Most methods used in these experiments have been described in detail in a recent book chapter (Mishra et al., 2016) and are summarized here in Fig. 1a. The starting somatic cells were skin fibroblasts. Skin samples were obtained from a newborn female marmoset, a 4-year-old male, a 7-year-old female and two different 13-year-old male animals. All samples were obtained from tissue samples of animals being euthanized at the SNPRC . The chimpanzee skin sample was obtained in 2011 from a stillborn animal, also at the SNPRC. Fibroblasts were derived from the skin samples as previously described (Mishra et al., 2016).

For all marmoset cell experiments and for derivation of chimpanzee iPS cells using retroviral vectors, fibroblasts were trypsinized and plated in polylysine-coated six-well plates (Corning, Tewksbury, MA, USA). The day following plating, cells were infected with a mixture of four pMXs retroviruses, each encoding one of the reprogramming factors OCT4, SOX2, KLF4 and c-MYC (Salk Institute GT3 core), and the infection was repeated once more the following day as previously described (Wu et al., 2009). For chimpanzee Sendai iPS clones, infection was carried out using the CytoTune-iPS Sendai Reprogramming Kit according to the manufacturer's protocol (Invitrogen). Chimpanzee Epstein–Barr virus nuclear antigen (EBNA) episomal iPS clones were generated using the Episomal iPSC Reprogramming Vectors (Invitrogen) according to the manufacturer's protocol. The plasmids were introduced into the fibroblasts via electroporation using a Nucleofector instrument (Lonza). Following infection or transfection, cells were treated over the next several weeks as previously described and as shown in outline in Fig. 1. Routine iPS cell maintenance was in E8 medium (Chen et al., 2011) supplemented with 10 % fetal bovine serum (GlobalStem, Gaithersburg, MD, USA). Culture plates were coated with Matrigel. Subculturing was performed with Accutase (BioExpress, Kaysville, UT, USA).

All iPS cell clones were grown in E8 medium until the cells were to be tested for their differentiation potential. As the cells approached confluency, in the 24 h preceding the protocol cells were incubated in E8 medium either with or without 0.5 % DMSO (Sigma Aldrich, St. Louis, MO, USA). The following day the medium was aspirated, and the cells were disassociated with Accutase. Cells were collected, centrifuged and suspended at a concentration of 3000 cells per 30 µL in the differentiation medium previously described (Qiu et al., 2015). The lid of a 96-well plate was used to create numerous 30 µL droplets, each containing 3000 cells. The lids were then inverted and placed in a humidified incubator at 37.5∘ C for 72 h and allowed to form embryoid bodies. After this time the cells were collected by flushing the lids with DMEM/F12 into a 15 mL conical tube and allowed to settle under gravity. The medium was then aspirated, and the embryoid bodies were resuspended in 2 mL of a second differentiation medium, also as previously described (Qiu et al., 2015), and plated in a nonadherent 35 mm dish. Cells were maintained in the same medium for 72 h. Following this period, cells were harvested for preparation of RNA.

Total RNA was isolated from cells using RNA Bee (Tel-Test, Friendswood, TX, USA) according to the manufacturer's instructions. A total of 2 µg of RNA was reverse-transcribed by using SuperScript II (Life Technologies). Quantitative polymerase chain reaction (qPCR) was conducted using SYBR Green detection and an ABI 7900HT system (Applied Biosystems, Foster City, CA, USA). Levels of messenger RNAs (mRNAs) are reported as cycles versus β-actin. The data analysis for this paper was generated using Microsoft Excel and the Real Statistics Resource Pack software (Release 4.3; copyright 2013–2015 Charles Zaiontz, www.real-statistics.com). Responses to differentiation and DMSO pretreatment were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test of multiple comparisons with a control.

For this study we selected animals of a range of ages spanning most of the life span of the marmoset (a newborn female, a 4-year-old male, a 7-year-old female and two 13-year-old male animals). We chose the 13-year-old animals as representing individuals within the senescent period of the life span of the marmosets in this colony (Fig. 1b). Multiple clones were derived from the older animals in order to test the possibility that there are defects in differentiation in some iPS cells derived from older donors. iPS cells were generated by forced expression of the Yamanaka reprogramming factors OCT4, SOX2, KLF4 and c-MYC by retroviral transduction (Fig. 1a). Colonies began to appear around day 8 and were clearly discernible and large enough for isolation by day 11 at all ages (Fig. 2a). To confirm proper reprogramming, we performed qPCR on the isolated clones to determine the expression levels of the pluripotency markers NANOG and endogenous OCT4. We found appropriate expression levels for all clones derived. In all cases the levels were much higher than those in skin fibroblasts, as previously reported (Wu et al., 2010). However there were more minor differences in expression of these pluripotency factors among the different iPS cell clones (Table 1) The frequency of iPS cell clone generation, colony formation and the rate of cell growth were all unaffected by donor age. Cellular morphology of all of the derived iPS cell clones was as expected for marmoset iPS cells (Fig. 2). The distinctive morphology (cell shape, nuclear / cytoplasmic ratio, cell attachment in colonies) has been shown to be a good indicator of reprogramming in marmoset iPS cells (Wu et al., 2010) and in pluripotent cells generally (Quintanilla Jr., 2013).

