An expedition in the jungle of pluripotent stem cells of non-human primates

Summary

For nearly three decades, more than 80 embryonic stem cell lines and more than 100 induced pluripotent stem cell lines have been derived from New World monkeys, Old World monkeys, and great apes. In this comprehensive review, we examine these cell lines originating from marmoset, cynomolgus macaque, rhesus macaque, pig-tailed macaque, Japanese macaque, African green monkey, baboon, chimpanzee, bonobo, gorilla, and orangutan. We outline the methodologies implemented for their establishment, the culture protocols for their long-term maintenance, and their basic molecular characterization. Further, we spotlight any cell lines that express fluorescent reporters. Additionally, we compare these cell lines with human pluripotent stem cell lines, and we discuss cell lines reprogrammed into a pluripotent naive state, detailing the processes used to attain this. Last, we present the findings from the application of these cell lines in two emerging fields: intra- and interspecies embryonic chimeras and blastoids.

This review delves into more than 180 primate embryonic and induced PSC lines from marmosets to orangutans. It covers derivation methods, molecular features, and the inclusion of fluorescent reporters. Comparisons with human pluripotent stem cells are discussed, highlighting pluripotent naive state reprogramming. Applications in chimeras and blastoids are also explored.

Introduction

For the past 30 years, pluripotent stem cell (PSC) lines from mice have served as a versatile tool for developmental biologists. The journey began with pluripotent embryonic stem (ES) cells, also known as ESCs, and later included induced pluripotent stem (iPS) cells (iPSCs). Leveraging genome editing and germline chimera technologies, mouse PSCs have offered invaluable insights into mammalian development and have played a crucial role in establishing animal models of human genetic diseases. Moreover, these cells have been instrumental for developing in vitro differentiation protocols, which were later adapted to human PSC cells, paving the way for advances in regenerative medicine. Mouse ESCs have been used as a source of donor nuclei in embryo reconstitution experiments with enucleated oocytes, resulting in the creation of cloned mice (Rideout et al., 2000; Wakayama et al., 1999). In recent research, mouse ESCs have been used to create pseudo-embryos known as blastoids (Li et al., 2019; Rivron et al., 2018; Sozen et al., 2019). ESCs and iPSCs have been successfully derived in other rodent species, including rats, Apodemus, and naked mole rats (Buehr et al., 2008; Lee et al., 2017; Li et al., 2008; Liao et al., 2008; Xiang et al., 2008).

ESCs started to be derived from embryos of human and non-human primates in the mid-1990s by Thomson and colleagues (Thomson et al., 1995, 1998) with iPSCs following from 2007 (Takahashi et al., 2007; Yu et al., 2007). The number and diversity of the cell lines generated have significantly altered our understanding of pluripotency. This is because they have underscored the variety of pluripotency states, the intricacy of their stabilization in culture, and the uniqueness of rodent PSCs. In essence, rodents ESCs and iPSCs self-renew in what is known as the naive state of pluripotency, a state similar to the pluripotent epiblast of pre-implantation embryos. This is due to the differentiation inhibitory properties of cytokines of the interleukin-6 (IL-6) family. When cultured with fibroblast growth factor 2 (FGF2) and activin A, these cells transition to another pluripotency state called the primed state, analogous to the pluripotent epiblast of post-implantation embryos (Nichols and Smith, 2009). Naive and primed states exhibit significant differences, including developmental potential, epigenetic profile, and energy production, which in turn affect their characteristics and function (Weinberger et al., 2016). One major criterion for distinguishing the naive and primed states of pluripotency involves the status of X chromosome activity: female PSCs in the naive state exhibit two active X chromosomes, whereas those in the primed state have undergone random inactivation of one of the two X chromosomes. This reflects the cell’s relative developmental maturity (Nichols and Smith, 2009). Moreover, only naive PSCs can generate systemic chimeras after injection into pre-implantation embryos and create blastoids after in vitro aggregation.

Unlike rodent PSCs, human PSCs show minimal or no response to IL-6 family cytokines. As a result, most ESCs and iPSCs have been derived using FGF2 and activin A to promote self-renewal, leading to their stabilization in the primed state of pluripotency. Rare naive ESCs have been generated from human blastocysts (Guo et al., 2016), which requires combinations of leukemia inhibitory factor (LIF) and chemical inhibitors of protein kinase C (PKC), mitogen-activated protein kinase (MEK), and glycogen synthase kinase 3β (GSK3β). However, these cell lines are reputed to be unstable. A more reliable method involves treating ESCs and iPSCs in the primed state with resetting factors to instate naive-type characteristics. Several combinations of genes, growth factors, and small molecules have been developed to stabilize the self-renewal of human PSCs in the naive state (reviewed in Collier and Rugg-Gunn, 2018; Li and Izpisua Belmonte, 2018; Weinberger et al., 2016).

Over the past 25 years, a large number of ESC and iPSC lines have been established in various non-human primate (NHP) species and using a large variety of technologies. There is a significant interest in NHP PSCs for several reasons. First, NHP PSCs serve as crucial tools for developing cell therapy strategies and conducting pre-clinical validations in species that are closely related to humans (Aron Badin et al., 2019; Baik et al., 2023; Hu et al., 2023; Rodriguez-Polo and Behr, 2022; Uyama et al., 2022; Wianny et al., 2022). Second, in lieu of the prohibitively expensive CRISPR strategies on NHP embryos requiring cloning of transgenic founders (Liu et al., 2019; Qiu et al., 2019), NHP PSCs could be used to generate systemic chimeras for studying primate development and establishing new models of human disease. Third, PSCs derived from NHP embryos present a viable alternative to human PSCs in jurisdictions where legal restrictions limit or outright prohibit research on human embryos and human ESCs. In this review article, we aim to consolidate our current understanding of the biology of ESCs and iPSCs in NHPs. We provide a comprehensive overview of ESC and iPSC lines established in various primate species. We discuss the wide range of applications for NHP ESCs and iPSCs and how they compare with their human counterparts.

Embryo-derived PSCs in NHPs

In this segment and the next, we deliver a comprehensive review of both ESC and iPSC lines that have been derived across diverse NHPs. These encompass the New World monkeys or Platyrrhini, which share a common ancestor with humans 37–41 million years ago (MYA), Old World monkeys or Catarrhini, whose common ancestor with human dates back to 29–33 MYA; and great apes, which share a common ancestor with humans approximately 20 MYA (Kuderna et al., 2023; Figure 1). More than 80 ESC lines and more than 100 iPSC lines have been established from Platyrrhini, Catarrhini, and great apes. To our knowledge, PSC lines are not available in prosimians, including tarsiers, lemurs, and lorises, whose common ancestor with humans dates back to approximately 75 MYA.

Figure 1.

Phylogenetic tree showing the evolutionary distance between human and the different NHP species discussed in the review (www.timetree.org; Kuderna et al., 2023).

ESC lines established in New World monkeys

ESC lines have been successfully established in the common marmoset (Callithrix jacchus). Thomson et al. (1996) first created ESC lines in 1996 using naturally fertilized marmoset embryos. Eight euploid CjESC lines, 46XX lines, and 46XY lines, were generated (Table 1). Two lines, named Cj11 and Cj62, underwent further analysis to demonstrate long-term chromosomal stability. These cells expressed cell-surface markers later associated with the primed state of pluripotency, including SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. Sasaki et al. (2005) reported three new female CjESC lines (46XX), CMESC 20, 30, and 40. These lines expressed the previously mentioned cardinal markers of pluripotency, maintained euploidy after extensive passaging on feeder cells in a culture medium supplemented with knockout serum replacement (KOSR) factors, and displayed the ability to differentiate into cells from the three primitive germ layers, ectoderm, mesoderm, and endoderm in experimental teratomas. Muller et al. (2009) later established a new female ESC line (46XX), cjes001, derived from a naturally fertilized marmoset embryo. Alongside classical pluripotency markers, cjes001 expressed primordial germ cell markers such as VASA, BOULE, germ cell nuclear factor (NR6A1), and synaptonemal complex protein 3 (SYCP3), suggesting an increased capacity for spontaneous germ cell differentiation. Kishimoto et al. (2021) reported 17 marmoset ESC lines, comprising 11 female and 6 male lines. Eleven lines were derived from naturally fertilized embryos, while six were generated from in vitro fertilized (IVF) embryos. Notably, 13 lines were established and propagated using feeder-free conditions on laminin 551 in MEF-conditioned medium supplemented with KSR and FGF2. Only 10 of the 17 original lines retained a normal karyotype after long-term passaging, irrespective of sex, IVF, or naturally fertilized embryos and culture on laminin or feeders. This report is the first to establish marmoset ESC lines using feeder-free conditions and IVF blastocysts.

