中，該人類多能性幹細胞對於鹼性磷酸酶(AP)、Oct-4、SSEA-4、TRA-1-60、TRA-1-81、NANOG、SOX2、NF-200、短尾突變型(brachyury)、ATBF1或MAP2為陽性，且表現GDF9、GATA4、HAND1
或TUJ-1
基因。又，於該人類多能性幹細胞中MYC被降低調控。
在另一態樣中，該人類多能性幹細胞為人類胚胎幹(hES)細胞，並形成類胚胎體(embryoid bodies)。
在另一態樣中，本發明係提供一種用於人類多能性幹細胞之增殖之培養基，其中，該培養基包含在培養基中形成滋養層之臍帶衍生幹細胞。在一態樣中，培養基中之該臍帶衍生幹細胞為人類臍帶間質幹細胞(HUCMSCs)。
在另一態樣中，本發明係提供一種用於增殖人類多能性幹細胞之套組，其中，該套組包括包含臍帶衍生幹細胞之培養基，及其使用說明書。在一態樣中，該套組之培養基中之該臍帶衍生幹細胞為人類臍帶間質幹細胞(HUCMSCs)。
本文所提供之多種具體細節係用以更通盤瞭解本發明。
機械性地移除並以RLT裂解溶液(lysis buffer)(Qiagen)處理培養於HUCMSCs或MEF滋養層上之未分化或已分化之hES細胞。依照製造商的說明書使用SuperScript III One-Step RT-PCR套組(invitrogen)合成第一條cDNA。表1顯示每對引子之序列、黏合溫度及產物大小。所有的PCR樣本係藉由含有0.5 μg/ml溴化乙啶(Sigma)之2%洋菜膠電泳分析。關於定量RT-PCR(qRT-PCR)，係於ABI StepOne Plus系統(Applied Biosystems)中使用FastStart universal SYBR green master(ROX,Roche,USA)基因表現試驗，以GAPDH
作為內部控制組。引子之序列及黏合溫度顯示於表1。
將人類臍帶樣本(長20公分，重20克)收集於含有Hanks’平衡鹽溶液(HBSS；Gibco/BRL 14185-052)之滅菌盒中。收集及使用人類臍帶之實驗流程為慈濟大學醫院人體試驗委員會所認可的。同意的書面紀錄已自陣痛前的懷孕婦女獲得。
以無Ca2+
及Mg2+
之磷酸鹽緩衝生理鹽水(Dulbecco’s PBS,Life Technology)清洗收集的人類臍帶組織。沿中線機械性地切開，且將臍帶動脈、靜脈及羊膜自華特氏膠(WJ)分離。接著將該膠切成小於0.5立方公分的片狀，以胰蛋白酶/EDTA(Sigma,St. Louis,USA)處理，並培養於37℃，95%空氣/5%CO2之潮濕環境中30分鐘。接著將該組織培養物培養於含有10%人類臍帶血清(CBS)及抗生素之Dulbecco’s Modified Eagle培養基(DMEM)中，並靜置五至七天以使該細胞自該組織培養物移行(migrate)。
藉由流式細胞儀分析MSC專一性表面標記之特性。將細胞以2mM磷酸鹽緩衝生理鹽水(PBS)分開，清洗並與已接合螢光素異硫氰酸鹽(Fluorescein isothiocyanate，FITC)或藻紅素(phycoerythrin，PE)之適當的抗體培養。該等抗體為CD1q、CD3、CD10、CD13、CD14、CD29、CD31、CD34、CD44、CD45、CD49b、CD49d、CD56、CD73、CD90、CD105、CD117、CD166、HLA-ABC及HLA-DR(BD,PharMingen)。接著，使用Becton Dickinson流式細胞儀(Vantage SE,Becton Dickinson,San Jose,CA)分析細胞。
在WJ組織片的第一次培養後，貼附自該組織培養物移行之帶有紡錘狀形態之生長細胞(第1A圖)。該等細胞以28小時的倍增時間(doubling time)快速分裂，且經歷多於25次繼代(相當於多於40次數量倍增)，無自發性的分化。該等細胞對於CD1q、CD3、CD34、CD45、CD56、CD117及HLA-DR為陰性，且對於CD10、CD13、CD29、CD44、CD73、CD90、CD166及HLA-ABC為陽性(第1B圖)。這些觀察證實分離自人類臍帶之WJ之細胞具有與間質幹細胞(MSCs)相同的表面標記。