We selected one iPS cell clone each from the newborn, 4-year-old and 7-year-old animals and three clones from each of the 13-year-old animals. Cells of each clone were subjected to a directed neural differentiation protocol previously employed in this lab (Qiu et al., 2015). After 7 days the differentiated cells were assayed for the expression levels of NCAD, POU3F1, SOX1, NKX6.6 and OLIG2 by qPCR. These genes were previously shown to be highly responsive to the differentiation protocol used and to provide useful markers of neural differentiation generally (Fig. 3a–e). They represent a collection of marker genes that are broadly useful in assessing differentiation potential, rather than differentiation into a specialized neural lineage. The results show that NCAD expression was significantly decreased (p < 0.05) in the average of the three clones from each of the 13-year-old marmosets when compared to the newborn. For POU3F1 expression was significantly decreased in the average of the three clones from 13-year-old marmoset #1 but not 13-year-old marmoset #2 when compared to the newborn. For SOX1 expression was significantly decreased in the clone from the 7-year-old and in the average of the three clones from each of the 13-year-old marmosets when compared to the newborn. For NKX6.1 expression was significantly decreased in the clone from the 7-year-old and in the average of the three clones from each of the 13-year-old marmosets when compared to the newborn. Furthermore, for OLIG2, a significant decrease was found in the clone from the 7-year-old and in the average of the three clones from each of the 13-year-old marmosets when compared to the newborn. Because we had previously shown that 24 h DMSO pretreatment may increase the differentiation potential of marmoset iPS cells, as well as human pluripotent cells (Chetty et al., 2013; Qiu et al., 2015), we repeated our differentiation protocol after allowing the cells to incubate for 24 h with 0.5% DMSO added to the normal growth medium. The results show that after pretreatment the clone from the 7-year-old showed a significant increase in POU3F1 expression compared to the same cells without pretreatment. The iPS cells from 13-year-old marmoset #1 showed significant increases for all five markers, while 13-year-old #2 showed a significant increase for four of them.

We next investigated the six clones generated from the two 13-year-old marmosets individually (Fig. 4a–e). The results show that NCAD expression was significantly increased by DMSO pretreatment in four out of the six clones; POU3F1 expression was significantly increased by DMSO pretreatment in four out of the six clones; SOX1 expression was significantly increased by DMSO pretreatment in four of the six clones; NKX6.1 expression was significantly increased by DMSO pretreatment in five out of the six clones; and for OLIG2 a significant increase by DMSO pretreatment was found for five of the six clones from the two 13-year-old marmosets.

Overall, these experiments show that generation of iPS cell clones was feasible in the case of all ages of marmosets tested and that differentiation defects, which were apparent as a function of age in the case of some of the derived cell clones, were mostly corrected by prior treatment with DMSO. While there were minor differences in the pluripotency characteristics of the clone derived from old animals, these did not correlate with the occurrence of defects in directed differentiation.

The chimpanzee is a NHP species of exceptional interest, and iPS cells from this species will be of great value in comparative in vitro studies. Many technologies are currently available for the generation of iPS cells. In order to investigate what effect vector choice might have on the differentiation potential of chimpanzee iPS cells, we generated multiple iPS cell clones using three different technologies: an integrating retrovirus, an RNA-based replicative virus (Sendai virus) and a replicating episomal plasmid vector based on the Epstein–Barr virus genome (EBNA). All three vectors were able to generate multiple clones in approximately the same time frame and with protocols similar to that of the previously mentioned retroviral generation method (Fig. 1a). In place of the retroviral infection step, cells were exposed to recombinant Sendai virus vectors or were transfected by electroporation with the episomal EBNA vectors. Upon isolation, clones from all vectors displayed normal growth characteristics and displayed typical morphologies (Fig. 5).

We then exposed the chimpanzee iPS cells generated from each vector to the same neural differentiation protocol as used for marmoset iPS cells. We assayed the same neural markers to quantify the level of differentiation exhibited by each clone (Fig. 6a–e). In each case we compared the extent of induction of the same differentiation markers as used for the marmoset cells and compared the induction with that observed in the retrovirally reprogrammed cells as our standard. For NCAD we found significantly decreased expression in one out of three Sendai virus clones and three out of four EBNA clones when compared to a retrovirally generated clone. For POU3F1 we found significantly decreased expression in none of three Sendai virus clones and two out of four EBNA clones when compared to a retrovirally generated clone. For SOX1 we found significantly decreased expression in one of three Sendai virus clones and one out of four EBNA clones when compared to a retrovirally generated clone. For NKX6.1 we found significantly decreased expression in two out of three Sendai virus clones and three out of four EBNA clones when compared to a retrovirally generated clone. Finally, for OLIG2 we found significantly decreased expression in none of three Sendai virus clones and four out of four EBNA clones when compared to a retrovirally generated clone. We once again repeated the differentiation with a 24 h pretreatment of 0.5 % DMSO in the growth medium and found that many of the markers that had previously shown previous decreases were now significantly increased. For NCAD we found significantly increased expression in one out of three Sendai virus clones and four of four EBNA clones when compared to differentiation without pretreatment. For POU3F1 we found significantly increased expression in one of three Sendai virus clones and two out of four EBNA clones when compared to differentiation without pretreatment. For SOX1 we found significantly increased expression in three out of three Sendai virus clones and three of four EBNA clones when compared to differentiation without pretreatment. For NKX6.1 we found significantly increased expression in the retroviral clone, one out of three Sendai virus clones and three out of four EBNA clones when compared to differentiation without pretreatment. Finally, for OLIG2 we found significantly increased expression in the retroviral clone, one out of three Sendai virus clones and four out of four EBNA clones when compared to differentiation without pretreatment.

Overall, therefore, we showed that chimpanzee iPS cells may be generated by multiple technologies and that clone-specific defects in differentiation under standardized conditions can be mostly eliminated by prior exposure to DMSO.