Table 1.

ESC lines in New World monkeys

Species	Name of cell lines	Culture medium	Cells differentiation analysis	Reference
Marmoset (C. jacchus)	Cj11.2, Cj25.1, Cj28, Cj33, Cj35, Cj36, Cj39, Cj62	DMEM culture medium/feeder-MEF	embryoid bodies	Thomson et al. (1996)
	CMESC 20, CMESC 30, CMESC 40	CMESC culture medium/feeder-MEF	embryoid bodies teratomas hematopoietic stem cells neurospheres	Sasaki et al. (2005)
	cjes001	CMESC culture medium/feeder-MEF	embryoid bodies teratomas	Müller et al. (2009)
	nos. 1–17	CMESC medium/feeder-MEF/iMatrix/Laminin551	embryoid bodies teratomas	Kishimoto et al. (2021)

ESC lines established in old world monkeys

Rhesus monkey

The first established ESCs in Rhesus (Macaca mulatta [RhESCs]) were derived from naturally fertilized embryos in 1995 (Thomson et al., 1995). Seven RhESC lines (R278, R366, R367, R394, R420, R456, and R460) were generated (Table 2). These cells lines exhibited hallmark pluripotency markers and formed teratomas comprising derivatives from all three germ layers. Mitalipov et al. (2006) reported an additional 18 novel RhESC lines (ORMES-1–ORMES-18) derived from embryos fertilized by intra-cytoplasmic sperm injection (ICSI). Each cell line expressed cardinal markers of pluripotency. The majority of these 18 cell lines maintained a normal karyotype (42XX and 42XY). ORMES-6 and ORMES-7 RhESCs demonstrated pluripotency in vitro by differentiating into neural, retinal, cardiac, and pancreatic lineages. Wianny et al. (2008) established a new female ESC line (42XX), LYON-ES1, from an ICSI rhesus embryo. Last, two novel RhESC lines, Pa2.2 and Pa3, were established by Wei et al. (2011) from parthenogenetic blastocysts. Both lines maintained a normal karyotype (42XX), even after 80 passages.

Table 2.

ESC lines in Old World monkeys

Species	Name of cell lines	Culture medium	Cells differentiation analysis	Reference
Rhesus monkey (M. mulatta)	R278, R366, R367, R394, R420, R456, R460	DMEM medium/feeder-MEF	embryoid bodies teratomas	Thomson et al. (1995)
	ORMES-1–ORMES-18	DMEM/DMEM-F12 medium/feeder-MEF	embryoid bodies neural, cardiac, retinal, pancreatic	Mitalipov et al. (2006)
	LYON-ES1	KO-DMEM medium/feeder-MEF	embryoid bodies teratomas	Wianny et al. (2008)
	Pa2.2, Pa3	DMEM-F12 medium/feeder-MEF	embryoid bodies teratomas	Wei et al. (2011)
Cynomolgus monkey (M. fascicularis)	CMK5, CMK6, CMK7, CMK9	DMEM medium and Ham’s nutrient mixture F-12/feeder-MEF	embryoid bodies teratomas	Suemori et al. (2001)
	CES-1–CES-8	CESM medium/feeder-MEF	teratomas	Chen et al. (2015)
Olive baboon (P. anubis)	BabESC-4, BabESC-15	DMEM medium /feeder-MEF	teratomas embryoid bodies	Simerly et al. (2009)
Yellow baboon (P. cynocephalus)	UT-1, UT-2, UT-3	DMEM medium /feeder-MEF	embryoid bodies teratomas	Chang et al. (2011)
African green monkey (C. aethiops)	AgMES	ESM medium/feeder-MEF	embryoid bodies teratomas	Shimozawa et al. (2010)
Rhesus- SCNT (M. mulatta)	CRES-1, CRES-2	DMEM-F12 medium /feeder-MEF	embryoid bodies teratomas	Byrne et al. (2007)
African green monkey- SCNT (C. sabaeus)	NT-ES1–NT-ES11	E8/mTeSR1/Matrigel	embryoid bodies teratomas	Chung et al. (2020)

Cynomolgus monkey

In 2001, cynomolgus monkey ESCs (CyESCs) were derived from the blastocysts of cynomolgus monkey (Macaca fascicularis) produced by IVF and ICSI. Four CyESCs lines, namely CMK5, CMK6, CMK7, and CMK9, were initially documented by Suemori et al. (2001), requiring KOSR and FGF2 for self-renewal. These cell lines maintained a diploid set of 42 chromosomes (42XX and 42XY), after 3–6 months in culture (Table 2). In addition to expressing traditional pluripotency markers, the CMK6 line expressed primordial germ cell markers VASA, NANOS, and PIWIL1 genes. This suggested an enhanced propensity for spontaneous germ cell differentiation (Yamauchi et al., 2009). In 2015, Chen et al. (2015) reported eight additional CyESC lines, CES-1–CES-8, from ICSI embryos. These lines were derived using conventional ESC media (i.e., FGF2 + KOSR on feeder cells) mixed with the fully defined, serum and feeder-free medium PSGro (CESM medium). The resulting CyESCs could be cultured for more than 60 passages while maintaining high expression levels of pluripotency markers (Chen et al., 2015). Notably, as with all other PSC lines described so far, these eight CyESCs lines were originally derived in the primed state of pluripotency. Two of them were subsequently converted to a naive-like state. Resetting of naive-type features will be discussed in section reprogramming of NHP PSCs to naive pluripotency.

Baboon

Simerly et al. (2009) developed two ESC lines, BabESC-4 and BabESC-15, from ICSI-derived blastocysts of the olive baboon (Papio anubis). These lines expressed pluripotency markers and maintained stable euploid female karyotypes (42XX) (Table 2). Subsequently, Chang et al. (2011) established three novel ESC lines from ICSI-derived yellow baboon blastocysts (Papio cynocephalus), including two male cell lines (UT-1 and -2; 42XY) and one female cell line (UT-3; 42XX). These lines displayed the cardinal features of pluripotency including cell-surface markers and teratoma formation.

African green monkey

Shimozawa et al. (2010) developed an ESC line, AgMES, from a blastocyst produced by ICSI in the African green monkey (Cercopithecus aethiops, also called Chlorocebus aethiops). This line displayed expressed the cardinal markers of pluripotency and maintained stable euploid female karyotype (60XX) (Table 2).

ESC lines established from somatic cell nuclear transfer embryos

NHP ESC lines have been derived from cloned embryos produced by somatic cell nuclear transfer (SCNT) in rhesus macaques and African green monkeys. Byrne et al. (2007) derived two nuclear transfer (nt)ESC lines from 20 male rhesus macaque SCNT blastocysts, termed CRES-1 and CRES-2 (Table 2). Both lines expressed the cardinal pluripotency markers and exhibited capabilities for cell differentiation in the three germ layers akin to fertilized embryo-derived ESC lines. CRES-2 exhibited a normal male rhesus macaque chromosome complement (42XY), while CRES-1 demonstrated both a loss of the Y chromosome in a small number of cells and a translocation involving the Y chromosome. In a more recent study, Chung et al. (2020) derived 11 ntESC lines from 30 male and female African green monkey (Chlorocebus sabaeus) SCNT blastocysts. These ntESC lines were first established on growth-inactivated mouse fibroblasts in a culture medium supplemented with FCS, KOSR, FGF2, and LIF. Following stabilization, the lines were propagated in E8/mTeSR1(1:9) supplemented with FGF2 on Matrigel-coated dishes. Among these ntESC lines, one called SCNT4 displayed a normal 60XY karyotype.