為了誘發成骨及成脂細胞分化，將HUCMSCs轉移(transfer)至成骨培養基(補充10% CBS、0.1 μmol/L地賽米松(dexamethasone)、10 mmol/L β-甘油磷酸、及50 μmol/L抗壞血酸鹽之DMEM)及成脂培養基(補充10% CBS、1 μmol/L地賽米松、5 μg/mL胰島素、0.5 mmol/L異丁基甲基黃嘌呤及60μmol/L吲哚美辛(indomethacin)之DMEM)三週。藉由茜紅染劑(Alizarin Red S)(Sigma,USA)染色來確認鈣化，以評估成骨潛力。為了確認成脂細胞分化，於顯微鏡下觀察細胞內脂肪小滴，且該脂肪小滴藉由油紅染劑(Oil-Red-O)之染色被區分。
HUCMSC之成脂細胞分化在以成脂培養基補充物培養兩週後可見。於第二週結束時，在細胞形態及中性脂肪空泡(neutral lipid vacuoles)之形成(幾乎所有細胞含有許多對油紅染劑陽性的脂肪小滴)有明顯的改變(第1C圖)。同樣地，成骨培養基所誘發之細胞分化使得經處理的細胞快速生長且含有被茜紅染劑強烈染色之鈣化基質，顯示培養三至四週之鈣沉澱(第1C圖)。於RT-PCR分析中可見成脂(PPARγ
)及成骨(骨橋蛋白，Osteopontin
)基因之表現。
將得自工業研究院生物醫學及裝置研究實驗室之TW1細胞株(P22，即，第22次繼代)先培養於MEFs，如供應者，即台灣食品工業發展研究(FIRDI)，所指導。將絲裂黴素-C(mitomycin-C)去活化後之MEFs或HUCMSCs作為滋養層細胞，用來培養hES細胞。hES細胞以每孔每9.4平方公分200,000顆細胞的密度種(plate)於六孔盤。hES細胞培養基包括80%(v/v)Knockout(KO)DMEM(商品名)、20%(v/v)KO血清取代物、2 mM L-麩醯胺酸、10 mM非必需胺基酸(全來自Invitrogen)、50 μM B-巰基乙醇(Sigma)、及4 ng/mL bFGF。
使用對於未分化之hES細胞有專一性之螢光標記抗體，藉由免疫細胞化學分類，該等抗體為SSEA-4、SSEA-1、TRA-1-60、TRA-1-81及Oct-4(ES細胞分類套組；Chemicon)。首先，將hES細胞與滋養層細胞培養於培養盤中之細胞培養玻片(chamber slide)(Nunc,Denmark)或滅菌蓋玻片(Assisten,Germany)。在繼代後三至七天的特定時間將hES細胞之群落進行免疫螢光染色。簡單來說，係以4%三聚甲醛固定細胞，接著進行幾個流程，包含通透(permeabilization)(0.1% Triton X-100)、阻斷(blocking)(4%正常山羊血清)、一抗處理(1：10-1：50稀釋)、清洗三次、螢光標記二抗處理、清洗多於三次、以蓋玻片覆蓋並封片(mounting)。使用ES細胞分類套組(Chemicon)進行鹼性磷酸酶(AP)染色。
轉移至HUCMSCs滋養層之hES細胞以36小時的倍增時間有效地形成群落並繼續增殖。該等群落之形態與培養於MEFs上之細胞群落的形態有些微不同(第2A及2B)圖。然而，培養於HUCMSCs上及培養於MEFs上之單一人類ES細胞之形態相同。該細胞維持圓形且小，具有高核：質比率，且明顯存在一至三個核仁(第2C及2D圖)。HUCMSCs滋養層上之TW1 hES細胞表現AP、Oct-4、SSEA-4、TRA-1-60及TRA-1-81標記(第3A-3E圖)。
於第40次繼代研究細胞之核形(karyotype)。於繼代後第七天，以0.1μg/ml乙醯甲基秋水仙素(colcemid)(Gibco)處理hES細胞4小時。清洗細胞後，以0.25%胰蛋白酶處理3-5分鐘或以膠原蛋白酶第IV型處理8分鐘，使用移液吸管收取細胞。接著將該等細胞固定並封片於玻片上。於評鑑合格的細胞遺傳實驗室使用標準G條紋法(G-banding)分析減數分裂中期(metaphases)。於第40次繼代，HUCMSCs滋養層上之TWI hES細胞顯示正常核型46，XX(第3F圖)。
