Non-Human Primate iPSC Generation, Cultivation, and Cardiac Differentiation under Chemically Defined Conditions

Abstract

Non-human primates (NHP) are important surrogate models for late preclinical development of advanced therapy medicinal products (ATMPs), including induced pluripotent stem cell (iPSC)-based therapies, which are also under development for heart failure repair. For effective heart repair by remuscularization, large numbers of cardiomyocytes are required, which can be obtained by efficient differentiation of iPSCs. However, NHP-iPSC generation and long-term culture in an undifferentiated state under feeder cell-free conditions turned out to be problematic. Here we describe the reproducible development of rhesus macaque (Macaca mulatta) iPSC lines. Postnatal rhesus skin fibroblasts were reprogrammed under chemically defined conditions using non-integrating vectors. The robustness of the protocol was confirmed using another NHP species, the olive baboon (Papio anubis). Feeder-free maintenance of NHP-iPSCs was essentially dependent on concurrent Wnt-activation by GSK-inhibition (Gi) and Wnt-inhibition (Wi). Generated NHP-iPSCs were successfully differentiated into cardiomyocytes using a combined growth factor/GiWi protocol. The capacity of the iPSC-derived cardiomyocytes to self-organize into contractile engineered heart muscle (EHM) was demonstrated. Collectively, this study establishes a reproducible protocol for the robust generation and culture of NHP-iPSCs, which are useful for preclinical testing of strategies for cell replacement therapies in NHP.

Controlling the Switch from Neurogenesis to Pluripotency during Marmoset Monkey Somatic Cell Reprogramming with Self-Replicating mRNAs and Small Molecules

“…Lastly, although rhLIF and Forskolin were not necessary for the derivation of marmoset iPSCs, they bolstered the reprogramming process by improving cell proliferation. The combination of IWR1, CHIR99021, and a Src inhibitor has already been used in the generation of porcine and human expanded potential stem cells [45], while in our hands, IWR1 and CHIR99021 have recently proven very helpful in the long-term maintenance of iPSCs from NHPs, namely, rhesus monkey and baboon [46]. Altogether, we showed in the present report as well as in our previous publication [46] that NHP pluripotent stem cells have cell culture requirements different from human cells.…”

“…The combination of IWR1, CHIR99021, and a Src inhibitor has already been used in the generation of porcine and human expanded potential stem cells [45], while in our hands, IWR1 and CHIR99021 have recently proven very helpful in the long-term maintenance of iPSCs from NHPs, namely, rhesus monkey and baboon [46]. Altogether, we showed in the present report as well as in our previous publication [46] that NHP pluripotent stem cells have cell culture requirements different from human cells. This may reflect variations in the relevance of specific signal transduction pathways for the maintenance of pluripotency in the different primate species.…”

A non-invasive method to generate induced pluripotent stem cells from primate urine

“…To what extent this reprogramming procedure works in other species is currently unclear, but as the Sendai virus is thought to infect all mammalian cells 40 it could be widely applicable. Additionally, iPSCs have been previously generated from many species, even avian species 11 , when using human reprogramming factors and culture conditions, albeit with over tenfold lower reprogramming efficiencies 41 , 42 . So, while in principle it should be possible to isolate iPSCs from many or even all mammals, variation in reprogramming efficiency with human factors and culture conditions to keep cells pluripotent with and without feeder cells 42 will considerably vary among species and will make it practically difficult to obtain and maintain iPSCs from some species.…”

“…Additionally, iPSCs have been previously generated from many species, even avian species 11 , when using human reprogramming factors and culture conditions, albeit with over tenfold lower reprogramming efficiencies 41 , 42 . So, while in principle it should be possible to isolate iPSCs from many or even all mammals, variation in reprogramming efficiency with human factors and culture conditions to keep cells pluripotent with and without feeder cells 42 will considerably vary among species and will make it practically difficult to obtain and maintain iPSCs from some species. Investigating the cause of this variation more systematically will be important to better understand pluripotent stem cells in general and to generate iPSCs from many species in practice.…”

“…Investigating the cause of this variation more systematically will be important to better understand pluripotent stem cells in general and to generate iPSCs from many species in practice. Recent examples of such fruitful investigations include the optimization of culture conditions for baboons 43 , and the optimization of feeder-free culture conditions for rhesus macaques and baboons 42 . A related aspect of generating iPSCs from different species is testing whether iPSCs from a given species are actually bona fide iPSCs.…”