Generation of Functional Platelets from Canine Induced Pluripotent Stem Cells

Abstract

Thrombocytopenia (TTP) is a blood disease common to canines and human beings. Currently, there is no valid therapy for this disease except blood transfusion. In this study, we report the generation of canine induced pluripotent stem cells (ciPSCs) from canine embryonic fibroblasts, and a novel protocol for creating mature megakaryocytes (MKs) and functional platelets from ciPSCs. The ciPSCs were generated using lentiviral vectors, and differentiated into MKs and platelets on OP9 stromal cells supplemented with growth factors. Our ciPSCs presented in a tightly domed shape and showed expression of a critical pluripotency marker, REX1, and normal karyotype. Additionally, ciPSCs differentiated into cells derived from three germ layers via the formation of an embryoid body. The MKs derived from ciPSCs had hyperploidy and transformed into proplatelets. The proplatelets released platelets early on that expressed specific MK and platelet marker CD41/61. Interestingly, these platelets, when activated with adenosine diphosphate or thrombin, bind to fibrinogen. Moreover, electron microscopy showed that the platelets had the same ultrastructure as peripheral platelets. Thus, we have demonstrated for the first time the generation of ciPSCs that are capable of differentiating into MKs and release functional platelets in vitro. Our system for differentiating ciPSCs into MKs and platelets promises a critical therapy for canine TTP and appears to be extensible in principle to resolve human TTP.

Derivation of Canine Induced Pluripotent Stem Cells

“…; Nishimura et al. ). When LIF and bFGF are removed from the culture, the iPSCs can differentiate into cells from all three germ layers and produce embryoid bodies in vitro .…”

Inducible Gata1 suppression expands megakaryocyte-erythroid progenitors from embryonic stem cells

“…Platelets and megakaryocytes generated in vitro from embryonic stem (ES) or induced pluripotent stem (iPS) cells are potentially useful for treating thrombocytopenia and for delivering pro-or antithrombotic proteins to sites of vascular injury (1)(2)(3)(4). However, such therapies are impeded by relatively low yields of megakaryocytes from standard ES/iPS cell differentiation protocols.…”

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

“…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.…”