分化前，收取hES細胞並將其重新懸浮於無bFGF之hES細胞培養基中。接著，將該hES細胞以懸浮液中之聚集物(aggregates)的形式培養於低貼附六孔盤。五天後，添加胎牛血清(FBS)(最終濃度為5%)。聚集之hES細胞通常於七至十天後形成類胚胎體(EB)，且成熟(囊狀)EBs隨後出現自20%至80%之已形成之EBs。接著，將所得固體或囊狀EB種於經洋菜膠處理(gelatin-treated)之細胞培養玻片或35毫米培養盤，用以進一步分化，隨後以相似的處理方式處理以貼附培養直接生長之細胞。固定後，使用對於三胚層有專一性之螢光標記抗體以免疫細胞化學研究該已分化的hES細胞，該等抗體為MAP2、用於外胚層之NF200(Chemicon)，用於中胚層之短尾突變型及用於內胚層用於內胚層之ATBF1(Santa Cruz))。
如生長於MEFs上之hES細胞，當培養於懸浮液中，與HUCMSC滋養層共同培養之hES細胞形成EBs。於該等EBs中，觀察到已表現三胚層之細胞分化。如免疫細胞化學所指出，該等細胞表現外胚層標記(MAP-2及NF-200)、中胚層標記(短尾突變型)及內胚層標記(ATBF1)(第4圖)。如RT-PCR所指出，收取自七至十天之EBs之細胞表現GDF9(與生殖細胞有關)、GATA4
(與內胚層有關)、Hand1
(與中胚層有關)及Tuj-1
(與外胚層有關)(第5圖)。
使用玻璃毛細管機械性分開hES細胞，成塊狀(pelleted)重新懸浮於含有基質膠(Matrigel)(BD Biosciences)之PBS(1：1)中，並注射至非肥胖性糖尿病-嚴重合併性免疫不全症(NOD-SCID)小鼠之背部皮下組織(n=17)或腎上腺(n=4)。使用血球計數器(hemecytometer)計數細胞及將細胞懸浮於多種濃度之含有基質膠之PBS(1：1)中。於注射前將hES細胞置於冰上少於45分鐘以維持理想生存率。藉由觸診追蹤畸胎瘤之形成，將所得腫瘤解剖、固定、包埋於石蠟中及處理，以用於組織學研究。
使用異體移植(xenograft)模式於體內調查長期培養於HUCMSC滋養層後之細胞發展潛力。在21次不同移植至NOD-SCID小鼠(n=18)及裸鼠(n=3)兩者之密集實驗中，於多於三個月的長期追蹤期間，並未觀察到畸胎瘤生長(表2及3)。然而，在之後的暫時(幾天)培養於MEF滋養層上清楚觀察到畸胎瘤。於轉移至HUCMSC滋養層前與後之衍生自hES細胞之畸胎瘤組織學並無不同(表2及第6圖)。
於裂解緩衝液(150 mM NaCl、50 mM Tris-HCl[pH 7.4]、1%乙基苯基聚乙二醇-40(Nonidet P-40)加上蛋白酶抑制劑混合物(cocktail)(Roche,Indianapolis,IN)中裂解細胞。於10%十二基硫酸鈉-聚丙烯醯胺電泳膠(SDS-PAGE)電泳移行(electrophorese)蛋白質，接著轉移至硝化纖維素膜(Hybond-C Super;Amersham,Little Chalfont,UK)。將該膜與抗c-myc(2μg/m)或抗α-肌動蛋白(1：10,000;Sigma-Aldrich)單株抗體培養。使用已接合HRP山羊抗小鼠IgG(Jackson Immno-Research Laboratories)作為二抗。使用增強型化學發光試劑(enhanced chemiluminescence)(ECL;Amersham)檢測已結合之抗體。
進一步檢測兩種不同滋養層上之hES中之關鍵多能性基因的表現。如RT-PCR所指出，未分化幹細胞之標記，如Oct-4
、Nanog
、及Sox2
快速表現(第7A及7B圖)。在經HUCMSC共同培養之hES細胞中觀察到同源盒基因(homeobox gene)OCT4及原致癌基因(proto-oncogene)MYC的低表現量(第7A及7B圖)。進一步經西方點墨法分析及定量RT-PCR證實此MYC之降低調控(第7C及7D圖)。