Induced PSCs in NHPs

iPSC lines established in New World monkeys

Wu et al. (2010) pioneered the creation of marmoset iPSC (cjiPSC) lines using skin fibroblasts reprogrammed with retroviral vectors expressing human OCT4, KLF4, SOX2, and c-MYC. Two cell lines were subjected to molecular and functional analysis, which involved the expression of key pluripotency markers, a euploid set of chromosomes, and differentiation into ectoderm, mesoderm, and endoderm derivatives in experimental teratomas (Table 3). Concurrently, Tomioka et al. (2010) generated several iPSC lines by introducing six human transcription factors including OCT4, SOX2, KLF4, c-MYC, NANOG, and LIN28 in marmoset fetal liver cells via retrovirus-mediated transduction. Two lines were reported to retain a normal karyotype and differentiated into derivatives of all three primary germ layers in vitro. Subsequent studies focused on generating iPSC lines using non-integrative methodologies suitable for preclinical study applications. Wiedemann et al. (2012) reported a new iPSC line generated by reprogramming adult marmoset bone marrow-derived cells using a quad-cistronic (OCT4, KLF4, SOX2, and c-MYC) excisable lentiviral vector (Wiedemann et al., 2012). Along this line, Yoshimatsu et al. (2021b) used an all-in-one episomal plasmid expressing OCT4, SOX2, KLF4, and c-MYC to generate a female (46XX) iPSC line from neonatal marmoset fibroblast named NM-iPS. Similarly, Debowski et al. (2015) devised a transposon-based, fully excisable piggyBac system that expressed six marmoset reprogramming factors, OCT4, SOX2, NANOG, KLF4, c-MYC, and LIN28, in a single construct. The resulting six iPSC lines, named DPZcj_iPSC1-6 (one male and five females), demonstrated euploidy for more than 80 passages and were morphologically and transcriptionally indistinguishable from marmoset ESCs. The same team subsequently introduced another protocol for generating transgene-free iPSC lines from marmoset fibroblasts, using self-replicating Venezuelan equine encephalitis (VEE) mRNAs in feeder-free culture conditions (Petkov and Behr, 2021). In contrast with earlier studies that used standard culture media occasionally supplemented with FGF2 and transforming growth factor β (TGF-β), activin, and NODAL for iPSC propagation, this study used a complex medium consisting of StemMACS iPS-Brew XF, tankyrase inhibitor IWR1, GSK3 inhibitor CHIR99021, SRC family kinase inhibitor CGP77675, LIF, and forskolin (an adenylyl cyclase activator) to foster the growth of primary colonies and reinforce self-renewal. The necessity of including small molecules in the reprogramming medium aligns with the findings of a previous study describing the derivation of iPSC lines from marmoset fibroblasts using StemRNA-NM reprogramming kit (Reprocell) and the NutriStem medium (Reprocell) (Nakajima et al., 2019). In this study, NutriStem required an additional supplementation with N2B27, LIF, forskolin, GSK3 inhibitor, MEK inhibitor PD0325901, and activin receptor-like kinase (ALK) inhibitor A83-01. This cocktail allowed the establishment of a unique cjiPSC line termed RNA-iPSC#1.

Table 3.

iPSCs lines in New World monkeys

Species	Name of cell lines	Methodology of reprogramming	Cells differentiation analysis	Reference
Marmoset (C. jacchus)	Cj15, Cj36, Cj37, Cj81, Cj88, CjA10, CjA18, CjB8	retroviral transduction (OCT4, KLF4, SOX2, c-MYC)	embryoid bodies teratomas neural cells	Wu et al. (2010)
	Liver iPS-A, Liver iPS-B, Liver iPS1, Liver iPS7	retroviral transduction (OCT4, SOX2, KLF4, c-MYC, NANOG, LIN28)	embryoid bodies teratomas	Tomioka et al. (2010)
	unspecified	lentivirus transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies teratomas	Wiedemann et al. (2012)
	NM-iPS	episomal vector (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies	Yoshimatsu et al. (2021b)
	DPZcj_iPSC1–DPZcj_iPSC6	nonviral transduction (piggyBac)/(OCT4/3, SOX2, KLF4, c-MYC, NANOG, LIN28)	embryoid bodies teratomas	Debowski et al. (2015)
	cjFFs	self-replicating VEE-mRNAs OCT4, KLF4, SOX2, and c-MYC (VEE-OKSiM)	embryoid bodies teratomas	Petkov and Behr (2021)
	RNA-iPSC#1	RNA-based reprogramming/Stemgent StemRNA-NM	embryoid bodies neural cells	Nakajima et al. (2019)

iPSC lines established in Old World monkeys

Rhesus monkey

Rhesus iPSC (RhiPSC) lines were initially established by Liu et al. (2008). They reprogrammed skin fibroblasts using retroviral vectors that expressed human OCT4, KLF4, SOX2, and c-MYC. They created five cell lines, named M1, M2, M3, M5, and M6. The M2 line demonstrated differentiation into ectoderm, mesoderm, and endoderm derivatives in experimental teratomas (Table 4). A similar method was used by Chan et al. (2010) to reprogram skin fibroblasts from a transgenic Huntington’s monkey, resulting in the generation of a RhiPSC line known as rHD-iPSC. Subsequent studies focused on generating iPSC lines using integration-free, virus-free techniques that would be more suitable for preclinical research applications. Sosa et al. (2016, 2017) developed two iPSC lines, riPSC89 and riPSC90, from rhesus embryonic fibroblast cells. They used self-replicating VEE-mRNAs expressing four reprogramming factors OCT4, KLF4, SOX2, and GLIS1. During the same period, Zhang et al. (2017) produced six iPSC lines (L1–L6) from rhesus skin fibroblasts using episomal vectors. These vectors expressed OCT4, SOX2, KLF4, L-MYC, LIN28, and small hairpin TP53 (shTP53). Five of these lines exhibited a normal diploid karyotype (42XY) and displayed the molecular and functional properties associated with primed pluripotency. Unlike the riPSC89 and riPSC90 cell lines, which were established on mouse feeder cells, the RhiPSC lines developed by Zhang and colleagues were cultured on vitronectin in a medium supplemented with FGF2, activin A, and CHIR99021/IWR-1-supplemented human ESC-conditioned medium. Concurrently, Hong et al. (2017) reported the generation of two iPSC lines using the Cre-excisable STEMCCA vector. One line (ZG32-3-4) was derived from rhesus macaque fibroblasts, while the other (ZG15-M11-10) was derived from bone marrow stromal cells. A third line (ZH26-HS41) was generated from bone marrow CD34⁺ cells using the CytoTune-iPS 2.0 Sendai reprogramming kit. Last, in a study conducted by Rodriguez-Polo et al. (2021, 2022), the excisable six factors-in-one piggyBac system expressing SOX2, OCT4, KLF4, c-MYC, NANOG, and LIN28 was used to generate four iPSC lines, namely DPZ_iRhpb#1–4. These lines, consisting of three males and one female, were established using StemMACS iPS-Brew XF, tankyrase inhibitor IWR1, GSK3 inhibitor CHIR99021, SRC family kinase inhibitor CGP77675, LIF, and forskolin. They exhibited euploidy for more than 50 passages and demonstrated indistinguishable transcriptional characteristics compared with rhesus ESCs (Rodriguez-Polo et al., 2021, 2022).

Table 4.

iPSC lines in Old World monkeys

Species	Name of cell lines	Methodology of reprogramming	Cells differentiation analysis	Reference
Rhesus monkey (M. mulatta)	M1, M2, M3, M5, M6	retrovirus transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies Teratomas	Liu et al. (2008)
	rHD-iPSC	retrovirus expressing rhesus monkey (OCT4, SOX2, KLF4)	embryoid bodies	Chan et al. (2010)
	riPSC89, riPSC90	self-replicating VEE-mRNAs expressing, OCT4, KLF4, SOX2, GLIS1	teratomas	Sosa et al. (2016) Sosa et al. (2017)
	L1–L6	integration- free episomal vectors (OCT3/4, SOX2, KLF4, L-MYC, LIN28, shTP53)	teratomas cardiomyocytes	Zhang et al.,2017
	ZG32-3-4, ZG15-M11-10, ZH26-HS41	Cre-excisable STEMCCA vector, CytoTune-iPS 2.0 Sendai reprogramming kit	hematopoietic cardiac, hepatic, neural	Hong et al. (2017)
	DPZ_iRhpb#1 toDPZ_iRhpb#4	non-viral piggyBac transposon system (SOX2, OCT4, KLF4, c-MYC, NANOG, LIN28)	embryoid bodies teratomas	Rodriguez-Polo et al. (2021) Rodriguez-Polo et al. (2022)
	FF5-6-5, FF5-6-6, FF5-12-6, AF9-7-12, AF9-7-16, AF9-9-9, AF9-9-15	retrovirus transduction (OCT4, KLF4, SOX2, c-MYC)	embryoid bodies teratomas	Okahara-Narita et al. (2012)
Cynomolgus monkey (M. fascicularis)	CyiPSCs1, CyiPSCs2, CyiPSCs3, M1, M2, M3, M4	lentiviral transduction (SIV-based polycistronic vector), expressing OCT4, KLF4, SOX2, c-MYC	embryoid bodies teratomas cardiomyocytes	Wunderlich et al. (2012) Wunderlich et al. (2014)
	S-1, H-1, H-2, H-3, H-4, H-5	amphotropic retroviral transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies teratomas	Shimozawa et al. (2013)
	cIPSC1, cIPSC2, cIPSC3	Sendai virus transduction (OCT4, SOX2, KLF4, c-MYC)	endothelial cells	Thoma et al. (2016)
	cmESF-iPS-c5, cmKF-iPSC-C5	Sendai virus transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies	Zhen et al. (2022a) Zhen et al. (2022b)
Pig-tailed macaque (M. nemestrina)	MnOFiPS-6, MnOFiPS-7, MnOFiPS-10	retrovirus transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies teratomas	Zhong et al. (2011)
	pig-tailed iPSCs	Sendai virus transduction (OCT4, KLF4, SOX2, L-MYC)	embryoid bodies teratomas cardiomyocytes	Roodgar et al. (2022)
Japanese macaque (M. fuscata)	J5F1, J9F2	Sendai virus transduction (OCT4, KLF4, SOX2, L-MYC)	embryoid bodies neurospheres	Nakai et al. (2018)
Olive baboon (P. anubis)	biPS-90-25, biPS-160-2, biPS-160-5	retrovirus transduction (OCT4, KLF4, SOX2, c-MYC)	teratomas dopaminergic neurons	Navara et al. (2013)
	biPSCs	Sendai virus transduction (KLF4, OCT3/4, SOX2, c-MYC)	embryoid bodies teratomas	Navara et al. (2018)
	biPSC-RED	CytoTune-iPS 2.0 Sendai reprogramming Kit	embryoid bodies teratomas erythroid cells	Olivier et al. (2019)
	DPZ-biPSC1–D4PZ-biPSC5	non-viral piggyBac transposon system (SOX2, OCT4, KLF4, c-MYC, NANOG, LIN28)	embryoid bodies teratomas	Rodriguez-Polo et al. (2019) Rodriguez-Polo et al. (2022)
African green monkey (C. sabaeus)	iPSCs19	episomal vectors (OCT4, SOX2, KLF4, LIN28, shTP53)	embryoid bodies teratomas	Chung et al. (2020)

