Derivation of Canine Induced Pluripotent Stem Cells

Abstract

Dogs and humans have many inherited genetic diseases in common and conditions that are increasingly prevalent in humans also occur naturally in dogs. The use of dogs for the experimental and clinical testing of stem cell and regenerative medicine products would benefit canine health and welfare and provide relevant animal models for the translation of therapies to the human field. Induced pluripotent stem cells (iPSCs) have the capacity to turn into all cells of the body and therefore have the potential to provide cells for therapeutic use and for disease modelling. The objective of this study was to derive and characterize iPSCs from karyotypically abnormal adult canine cells. Aneuploid adipose-derived mesenchymal stromal cells (AdMSCs) from an adult female Weimeraner were re-programmed into iPSCs via overexpression of four human pluripotency factors (Oct 4, Sox2, Klf4 and c-myc) using retroviral vectors. The iPSCs showed similarity to human ESCs with regard to morphology, pluripotency marker expression and the ability to differentiate into derivatives of all three germ layers in vitro (endoderm, ectoderm and mesoderm). The iPSCs also demonstrated silencing of the viral transgenes and re-activation of the silent X chromosome, suggesting full reprogramming had occurred. The levels of aneuploidy observed in the AdMSCs were maintained in the iPSCs. This finding demonstrates the potential for generating canine induced pluripotent stem cells for use as disease models in addition to regenerative medicine and pharmaceutical testing.

Feeder‐independent canine induced pluripotent stem cells maintained under serum‐free conditions

“…In the current report, we established stable and long‐term cultures of ciPSCs using a doxycycline‐inducible system. ciPSCs had not been previously maintained long‐term (Baird et al, ; Gonçalves et al, ; Luo et al, ), and even canine embryonic stem cells could be cultured for a maximum of 40 passages (Hatoya et al, ; Hayes et al, ; Schneider, Wolf, Braun, Kolb, & Adler, ; Vaags et al, ; Wilcox et al, ). Whitworth et al () reported that ciPSCs could be passaged over 40 times, but their lines did not form a teratoma after transplantation into an immunodeficient mouse.…”

“…Previously, derived ciPSCs cultured in medium supplemented with bFGF and LIF formed complete teratoma that also possessed up‐regulated transgene expression (Koh et al, ; Vaags et al, ), implying that teratoma formation requires transgene expression as well as active bFGF and LIF signaling in ciPSCs; perhaps our ciPSCs may have formed teratomas if they were cultured with bFGF plus LIF and if doxycycline was administered to the mice to maintain the expression of transgenes after transplantation. Several groups reported that the expression of transgenes is maintained in ciPSCs (Baird et al, ; Gonçalves et al, ; Luo et al, ; Nishimura et al, ). Conversely, Whitworth et al () reported that transgene expression in their ciPSCs was completely silenced, suggesting that the long‐term maintenance of their ciPSCs without exogenous expression might be attributed to achievement of reprogramming at the same level as that of canine embryonic stem cells using mitogen‐activated protein kinase inhibitor and glycogen synthase kinase 3β inhibitor (Baird et al, ; Gonçalves et al, ; Luo et al, ; Nishimura et al, ).…”

“…Several groups reported that the expression of transgenes is maintained in ciPSCs (Baird et al, ; Gonçalves et al, ; Luo et al, ; Nishimura et al, ). Conversely, Whitworth et al () reported that transgene expression in their ciPSCs was completely silenced, suggesting that the long‐term maintenance of their ciPSCs without exogenous expression might be attributed to achievement of reprogramming at the same level as that of canine embryonic stem cells using mitogen‐activated protein kinase inhibitor and glycogen synthase kinase 3β inhibitor (Baird et al, ; Gonçalves et al, ; Luo et al, ; Nishimura et al, ). Yet, a previous report from Wilcox et al () noted that canine embryonic stem cells could not be maintained long‐term in medium used by Whitworth et al Thus, the factors necessary for culturing pluripotent canine stem cells are still under debate, leaving room for the development of an optimized ciPSC medium.…”

“…Here, we sought to establish stable and highly proliferating ciPSCs lines. Previous reports of ciPSC derivation required co‐culture with feeder cells (Baird, Barsby, & Guest, ; Gonçalves et al, ; Luo et al, ; Whitworth, Ovchinnikov, & Wolvetang, ) as well as both bFGF and LIF to maintain their undifferentiated status (Baird et al, ; Gonçalves et al, ; Luo et al, )—although the maintenance of ciPSCs without bFGF, but not without LIF, has been reported (Whitworth et al, ). Conversely, successful maintenance of ciPSCs in serum‐free medium has not been report.…”

Generation of American mink induced pluripotent stem cells: a protocol

“…В 2015 г. нами получены ИПСК американской норки, и, таким образом, появилась уни-кальная возможность сравнения полученных из эмбриона и индуцированных плюрипотентных клеток на новом, сравнительно мало исследованном виде (Menzorov et al, 2015). Ключевые транскрипционные факторы, по-зволяющие индуцировать плюрипотентность в геноме дифференцированных клеток, достаточно консервативны, что позволяет использовать для репрограммирования диф-ференцированных клеток различных видов гены человека, например для получения ИПСК собаки (Baird et al, 2015). Так, представленная система репрограммирования генома с минимальными модификациями была успешно при-менена нами для получения ИПСК как человека и мыши (неопубл.…”