第1圖顯示HUCMSC之形態、免疫表現型(immunotyping)及體外分化。生長自組織培養物之華特氏膠細胞為似纖維母細胞的，且帶有紡錘狀的型態(第1A圖)。流式細胞儀快速地區分HUCMSCs顯示其對於CD1q、CD3、CD34、CD45、CD56、CD117及HLA-DR為陰性，以及對於CD10、CD13、CD29、CD44、CD73、CD90、CD166及HLA-ABC為陽性(第1B圖)。待成脂細胞分化後，該細胞形成中性脂肪空泡，並含有複數對於油紅染劑為陽性的脂肪小滴(第1C圖，上欄)。於成骨細胞培養基中，該細胞延展而形成礦化基質，該礦化基質於三至四週的培養(cultivation)後被茜紅染劑顯著地染色(第1C圖，下欄)。藉由RT-PCR分析(以GAPDH
作為陽性控制組)顯示對於成脂(PPARγ)及成骨(骨橋蛋白，osteopontin)細胞分化具有專一性之基因表現(第1D圖)。第1A圖左欄之比例尺表示1000μm，而第1A圖及第1C圖右兩欄之比例尺表示100μm。
第2圖顯示生長於HUCMC及MEF滋養層上之未分化人類ES細胞群落之形態。生長於HUCMC上之hES群落圖(第2A圖)及生長於MEF上之hES群落圖(第2B圖)。生長於HUCMC上之群落放大圖顯示典型的hES細胞形態具有高核：質比率(第2C及2D圖)。第2A及2B圖之比例尺表示1000μm，而第2C圖及第2D圖之比例尺表示100μm。
第3圖顯示生長於HUCMC上之人類ES細胞表現型(phenotype)。使用專一性抗體之該hES群落之免疫染色(immnostaining)顯示鹼性磷酸酶(第3A圖)、Oct-4(第3B圖)、SSEA-4(第3C圖)、TRA-1-60(第3D圖)、及TRA-1-81(第3E圖)的高度表現。於HUCMC上持續培養並繼代20次後，在ES細胞中觀察到代表性的正常染色體核型(46,XX)(第3F圖)。比例尺表示1000μm。
第4圖顯示衍生自hES之類胚胎體(EB)之螢光免疫染色。相位差顯微鏡下顯示之類胚胎體(第4A圖)，及使用NF-200抗體(第4B圖)、短尾突變型抗體(第4C圖)、ATBF1(第4D圖)、及MAP2(第4E圖)之免疫染色。比例尺表示1000μm。
第5圖顯示於不同滋養層上之hES細胞之不同分化標記的表現。第5A圖為藉由RT-PCR的手段說明對於生殖細胞(GDF9
)、內胚層(GATA4
)、中胚層(HAND1
)及外胚層(TUJ-1
)有專一性之基因表現。GAPDH
作為控制組。第5B圖說明第5A圖所顯示之基因表現的半定量分析。
第6圖顯示當滋養層細胞由HUCMSC變為MEF之發展自hES細胞的畸胎瘤。第6A圖顯示待NOD-SCID小鼠上之滋養層由HUCMSC變為MEF後，畸胎瘤(箭頭指示)快速發展自hES細胞。組織切片顯示黑色素細胞(melanocyte)之帶狀物，其具有似視網膜(retina-like)結構(第6B圖)、似神經管(neurotube-like)結構(第6C-6E圖)、成牙上皮(odontogenic epithelium)(第6F圖)、神經上皮(第6G圖)、未成熟的軟骨(第6H圖)及未成熟的鱗狀上皮(squamoid epithelium)(第6I圖)。比例尺表示100μm。
第7圖顯示培養於HUCMSC及MEF上之hES細胞的多能性基因表現。第7A圖說明培養於HUCMSC(WJ)及MEF上之hES細胞(ES)之OCT4、NANOG、SOX2
及MYC
之RT-PCR分析，並以GAPDH作為內部控制組。第7B圖說明第7A圖所顯示之基因表現的半定量分析。第7C圖說明藉由西方點墨法分析測量C-MYC蛋白質。第7D圖說明藉由RT-PCR測量之MYC
的mRNA等級，係以該內部控制組之倍數顯示。EB表示類胚胎體。
Claims (14)