Cynomolgus monkey

Just as with humans and rhesus monkeys, the first iPSC lines in cynomolgus monkeys were created using retroviral and lentiviral vectors. In 2012, Okahara-Narita et al. generated seven CyiPSC lines using retrovirus-mediated transduction of human OCT4, SOX2, KLF4, and c-MYC (Table 4). In parallel, Wunderlich et al. (2012, 2014) established seven CyiPSC lines (CyiPSCs1-3; M1-M4) using a Simian immunodeficiency virus (SIV)-based polycistronic vector, which expressed codon-optimized human reprogramming factors OCT4, SOX2, KLF4, and c-MYC. Concurrently, Shimozawa et al. (2013) produced six CyiPSC lines using amphotropic retroviral vectors that expressed cynomolgus monkey cDNAs for the genes OCT4, SOX2, KLF4 and c-MYC. In their study, one line (S-1) was derived from newborn skin, whereas the other five lines came from fetal liver (H-1, H-2, H-3, H-4, and H-5). Subsequent studies used integration-free techniques to develop new CyiPSCs: Thoma et al. (2016) generated CyiPSCs from kidney fibroblasts of female cynomolgus monkeys using Sendai viruses expressing OCT4, SOX2, KLF4, and c-MYC. These new lines were initially developed on feeder cells, but were later adapted to grow on Matrigel; Zhen et al. (2022a, 2022b) also used the same somatic cell source and reprogramming method to create two additional CyiPSC lines, one from an ear skin fibroblast, cmESF-iPS-c5 (42XX), and the other from a kidney fibroblast, cmKF-iPSC-C5 (42XY).

Pig-tailed and Japanese macaques

In 2011, Zhong et al. generated iPSCs from oral fibroblasts (OF) of pig-tailed macaque (Macaca nemestrina) using retroviral vectors that encoded OCT4, SOX2, KLF4, and c-MYC (Table 4). These iPSC lines (MnOFiPSCs) were established on MEF- or human fetal fibroblast-derived feeder cells in a medium supplemented with FGF2. Maintenance in feeder-free conditions required further supplementation with MEK inhibitor PD0325901, and either insulin growth factor or a high concentration of FGF2 (200 ng/mL). Roodgar et al. (2022) generated iPSCs from peripheral blood mononuclear cells (PBMCs) of an adult pig-tailed macaque (Macaca nemestrina) using Sendai viruses that encoded OCT4, SOX2, KLF4, and c-MYC. These iPSCs were maintained in a feeder-free condition on recombinant laminin 511-coated plates and were cultured using E8 medium supplemented with the tankyrase inhibitor XAV939. Nakai et al. (2018) generated iPSCs from skin fibroblasts of two female Japanese macaque (Macaca fuscata) with Sendai virus expressing OCT4, SOX2, KLF4, and L-MYC, or plasmid vectors expressing OCT4, SOX2, KLF4, shTP53, LIN28, and EBNA1. These iPSCs, referred to as jm-iPSC, were established under feeder-free culture conditions, but feeder cells were found to be essential for their long-term maintenance. Two jm-iPSC lines, J5F1 (obtained with Sendai virus) and J9F2 (obtained with plasmid vectors) were shown to maintain a stable euploid karyotype (42XY). However, a genomic integration of OCT4, L-MYC, LIN28, and EBNA1 was identified in J9F2 cell.

Baboon

Navara et al. (2013) led the way in the creation of iPSC lines from the olive baboon, termed BabiPSC. They initially used skin fibroblasts reprogrammed with retroviral vectors, expressing human OCT4, SOX2, KLF4, and c-MYC. They successfully established three male euploid cell lines on mouse feeder cells: biPS-90-25, biPS-160-2, and biPS-160-5 (Table 4). Later, the same team developed BabiPSC lines from PBMCs of an adult male olive baboon (Navara et al., 2018). This was achieved using Sendai virus transduction of human KOS (a single vector combining KLF4, OCT4, and SOX2), c-MYC, and KLF4. They found that self-renewal stability was significantly enhanced by culturing the iPSCs in Pluristem medium. One cell line was shown to maintain a stable euploid karyotype (42XY). Following a similar pathway, Olivier et al. (2019) produced equivalent results using mobilized peripheral blood (MBP)-CD34⁺ cells of an adult male baboon and the CytoTune-iPS 2.0 Sendai Reprogramming Kit. In a parallel effort, Rodriguez-Polo et al. (2019, 2022) established a reliable protocol for generating BabiPSC lines from an adult female olive baboon using the reversible six factor-in-one piggyBac system and monkey cDNAs (SOX2, OCT4, KLF4, c-MYC, NANOG, and LIN28). This approach led to the development of five BabiPSC lines, named DPZ-biPSC1–5. Notably, three of these lines (DPZ_biPSC1, 4, and 5) were successfully adapted to feeder-free conditions using Geltrex and cultured in E8 medium (Rodriguez-Polo et al., 2019, 2022).

African green monkey

Chung et al. (2020) reported the establishment of two iPSC lines from female African green monkey (C. sabaeus) skin fibroblasts (Table 4). This was achieved using episomal vectors expressing OCT4, SOX2, KLF4, LIN28, and shTP53. iPSCs were established on mouse feeder cells in a medium supplemented with FGF2, KOSR, and LIF. One line, iPSC19, displayed a normal karyotype (60XY).

