Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.
In this review the distinct aspects of somatic cell reprogramming are discussed. The molecular mechanisms of generation of induced pluripotent stem (iPS) cells from somatic cells via the introduction of transcription factors into adult somatic cells are considered. Particular attention is focused on the generation of iPS cells without genome modifications via the introduction of the mRNA of transcription factors or the use of small molecules. Furthermore, the strategy of direct reprogramming of somatic cells omitting the generation of iPS cells is considered. The data concerning the differences between ES and iPS cells and the problem of epigenetic memory are also discussed. In conclusion, the possibility of using iPS cells in regenerative medicine is considered.
Òåõíîëîãèè ðåïðîãðàììèðîâàíèÿ êëåòîê ÷åëîâåêà ÿâëÿþòñÿ ïåðñïåêòèâíûì íàïðàâëå-íèåì ðàçâèòèÿ êëåòî÷íîé áèîëîãèè ñ äàëüíåé ïåðñïåêòèâîé èñïîëüçîâàíèÿ äàííûõ ìåòîäîâ äëÿ êëåòî÷íî-çàìåùàþùåé òåðàïèè â êëèíè÷åñêîé ïðàêòèêå. Îäíîé èç ïðîáëåì, âñòàþùèõ íà ïóòè èññëåäîâàòåëåé, ÿâëÿåòñÿ íèçêàÿ ýôôåêòèâíîñòü ðåïðîãðàììèðîâàíèÿ è èñïîëüçîâà-íèå íåaeåëàòåëüíûõ îíêîãåíîâ â ïåðâîíà÷àëüíûõ ìåòîäèêàõ.  äàííîé ðàáîòå íàìè áûë ïðåä-ëîaeåí àëüòåðíàòèâíûé ìîäèôèöèðîâàííûé ìåòîä ðåïðîãðàììèðîâàíèÿ ôèáðîáëàñòîâ êîaeè ÷åëîâåêà. Áûëî ïîêàçàíî, ÷òî èíäóêöèÿ òåëîìåðàçíîé àêòèâíîñòè óâåëè÷èâàåò âûõîä ðåïðîã-ðàììèðîâàííûõ êëåòîê è ïîçâîëÿåò èñêëþ÷èòü îêîíãåí c-Myc èç èñïîëüçóåìîãî íàáîðà ãåíîâ. Êëþ÷åâûå ñëîâà: òåëîìåðàçà, ðåïðîãðàììèðîâàíèå, ôèáðîáëàñòû êîaeè ÷åëîâåêà.Îäíèì èç ñàìûõ ïåðñïåêòèâíûõ è ðàçâèâàþùèõ-ñÿ íàïðàâëåíèé â ñîâðåìåííîé êëåòî÷íîé áèîëîãèè ÿâëÿåòñÿ ðåïðîãðàììèðîâàíèå ñîìàòè÷åñêèõ êëåòîê ÷åëîâåêà. Ïðè ïîìîùè ìåòîäèê ðåïðîãðàììèðîâà-íèÿ èññëåäîâàòåëè íàäåþòñÿ ïîëó÷àòü ïàöèåíò-ñïå-öèôè÷åñêèå êëåòêè ðàçëè÷íûõ òèïîâ òêàíåé, êîòî-ðûå ìîaeíî áûëî áû èñïîëüçîâàòü â çàìåñòèòåëüíîé êëåòî÷íî-òêàíåâîé òåðàïèè. Ïîìèìî ðàçðàáîòàííûõ ìåòîäîâ ïîëó÷åíèÿ ïàöèåíò-ñïåöèôè÷åñêèõ ïëþðè-ïîòåíòíûõ ñòâîëîâûõ êëåòîê ïîñðåäñòâîì ïåðåíîñà ÿäåð â îîöèòû (somatic cell nuclear transfer, SCNT) [1, 2], à òàêaeå ïðè ïîìîùè ñëèÿíèÿ ñîìàòè÷åñêèõ êëå-òîê ñ ýìáðèîíàëüíûìè ñòâîëîâûìè êëåòêàìè (ÝÑÊ) [3] â 2006-2007 ãã. áûë ðàçðàáîòàí íîâûé ïîäõîä ê ðå-ïðîãðàììèðîâàíèþ êëåòîê ÷åëîâåêà: ïðè ïîìîùè ëåíòèâèðóñíûõ òðàíñôåêöèé êëþ÷åâûõ ãåíîâ ïëþ-ðèïîòåíòíîñòè Oct4, Sox2, KLF4, c-Myc (KMOS) óäà-ëîñü ïîëó÷èòü ðåïðîãðàììèðîâàííûå êëåòêè, íàçâàí-íûå ïîçäíåå èíäóöèðîâàííûìè ïëþðèïîòåíòíûìè ñòâîëîâûìè êëåòêàìè (ÈÏÑ êëåòêè) [4,5]. ÈÏÑ êëåò-êè ïîõîäèëè íà ÝÑÊ ïî ìíîãèì õàðàêòåðèñòèêàì, âêëþ÷àÿ ïðîôèëè ãåííîé ýêñïðåññèè, ìîðôîëîãèþ, õàðàêòåð ìåòèëèðîâàíèÿ ÄÍÊ, ñïîñîáíîñòü äàâàòü íà÷àëî âñåì òðåì çàðîäûøåâûì ëèñòêàì in vitro è ôîðìèðîâàíèå çðåëûõ òåðàòîì â èììóííîäåôèöèò-íûõ ìûøàõ.Îäíîé èç ïðîáëåì ïîäîáíîãî ìåòîäà ÿâëÿåòñÿ íèç-êàÿ ýôôåêòèâíîñòü ðåïðîãðàììèðîâàíèÿ.  ðàííåé èñõîäíîé ìåòîäèêå òðàíñôåêöèåé ÷åòûðüìÿ ôàêòî-ðàìè KMOS ðåïðîãðàììèðîâàíèþ ïîäâåðãàëèñü òîëü-êî ïîðÿäêà 0,01-0,1% òðàíñôèöèðîâàííûõ êëåòîê. Äðóãîé ÿâíîé ïðîáëåìîé ïîäîáíîãî ìåòîäà ðåïðîã-ðàììèðîâàíèÿ ÿâëÿåòñÿ èñïîëüçîâàíèå îíêîãåíà c-Myc â ñîñòàâå íàáîðà ðåïðîãðàììèðîâàíèÿ KMOS. Ýêçî-ãåííàÿ ñâåðõýêñïðåññèÿ ýòîãî ãåíà ìîaeåò âûçûâàòü àêòèâàöèþ ãåíîâ îíêîñóïðåññîðîâ, íàïðèìåð Cdkn1a, Cdkn2a, p53, êîòîðûå âîâëå÷åíû â ðàçëè÷íûå ïóòè äèôôåðåíöèðîâêè è ïîäàâëåíèÿ ïðîëèôåðàöèè. Òàê-aeå èñïîëüçîâàíèå ýòîãî îíêîãåíà íåãàòèâíî âëèÿ-åò íà âîçìîaeíîñòü ïðèìåíåíèÿ ìåòîäà â êëèíèêå, ïîñêîëüêó íå èñêëþ÷åíà âåðîÿòíîñòü ðåàêòèâàöèè âñòðîåííîãî ãåíà ñ ïîñëåäóþùåé çëîêà÷åñòâåííîé òðàíñôîðìàöèåé ðåïðîãðàììèðîâàííûõ êëåòîê [6]. Ïîêàçàòåëüíî, ÷òî ó õèìåðíûõ ìûøåé, ïîëó÷åííûõ èç ÈÏÑ êëåòîê áåç ââåäåíèÿ c-Myc, íå îáðàçîâûâà-ëîñü îïóõîëè ïîñëå ðîaeäåíèÿ, òîãäà êàê ïðèáëèçè-òåëüíî 15% aeèâîòíûõ, ïîëó÷åííûõ îò ÈÏÑ êëåòîê ñ ýêçîãåíîì c-Myc, ñòðàäàëè îíêîëîãè÷åñêèìè çàáî-ëåâàíèÿìè [7]. òî aeå âðåìÿ èçâåñòíî, ÷òî âî âðåìÿ ðåïðîãðàì-ìèð...
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