- 一種無致瘤性增殖人類胚胎幹細胞之方法，係包括：培養人類臍帶間質幹細胞多於25次繼代，且無自發性的分化；以及共同培養該人類胚胎幹細胞與經培養多於25次繼代之人類臍帶間質幹細胞，其中該人類胚胎幹細胞之增殖不會形成畸胎瘤。
- 如申請專利範圍第1項所述之方法，其中，該人類臍帶間質幹細胞係於培養基中形成滋養層，以增殖該人類胚胎幹細胞。
- 如申請專利範圍第1項所述之方法，其中，該人類臍帶間質幹細胞衍生自人類臍帶之華特氏膠(Wharton’s jelly)。
- 如申請專利範圍第1項所述之方法，其中，該人類臍帶間質幹細胞係用以維持該人類胚胎幹細胞處於未分化狀態。
- 如申請專利範圍第1項所述之方法，其中，該人類臍帶間質幹細胞對於CD10、CD13、CD29、CD44、CD73、CD90、CD166及HLA-ABC之一者或多者為陽性。
- 如申請專利範圍第1項所述之方法，其中，該人類臍帶間質幹細胞對於CD1q、CD3、CD34、CD45、CD56、CD117及HLA-DR之一者或多者為陰性。
- 如申請專利範圍第1項所述之方法，其中，該人類臍帶間質幹細胞具有成骨或成脂細胞分化能力。
- 如申請專利範圍第1項所述之方法，其中，該人類胚胎幹細胞對於鹼性磷酸酶(AP)、Oct-4、SSEA-4、TRA-1-60、TRA-1-81、NANOG、SOX2及分化標記之一者或多者為陽性，且其中，該分化標記為NF-200、短尾突變型、ATBF1及MAP2之一者或多者。
- 如申請專利範圍第1項所述之方法，其中，該人類胚胎幹細胞表現一種或多種分化基因，且其中，該分化基因為GDF9 、GATA4 、HAND1 或TUJ-1 基因。
- 如申請專利範圍第1項所述之方法，其中，於該人類胚胎幹細胞中MYC被降低調控。
- 如申請專利範圍第1項所述之方法，其中，該人類胚胎幹細胞形成類胚胎體。
- 一種用於增殖人類胚胎幹細胞之培養基，係包括人類臍帶間質幹細胞，其中，該人類臍帶間質幹細胞係培養多於25次繼代，且無自發性的分化。
- 如申請專利範圍第12項所述之培養基，其中，該人類臍帶間質幹細胞係於該培養基中形成滋養層。
- 一種用於增殖人類胚胎幹細胞之套組，係包括包含人類臍帶間質幹細胞之培養基，及其使用說明書，其中，該人類臍帶間質幹細胞係培養多於25次繼代，且無自發性的分化。
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| US20160187317A1 (en) * | 2013-08-01 | 2016-06-30 | Agency For Science, Technology And Research | Method of identifying adipose stem cells |
| USD748462S1 (en) | 2014-08-11 | 2016-02-02 | Auxocell Laboratories, Inc. | Centrifuge clip |
| US9993748B2 (en) | 2014-08-11 | 2018-06-12 | Auxocell Laboratories, Inc. | Centrifuge clip and method |
| JP7108537B2 (ja) * | 2016-04-27 | 2022-07-28 | ロート製薬株式会社 | Ｃｄ２０１、ｃｄ４６、ｃｄ５６、ｃｄ１４７及びｃｄ１６５からなる群より選択される少なくとも１種の細胞表面マーカーを発現する間葉系幹細胞及びその調製方法、並びに上記間葉系幹細胞を含む医薬組成物及びその調製方法 |
| TWI649422B (zh) * | 2016-12-30 | 2019-02-01 | 中國醫藥大學 | 成體多功能嗅幹細胞、其分離方法及其應用 |
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| EP2514817A1 (en) | 2012-10-24 |
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