iPSC lines established in great apes

Chimpanzee and bonobo

iPSC lines have been successfully established in several Ape species including Chimpanzees (Pan troglodytes), Bonobos (Pan paniscus), Gorillas (Gorilla gorilla), and Orangutans (Pongo abelii) (Table 5). Marchetto et al. (2013) reported the establishment of two iPSC lines from chimpanzee skin fibroblasts, derived using retroviral vectors expressing human OCT4, SOX2, KLF4, and c-MYC. These ChiPSC lines, designated as PR00818 (48XY) and PR01209 (48XY), were cultured in feeder-free conditions in mTeSR medium. Using identical methodology, this group also developed two iPSC lines from male bonobos (BoiPSCs: AG05253 and PR01086), which demonstrated comparable pluripotency characteristics. In their study, Wunderlich et al. (2014) also reported the establishment of two Bonobo iPSC lines (B1 and B2) using an HIV-based polycistronic vector, which expressed the codon-optimized human reprogramming factors OCT4, SOX2, KLF4, and c-MYC. Fujie et al. (2014) moved toward integration-free somatic cell reprogramming and used a temperature-sensitive Sendai virus vector expressing human OCT4, SOX2, KLF4, and c-MYC reprogramming factors to create four iPSC lines (48XX) from chimpanzee peripheral blood cells, named C101, C201, C205, and C402. In a related study, Gallego Romero et al. (2015) developed seven iPSC lines (C1–C7) from chimpanzee skin fibroblasts using episomal vectors carrying five reprogramming factors (human OCT4, SOX2, KLF4, L-MYC, and LIN28), and shRNA against TP53. These lines were successfully maintained in culture for a minimum of 60 passages in both feeder and feeder-free conditions. They displayed normal karyotypes (four 46XX lines and three 46XY lines). They met all the established criteria for pluripotency. Mora-Bermudez et al. (2016) developed another iPSC line (Sandra A) from leukocytes of a female chimpanzee using the same episomal vectors as the previous study. The report does not contain a detailed characterization of the Sandra A iPSC line, but the cells were successfully used to produce brain organoids. Similarly, Kitajima et al. (2020) developed five iPSC lines from skin fibroblasts of three chimpanzee individuals (one male and two females) using the same reprogramming strategy. On this later study, the five lines, named 0363M-1, 0363M-2, 0274F-1, 0274F-2, and 0138F-1, were cultured under feeder-free condition in StemFit medium and were shown to maintain a normal karyotype (48XX or 48XY) for more than 20 passages. In another study by Lin et al. (2020), three iPSC lines from chimpanzee skin fibroblasts, named SF-1E6F-01 and SF-2E6F-01 lines, as well as one from adult testis fibroblasts, named TF-1E6FG-02, were generated with the same five factor/shTP53 strategy. All of these cell lines were cultured on feeder cells in chemically defined N2B27 medium supplemented with a MEK inhibitor (PD0325901) and a GSK3β inhibitor (CHIR99021). SF-1E6F-01 and SF-2E6F-01 displayed a normal 48XX karyotype. Ultimately, Roodgar et al. (2022) generated iPSCs from the PBMCs of an adult chimpanzee using Sendai viruses that encoded OCT4, SOX2, KLF4, and c-MYC. These iPSCs were maintained in a feeder-free condition on recombinant laminin 511 and were cultured using E8 medium supplemented with the Tankyrase inhibitor XAV939. They exhibited the hallmark molecular signature and differentiation potential of pluripotent cells.

Table 5.

iPSC lines in great apes

Species	Name of cell lines	Methodology of reprogramming	Cells differentiation analysis	Reference
Chimpanzees (P. troglodytes)	PR00818, PR01209,	retroviral transduction (KLF4, OCT4, SOX2, and c-MYC)	embryoid bodies teratomas	Marchetto et al. (2013)
Bonobos (P. paniscus)	AG05253, PR01086	retroviral transduction (KLF4, OCT4, SOX2 and c-MYC)	embryoid bodies teratomas	Marchetto et al. (2013)
	B1, B2	virus (HIV)-based polycistronic vector (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies teratomas	Wunderlich et al. (2014)
Chimpanzees (P. troglodytes)	C101, C201, C205, C402	Sendai virus transduction (OCT4, SOX2, KLF4 and c-MYC)	embryoid bodies teratomas	Fujie et al. (2014)
	C1, C2, C3, C4, C5, C6, C7	episomal plasmids vector (OCT4, SOX2, KLF4, LIN28, L-MYC, shTP53)	embryoid bodies teratomas hepatocytes, cardiomyocytes	Gallego Romero et al. (2015)
	Sandra A	episomal plasmid vectors (OCT4, SOX2, KLF4, L-MYC, LIN28, shTP53)	brain organoids	Mora-Bermudez et al. (2016)
	0363M-1, 0363M-2, 0274F-1, 0274F-2, 0138F-1	episomal plasmid vectors (OCT4, SOX2, KLF4, L-MYC, LIN28, shTP53)	embryoid bodies neurospheres	Kitajima et al. (2020)
Chimpanzees (P. troglodytes)	SF-1E6F-01, SF-2E6F-01, TF-1E6FG-02	episomal plasmid vectors (OCT4, SOX2, KLF4, L-MYC, LIN28, shTP53)	embryoid bodies teratomas neurospheres	Lin et al. (2020)
	Chimp-iPSCs	Sendai virus transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies teratomas	Roodgar et al. (2022)
Gorilla (G. gorilla)	G1–G5	retroviral transduction (OCT4, SOX2, NANOG, LIN28)	embryoid bodies teratomas	Wunderlich et al. (2014)
Orangutans (P. abelii)	KB10973, KB10460	retroviral transduction (OCT4, SOX2, KLF4, c-MYC)	embryoid bodies teratomas	Ramaswamy et al. (2015)
	Toba	episomal plasmid vectors (OCT4, SOX2, KLF4, L-MYC, LIN28, shTP53)	embryoid bodies teratomas	Mora-Bermudez et al. (2016)
	unspecified	CytoTune-iPS 2.0 Sendai Reprogramming kit	embryoid bodies neural cells	Geuder et al. (2021)

Gorilla and orangutan

Wunderlich et al. (2014) reported the first successful generation of five iPSC lines from a female gorilla (G. gorilla) (GoiPSCs: G1–G5; 48XX). These lines were derived from endothelial cells, using human OCT4, SOX2, NANOG, and LIN28 expressed through lentiviral vectors (Table 5). Ramaswamy et al. (2015) reported the successful generation of two iPSC lines, named KB10973 (male) and KB10460 (female). These lines were derived from cryopreserved skin fibroblasts of two Sumatran orangutans (P. abelii), using human OCT4, SOX2, KLF4, and c-MYC expressed through retroviral vectors. Both iPSC lines maintained a normal karyotype. In a subsequent study, Mora-Bermudez et al. (2016) developed another iPSC line, named Toba, from leukocytes of a female orangutan, using episomal vectors carrying five reprogramming factors (human OCT4, SOX2, KLF4, L-MYC, and LIN28), and shRNA against TP53. The report does not provide a detailed characterization of the Toba iPSC line, but the cells were successfully used to produce brain organoids. Finally, in a recent study, Geuder et al. (2021) produced iPSCs in orangutan and gorilla. This time, the source of the cells was urine epithelial cells, and reprogramming was achieved using the CytoTune-iPS 2.0 Sendai Reprogramming kit. The newly generated iPSCs were expanded on GelTrex in feeder-free condition, with StemFit medium supplemented with FGF2. All the gorilla and orangutan iPSC lines exhibited the hallmark molecular signature and differentiation potential of pluripotent cells.

Comparison between human and ape PSCs

It has been reported that producing high-quality iPSC lines is more challenging in chimpanzees than in humans and mice (Gallego Romero et al., 2015; Mora-Bermudez et al., 2016). To address this, a shTP53 vector has been incorporated into the reprogramming cocktail of several established chimpanzee iPSC lines to enhance the derivation efficiency, as previously demonstrated in humans (Marion et al., 2009). The focus then shifts to the question of how closely ESC and iPSC lines established in NHPs resemble those derived from humans under similar or identical reprogramming conditions. In an effort to tackle this, Wunderlich et al. (2014) measured gene expression using mRNA sequencing across a variety of iPSC lines: five gorilla iPSCs (G1–G5), two bonobo iPSCs (B1 and B2), three cynomolgus monkey iPSCs (M1–M3), and three human iPSCs (H1–H3). Additionally, they included six ESC lines, three derived from human embryos and three from cynomolgus monkey embryos. Notably, all 23 cell lines were cultivated under identical conditions, including feeder cells and FGF2 supplementation. They found that, within a given species, the average gene expression variability between cell lines derived from the same individual was as significant as the variability between cell lines from different individuals. This suggests that the gene expression differences between cell lines from the same species primarily results from non-genetic variations. Furthermore, it was observed that the expression distance between different primate species was more than three-times larger than the average distance between different ESC/iPSC lines derived from the same species. Thus, the genetic differences between species seems to significantly influence PSC gene expression. Gallego Romero et al. (2015) corroborated these findings using a different set of chimpanzee and human PSC lines, drawing similar conclusions. Specifically, they compared gene expression profiles across seven chimpanzee iPSC lines (C1–C7) and seven human iPSC lines, all of which were generated in the same laboratory from either fibroblasts (both chimpanzee and human) or lymphoblastoid cell lines (human). Greater dispersion was noted between the two species, while less dispersion was observed between human lines of different ethnic origins and minimal or no dispersion between human lines with different cell origins. Further analysis of differentially methylated regions between the iPSCs of both species yielded similar results. Collectively, these observations strongly suggest that human and chimpanzee iPSC lines substantially differ, irrespective of somatic cell types of origin. Last, Geuder et al. (2021) confirmed these findings using a homogeneous set of iPSC lines: three from gorillas, four from orangutans, and nine from humans. All these lines were derived from primary urinary cells in the same laboratory and cultivated under identical conditions. They found that the average distance was twice as large between gorilla, orangutan, and human iPSCs as it was between iPSCs from the same species or the same individuals. These three studies together highlight species-specific gene expression profiles across iPSC lines of hominids. However, it is important to note that none of these studies took into account the high genetic diversity among great apes. This diversity is known to be significantly higher in chimpanzees, gorillas, and orangutans than in humans (Prado-Martinez et al., 2013).

Reporter cell lines

In this segment, we deliver a comprehensive review of both ESC and iPSC lines that have been engineered to express gene reporters. These reporter lines can be used for tracking cells, or their differentiated progeny, in a wide range of experimental situations, both in vivo and in vitro. Wianny et al. (2008) advanced this field by creating a fluorescent reporter cell line derived from the LyonES1 RhESCs (Table 2). They achieved this by stably expressing a green fluorescent protein fused to a tau domain (tauGFP reporter) using a SIV-based lentiviral vector. The expression of tauGFP was control by the EF1α promoter. Upon in vitro differentiation, these cells produced neural precursors, which subsequently differentiated into neuronal and astroglial pathways post-grafting into the rat brain. This result underlines the applicability of LYON-ES1-tauGFP cells in neurodevelopmental studies. In a later study, Hong et al. (2017) modified the ZG15-M11-10, ZG32-3-4, and ZH26-HS41 RhiPSC lines (see Table 4) by inserting a truncated CD19 (hΔCD19) or GFP into the adeno-associated virus integration site 1, which functions as a safe harbor. Stable expression of ΔCD19 or GFP was demonstrated in vitro after differentiation to hematopoietic stem and progenitor cells, cardiomyocytes, hepatocytes, and neural progenitors. In addition, the majority of cells present in teratomas expressed ΔCD19 or GFP, including mature cells derived from all three germ layers.

Expanding on the knock-in methodology to marmoset PSCs, Yoshimatsu et al. (2019) modified the female CMES40 CjESC line (Table 1) and the unpublished male CjESC line, DSY127. A single copy of enhanced GFP (EGFP) was inserted into the β-actin (ACTB) locus. This resulted in ubiquitous EGFP expression. The EGFP transgene was engineered with heterotypic lox sites, allowing for straightforward replacement with a red fluorescent protein via Cre recombinase-mediated cassette exchange. Thus, this cell line, called BR29, is a versatile marmoset ESC line capable of stable transgene knock-in and expression. Yoshimatsu et al. (2021a) modified the female CMES40 CjESC line by inserting a mCerulean3 fluorescent reporter gene into the OCT4/POU5F1 locus, resulting in a OCT4-2A-mCerulean3 knock-in reporter allele (CMES40-OC cell line). The CMES40-OC cell line is useful for identifying undifferentiated PSCs within a population of differentiated cells and, if necessary, eliminating them by cell sorting before grafting.

Reprogramming of NHP PSCs to naive pluripotency

Primed- to naive-state conversion

Most of the NHP PSC lines reported above have been established either on mouse feeder cells or in matrix-coated dishes in feeder-conditioned medium, with occasional supplementation of FGF2 and KSR. These are conventional conditions known to support the self-renewal of human PSCs in the primed state of pluripotency. Subsequently, NHP ESCs and iPSCs in the prime state have been treated with resetting factors to instate naive-type characteristics. Fang et al. (2014) pioneered this approach in the rhesus monkey. They cultured conventional, primed RhiPSCs in a medium supplemented with growth factors LIF and FGF2, as well as inhibitors PD0325901 (MEK), CHIR99021 (GS3β), SB203580 (p38MAPK), and SP600125 (JNK) (medium known as 4i/L/b). This resulted in the gradual transformation of the cell population into dome-shaped colonies, a characteristic of naive cells. This combination of growth factors and small molecules has been known to facilitate the conversion of human PSCs from the primed to the naive state (Gafni et al., 2013). Along with the traditional pluripotency markers OCT4, NANOG, and TRA-1-80, these colonies expressed naive markers such as TBX3, FOXA2, NR5A2, PRDM14, LIFR, KLF5, and REX1. Moreover, female iPSCs displayed an X chromosome reactivation state, as indicated by the loss of H3K27me3 foci in the nuclei and a significant decrease in XIST expression levels, thereby validating the naive state. Notably, two other small molecules, the ROCK inhibitor Y27632 and the PKC inhibitor Gö6983, which are known to be beneficial for the survival and maintenance of human naive ESCs and iPSCs (Gafni et al., 2013), led to pronounced differentiation in rhesus naive iPSCs. This suggests that the requirements for signal regulation to establish naive pluripotency differ between humans and rhesus monkeys.

Concurrently, Chen et al. (2015) developed naive-like ESCs in the cynomolgus monkey. They cultivated conventional, primed CyESCs in a medium supplemented with LIF, FGF2, TGF-β1, BRAF inhibitor SB590885, and inhibitors of MEK, GS3β, p38MAPK, JNK, SRC, and ROCK (known as NHSM formulation, suitable for resetting naive characteristics in human PSCs) (Gafni et al., 2013). Notably, the NHSM formulation required further supplementation with vitamin C (referred to as NHSMV) to be effective on CyESCs. This culture regimen converted the CyESC population into dome-shaped colonies that could be easily propagated after single-cell dissociation, for up to 27 passages, while maintaining a normal karyotype. Culture of CyESCs in NHSMV resulted in the transcriptional activation of naive pluripotency markers such as the LIF receptor (LIFR), STAT3, and STAT3 target genes KLF4 and GBX2. Additionally, there was a noticeable downregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the histone methyltransferase EZH2, as well as an apparent loss of H3K27me3 foci and downregulation of XIST expression levels. These alterations further substantiate the conversion of CyESCs from a primed to a naive state, using a culture regimen originally developed for this conversion in humans, but modified to be effective on CyESCs. However, it is worth noting that a rhesus ESC line, LyonES1, when cultured in NHSMV, failed to acquire naive-like characteristics. This suggests that not every NHP PSC line is receptive to reprogramming induced by NHSMV (Aksoy et al., 2021). In another cynomolgus study, Honda et al. (2017) used a doxycycline (D)-inducible transient expression of KLF2 and NANOG, in conjunction with LIF (L), GS3Kβ and MEK inhibitors (referred to as 2iLD), to reprogram CMK6 CyESCs toward a naive state. Despite this protocol’s effectiveness in resetting naive-like features in humans (Takashima et al., 2014), it was unable to support naive pluripotency in CMK6 CyESCs. Ultimately, stable naive-like pluripotency was achieved using doxycycline, LIF, GS3β inhibitor, forskolin, and Kenpaullone, a GSK3β and cyclin-dependent kinase inhibitor (referred to as K3cLD). Supplementation of this mixture with BRAF and PKC inhibitors (termed K5cLD) was found to further reinforce self-renewal. K5cLD-CMK6 cells exhibited characteristic features of naive pluripotency as originally defined in mice and humans: higher expression of genes whose transcriptional activity increase in naive-type PSCs, including GDF3, DNMT3L, DPPA5, TBX3, TFCP2L1, CDH1, and KLF5; alterations of mitochondria morphology, from an elongated to a round shape with sparse and irregular cristae; resistance to 2-deoxyglucose, an inhibitor of glycolysis, and higher oxygen consumption rate, indicating an upregulation of mitochondrial respiration. In a recent study, Li et al. (2023) attempted to convert a newly established CyESC line to naive pluripotency using four protocols originally developed for naive human PSCs: PXGL (Guo et al., 2017), 5i/L/A (Theunissen et al., 2014), LCDM (Yang et al., 2017), and 4CL (Li et al., 2023; Mazid et al., 2022). Only the 4CL protocol resulted in CyESCs exhibiting naive characteristics, displayed elevated levels of KLF17, and chromosomal stability. This finding reinforces the notion that NHP and human PSCs do not equally respond to naive conversion protocols.

The conversion from the primed to the naive state was also explored in marmoset (Shiozawa et al., 2020), using the female CMES40 CjESC line (Sasaki et al., 2005) and the unpublished male CjESC line, DSY127. The conversion to a naive-like state was initiated by overexpressing six specific genes (OCT4, KLF2, KLF4, SOX2, c-MYC, and NANOG) via a doxycycline-inducible six factors-in-one piggyBac vector in a medium supplemented with LIF, forskolin, MEK (PD0325901), GSK3β β (CHIR99021), and ALK inhibitors (medium known as NBK5/2iLD). Upon conversion, the CjESCs exhibited increased expression of naive markers ESRRB, DPPA3, and KLF5, while the expression of primed markers LEFTY1 and LEFTY2 decreased. Additionally, the converted CjESCs demonstrated dependency on JAK activity to inhibit differentiation, decreased glycolytic capacity, and increased mitochondrial respiration demonstrating a primed to naive conversion. Moreover, the converted female CMES40 CjESCs exhibited reactivation of the second X chromosome, which further validates their transition to a naive-like state. When the transgenes were turned off via removal of doxycycline, the converted cells largely maintained their naïve-like gene expression. This suggests a stable resetting of naive-like characteristics.

The conversion from the primed to the naive was also investigated in three SCNT embryo-derived cell lines from the African green monkey, namely SC2, SC5, and SC6 (De Los Angeles et al., 2019). These cell lines were first transitioned from FGF2/KOSR-supplemented medium to N2B27 basal medium supplemented with LIF, the MEK inhibitor PD0325901, the tankyrase inhibitor XAV939, and the histone deacetylase inhibitor valproic acid. Subsequently, they were shifted to the N2B27 medium supplemented with PD0325901, the PKC inhibitor Gö6983, the GSK3 inhibitor CHIR99021, XAV939, and LIF, a regimen referred to as PGCXL. Of these, two cell lines, SC2 and SC5 were successfully propagated for 10 months under naive-like culture conditions. In comparison with the original ntESCs, the converted SC2 and SC5 cells displayed elevated levels of KLF4, increased nuclear staining for TFE3, and augmented mitochondrial membrane depolarization, which are all indicative of a naive state.

Alignment of naive PSCs with epiblasts of NHP pre-implantation embryos

One indirect approach to evaluate the extent of reprogramming to a naive pluripotency state involves comparing the transcriptome of reprogrammed cells with that of the embryo epiblast from the same species at various developmental pre- and post-implantation stages. This approach has been implemented in several studies, yielding diverse results. In their study, Nakamura et al. (2016) examined the single cell transcriptome of cynomolgus pre- and early post-implantation embryos, spanning embryonic day (E)6–7 (blastocysts) to E16–17 (gastrulating embryos) equivalent to Carnegie stages 1–5 in human development. They then studied the relationship between CyESCs, both before and after reprogramming with the NHSMV protocol as described by Chen et al. (2015), and cells of the ICM/EPI lineage. They found that, after NHSMV-induced reprogramming, the CyESCs’ transcriptome remained nearly unchanged from their original primed state, mirroring the gastrulating epiblast of E16/E17 embryos. In contrast, Shiozawa et al. (2020) observed in their study on marmoset naive ESCs that the transcriptome of naive CjESCs (DSY127 and CMES40) approached, without fully matching, that of cells of the ICM/EPI lineage of marmoset pre-implantation E6/E7 embryos. Recently, Bergmann et al. (2022) reprogrammed marmoset ESCs to naive-like pluripotency using a FGF2/KSR-based culture medium supplemented with MEK and WNT inhibitors, LIF, activin A, and ascorbic acid, a formulation designated as PLAXA. Before and after the conversion from primed to naive state, these cells were analyzed by single-cell RNA sequencing. The generated data were then compared with the single-cell transcriptome of marmoset blastocysts at E6, E15, and E20, equivalent to Carnegie stages 1, 5, and 6, respectively. Interestingly, CjESCs in the primed state mapped to the anterior domain of the E15–E20 embryonic disk, indicative of their late developmental stage. In contrast, CjESCs-PLAXA were found aligned with the ICM/epiblast cells of E6 embryos, indicative of a much more immature developmental stage.

A pressing question in this field pertains to whether certain reprogramming protocols yield results more consistent with naive pluripotency in vivo than others. Aksoy et al. (2021) directly addressed this question with RhESCs LyonES1 cells using the previously described 4i/L/b (Fang et al., 2014) and NHSMV (Chen et al., 2015) protocols. They also used five other protocols initially developed to reprogram human PSCs: ENHSM (Bayerl et al., 2021), TL2i (Chen et al., 2015), t2iLGöY (Takashima et al., 2014), TLCDK8/19i (Lynch et al., 2020), and LCDM/EPS (Yang et al., 2017). Comparing the resulting transcriptomes with those of cynomolgus embryos between stages E6 and E17 unveiled significant variation. One cell line (RhESC-NHSMV) exhibited strong similarities to the epiblast of the gastrulating embryo, four lines (RhESC-t2iLGöY, RhESC-LCDM/EPS, RhESC-ENHSM, and RhESC-TLCDK8/19i) were more akin to the epiblast of the pre-implantation embryo, and two (RhESC-4i/L/b and RhESC-TL2i) matched the early epiblast. These findings indicate that, similar to human, various naive-like states exhibiting different degrees of proximity to the early epiblast, can be generated from the same RhiPSC line.

Chimeras and blastoids with NHP PSCs

Intraspecies chimerism in cynomolgus monkeys

Germline chimerism, also known as systemic chimerism, pertains to the ability of PSCs to partake in the formation of all tissues and organs of both the fetus and the adult organism post-injection into pre-implantation embryos. Thus far, systemic chimerism has been documented solely in rodents and is deemed the definitive test for pluripotency, as it offers irrefutable proof that the injected cells possess the potential to generate fully functional cells in every organ and tissue. Consequently, a vital question arises: can ESCs and iPSCs from NHPs generate systemic chimeras in a comparable experimental model? Chen et al. (2015) reported the first, and so far only, chimeric fetuses in NHPs using CyESCs (XY) as donor cells and cynomolgus morulae as host embryos. In rodents, only naive PSCs are capable of colonizing pre-implantation embryos to produce chimeric fetuses (Nichols and Smith, 2009). Accordingly, they used GFP-expressing CyESCs, which had previously been reprogrammed to the naive state using the NHSMV culture protocol described earlier. Following the injection of NHSMV-CyESC-GFP cells into pre-implantation embryos and subsequent transfer into the uterus of female monkeys, two fetuses, one male and one female, were recovered after 100 days of development. All tissues examined exhibited broadly distributed GFP⁺ cells. The proportion of CyESC-derived progeny seemed to fluctuate considerably among different tissues and organs, from 1% in the heart and lungs to 10%–18% in the pancreas, bone marrow, and bladder. PCR analysis of proviral DNA, sex determining region Y gene analysis in the female fetus, and microsatellite parentage analysis of genomic DNA confirmed the presence of CyESC-derived progeny. In multiple tissues and organs, CyESC-derived progeny was found to express differentiation markers consistent with those of host cells, including MAP2⁺ neurons, MBP⁺ oligodendrocytes, and GFAP⁺ astrocytes in the brain; cTnT⁺ cardiomyocytes in the heart; and PDX1⁺ pancreatic progenitors, insulin⁺ β-cells, lucagon⁺ α-cells, and CK19⁺ ductal cells in the pancreas. Notably, sporadic GFP⁺/VASA⁺ cells were detected in the testis, suggesting germline colonization by CyESCs. Despite these promising initial results, several questions persist, such as why the proportion of CyESC-derived progeny varies dramatically among different tissues and organs. This variance might reflect the elimination of a portion of CyESC-derived cells in some tissues during development. Further investigation is needed to determine whether all cells differentiate strictly in accordance with the colonized organ, and whether any off-target differentiation occurs. Moreover, cell fusions between donor and host cells could occur throughout organogenesis, producing hybrid cells with confusing phenotypes. This possibility needs to be thoroughly examined. Despite these limitations, these preliminary findings have set the ground for the production of systemic chimeras in NHPs.

Interspecies chimeras with NHP PSCs

Interspecies chimeras have been proposed as an alternate paradigm for exploring the chimeric competence of human PSCs, to study early human developmental processes, and to address their potential applications in regenerative medicine (James et al., 2006). Although the creation of interspecific chimeras with human PSCs is beyond the scope of our review article, we believe it is pertinent to discuss this topic in the context of NHP PSCs and to examine the advantages offered by NHP PSCs in this research area. The first study of interspecies chimeras involving NHP PSCs was conducted in 2011. In this study, Simerly et al. (2011) explored the capability of rhesus macaque and baboon ESCs to colonize mouse embryos, either through aggregation with cleavage-state embryos or through injection into blastocysts. However, most of the NHP cells were rapidly eliminated. After the embryos transferred into surrogate hosts, no progeny cells derived from rhesus or baboon (RhESC or BaESC) were observed in any of the fetuses examined (Simerly et al., 2011). In hindsight, this result was not surprising, as the injected RhESCs and BaESCs were in a primed pluripotent state; mouse EpiSCs are unable to colonize pre-implantation mouse embryos unless they are selected for rare subpopulations resembling very early post-implantation epiblast (Han et al., 2010). From the early 2010s, the development of methods for reprogramming human and NHP PSCs into a naive state opened up new avenues for chimera research. A pioneering study by Fang et al. (2014) involved injecting naïve-like, 4i/L/b-reprogrammed RhiPSCs into mouse embryos. By using an anti-HuN antibody that also recognized the nuclei of NHP cells, they found RhiPSC-derived cells scattered throughout various organs, including the intestine, liver, heart, and brain, in two fetuses on the 16th day of development. However, whether these rhesus cells differentiated in conjunction with the host cells was not thoroughly examined. Honda et al. (2017) conducted a similar study using K5cLD-CMK6 CyESCs. Despite these cells exhibited naive-like markers, including transcriptional reconfiguration and acquisition of mitochondrial respiration typical of naive PSCs, no CyESC-derived progeny was observed in post-implantation embryos and fetuses following injection into mouse blastocysts and subsequent transfer to surrogate mothers. In a later study, Fu et al. (2020) developed a novel culture protocol for CyESCs, combining 50% PSC culture medium supplemented with FGF2, activin A, and GSK3β inhibitor CHIR99021, with 50% pig embryo culture medium. After injecting these cells into pig embryos and transferring them to swine surrogates, GFP⁺ cells were observed in various organs in three E25–30 fetuses (out of 59) and two newborns (out of 10) (Fu et al., 2020). Some CyESC-derived cells expressed lineage markers, including endoderm marker FOXA2, the mesoderm marker TBX6, and the ectoderm marker SOX1. However, the rate of chimerism was very low, estimated at one monkey cell per 1,000– to 10,000 pig cells, based on mitochondrial DNA analysis. The reasons for such a low chimerism rate are unclear.

NHP PSC embryo colonization ability

The limited number of chimeric fetuses obtained after injecting NHP PSCs into mouse or pig embryos, followed by intrauterine transfer, display a notably low chimerism rate. This rate ranges from a few percent in mice to less than 0.1% in pigs. Similar results have been obtained with human PSCs, albeit with significant fluctuations across studies (Gafni et al., 2013; Hu et al., 2020; Taei et al., 2020; Theunissen et al., 2016; Wu et al., 2017). These percentages are considerably lower than those typically observed with mouse ESCs after injection into mouse or rat embryos. Various factors may account for this discrepancy. Intuitively, one might attribute the low colonization efficiency primarily to the phylogenetic distance between the donor species providing the PSCs and the recipient species providing the host embryos. However, it is worth noting that chimeric fetuses obtained by Chen et al. (2015) involving the injection of cynomolgus ESCs into cynomolgus embryos resulted in a chimerism rate barely higher than rates achieved after injection into mouse embryos in the study of Fang et al. (2014). Moreover, these rates were significantly lower than those commonly observed in mouse PSCs/mouse embryo chimera experiments. This suggests that the phylogenetic distance may not be the sole factor contributing to low interspecies chimerism rates in these assays. Given these observations, we believe it is important to reconsider the nature of the NHP PSCs used in these experiments. Could particular characteristics of these cells be contributing to the low chimerism rates observed in the interspecies chimera assay? Aksoy and colleagues delved into this question in details, investigating the ability of rhesus ESCs (RhESCs; LyonES1 line) (Wianny et al., 2008) and human iPSCs (hiPSCs) to colonize cynomolgus and rabbit embryos after reprogramming to a naive-like pluripotent state. They found that, regardless of host embryo species or reprogramming protocols used, RhESCs and hiPSCs displayed an inability to survive and proliferate within the host embryo post-injection. In contrast, mouse ESCs demonstrated a high proliferation capability and naturally avoided cell death and differentiation in the same experimental context (Aksoy et al., 2021). In a recent report, Roodgar et al. (2022) corroborated and expanded these findings on apoptosis in chimpanzee and pig-tail macaque iPSCs after injection into rhesus embryos. They demonstrated that the ectopic expression of BCL2 significantly enhanced the efficiency of interspecies blastocyst formation in vitro (Roodgar et al., 2022). Collectively, these results strongly suggest that currently available naive NHP PSCs have significantly lower innate competence for embryo colonization and chimerism than naive mouse PSCs.

NHP PSC blastoids

Blastoid technology offers new possibilities for studying human embryonic development. However, the pursuit of this goal is stymied by ethical limitations placed on human embryo research, in particular, the near-universal ban on cultivating embryos beyond the 15th day of development. Within this restrictive regulatory landscape, NHPs offer a substantial advantage, as they do not face the same constraints. Capitalizing on this, Li et al. (2023) made use of 4CL naive CyESCs to generate cynomolgus blastoids, using a modified two-step protocol that was originally developed for naive human PSCs. In short, aggregates of 4CL naive CyESCs were treated with a hypoblast differentiation medium for 2 days, followed by a modified trophoblast differentiation medium for 5–7 days. Key modifications of the trophoblast differentiation medium involved an increased concentration of MEK inhibitor PD03225901, addition of ALK5 inhibitor A83-01, KOSR, and bovine serum albumin to enhance the formation of the blastoid cavity and the growth of the inner cell mass. The newly derived blastoids seemed to be morphologically similar to cynomolgus monkey blastocysts on E8 and E9, and they exhibited the three main lineages, epiblast, hypoblast, and trophoblast. After extended in vitro culture for 5–8 days, some blastoids gradually developed into embryonic disks with typical embryonic structures, including a yolk sac, chorionic cavity, amnion cavity, and primitive streak. Their morphology then closely resembled that of implanted human embryos at Carnegie stage 7. Single-cell transcriptomics verified differentiation to three germ layers, ectoderm, mesoderm, and definitive endoderm, as well as to visceral endoderm and primordial germ cells. Consequently, these monkey blastoids demonstrated the capacity for further development to the peri-gastrulation stage. Additionally, after the transfer of E7 blastoids to surrogate mothers, implantation in the uterus was observed, as evidenced by ultrasound imaging and a significant increase in chorionic gonadotropin and progesterone serum levels. However, the gestation sacs disappeared without any further development, which indicates that the transferred blastoids do not possess all the characteristics of natural embryos.

Concluding remarks

Over the past three decades, more than 180 PSC lines have been established from NHPs, and have been used in a variety of experimental settings including embryo chimeras and blastoids (Figure 2). These cell lines serve as unique repositories of primate genetic information and provide an invaluable resource for preclinical studies and biotechnological applications. Furthermore, they are of significant importance in studying primate embryonic development, including human development the research of which is limited by regulatory restrictions. A critical question arising in this context is the extent to which NHP PSC lines can accurately model their human counterparts. Transcriptome data from macaque PSC lines have revealed certain differences compared with human cell lines (Gallego Romero et al., 2015; Geuder et al., 2021; Wunderlich et al., 2014). Furthermore, the derivation of iPSCs in apes has proven to be challenging, sometimes requiring more complex gene cocktails than those commonly used for reprogramming human somatic cells (Gallego Romero et al., 2015; Mora-Bermudez et al., 2016). The culture media used for conversion from the primed to the naive state sometimes needs adjustment from the media initially described for humans, suggesting the existence of species-specific characteristics (Chen et al., 2015; Fang et al., 2014; Honda et al., 2017; Li et al., 2023). Nevertheless, when propagated in the primed pluripotent state, human and NHP PSCs share all defining features, highlighting the potential of NHP PSCs as models for studying human development and disease. Of note, Old World monkeys exhibit a high degree of genetic diversity, potentially exhibiting up to three times more variation in macaques than in humans (Warren et al., 2020; Yuan et al., 2012). This is also true for chimpanzees, gorillas, and orangutans, despite their small population sizes and relatively confined habitats (de Manuel et al., 2016; Locke et al., 2011; Prado-Martinez et al., 2013). However, we lack information about the geographical origins of the animals used to derive ESCs and iPSCs. This knowledge gap raises crucial questions about how this genetic diversity could impact the biology of PSCs, including their performance in chimerism or blastoid experiments. As we explore the potential benefits of PSCs derived from NHPs, it is imperative that future research considers this question.

Figure 2.

Summary of NHP PSC lines and their applications.

In addition to the use of NHP PSCs for preclinical development of cell therapy strategies, an area beyond the scope of this review, a prominent area of interest involves the generation of somatic and germline chimeras for studying primate development and establishing new models of human disease. Current methods involve TALEN- and CRISPR-Cas9-mediated genome editing in zygotes, procedures that are laborious and expensive in the context of NHPs (Chu et al., 2019; Kumita et al., 2019; Liu et al., 2014; Niu et al., 2014; Qiu et al., 2019; Zhang et al., 2018). Conversely, creating chimeric embryos using naive PSCs with well-characterized gene alterations could potentially pave the way for more economical and less time-consuming strategies. This could include cloning individuals using gene-edited NHP PSCs as sources of donor nuclei (da Silva and Martins, 2023; Rideout et al., 2000). Interspecies chimera is another research field where NHP PSCs are expected to play an important role, particularly for studying chimerism in the brain and the germline, without the ethical dilemmas associated with using human PSCs. In this context, the use of Apes PSCs is especially interesting because of their close relation to humans. More generally, NHP PSC lines will be extremely useful for comparatively studying primate development as well as their evolution (Kanton et al., 2019). The increasing genomic resources available is essential to this goal (Kuderna et al., 2023).