Karyotype analysis.

<p>CMK6_SFF and CMK970 cells retain the normal karyotype, male 40 XY. Red arrows indicate the Y chromosome.</p

A Single-Cell and Feeder-Free Culture System for Monkey Embryonic Stem Cells

<div><p>Primate pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), hold great potential for research and application in regenerative medicine and drug discovery. To maximize primate PSC potential, a practical system is required for generating desired functional cells and reproducible differentiation techniques. Much progress regarding their culture systems has been reported to date; however, better methods would still be required for their practical use, particularly in industrial and clinical fields. Here we report a new single-cell and feeder-free culture system for primate PSCs, the key feature of which is an originally formulated serum-free medium containing FGF and activin. In this culture system, cynomolgus monkey ESCs can be passaged many times by single-cell dissociation with traditional trypsin treatment and can be propagated with a high proliferation rate as a monolayer without any feeder cells; further, typical PSC properties and genomic stability can be retained. In addition, it has been demonstrated that monkey ESCs maintained in the culture system can be used for various experiments such as <i>in vitro</i> differentiation and gene manipulation. Thus, compared with the conventional culture system, monkey ESCs grown in the aforementioned culture system can serve as a cell source with the following practical advantages: simple, stable, and easy cell maintenance; gene manipulation; cryopreservation; and desired differentiation. We propose that this culture system can serve as a reliable platform to prepare primate PSCs useful for future research and application.</p></div

Directional differentiation to neuroectodermal cell lineages.

<p>CMK6_SFF cells were treated in the MT-CDM medium in the presence or absence of 10 µM SB, 1 µM PD, and 1 µM DM for 4 days. A combination of SB and PD and/or DM in the MT-CDM medium generated the cells that highly expressed the neuroectoderm markers Sox1 and/or Pax6. SB, SB431542 (TGF-β inhibitor). PD, PD0325901 (MEK inhibitor). DM, dorsomorphin (BMP inhibitor). <i>Scale bar</i> = 100 µm.</p

Morphology of monkey ESCs grown under the MT-fCFA culture condition.

<p>A, B. CMK6_SFF (A) and CMK970 (B) cells maintained in the single-cell and feeder-free culture system with the MT-fCFA medium grow as a uniform monolayer with a high proliferation rate. Each cell shows an undifferentiated morphology with a high nucleus-to-cytoplasm ratio. C. CMK6 cells maintained under the conventional culture condition with MEF feeders. <i>Scale bar</i> = 100 µm.</p

Neuronal differentiation.

<p>CMK6_SFF cells were differentiated into cortical neurons. A. Immunocytochemical analysis. Scale bar = 50 µm. B–D. NMDA-induced Ca²⁺ influx. NMDA (with 10 µM glycine) induced a concentration-dependent increase in [Ca²⁺]_i (B). MK-801 (C, NMDA receptor antagonist) and ifenprodil (D, NR2B-specific antagonist) decreased NMDA (10 µM)-induced Ca²⁺ influx.</p

Clonal isolation after gene transfer in CMK6_SFF cells.

<p>The plasmid pCAG-GFP2, which carries the CAG promoter-driven GFP2 green fluorescent protein cDNA and a neomycin resistance gene, was used for the gene transfer experiment. CMK6_SFF cells were transfected with pCAG-GFP2 using Human Stem Cell Nucleofector Solution 1 (Amaxa) and the A-013 program following the manufacturer's instructions. After 7–10 days of the gene transfer, G418-resistant colonies derived from single cells were selected and propagated in the MT-fCFA medium containing 80 µg/ml G418.</p

Teratoma formation.

<p>Teratomas consisting of the three germ layers developed from CMK6_SFF and CMK970 cells under the MT-fCFA culture condition. Cells were injected into the testis and subcutaneous space of SCID mice. After 8–11 weeks, the fate of the cells was analyzed. Hematoxylin and eosin staining showed differentiation into various tissues, including neural cells (ectoderm), chondrocyte (mesoderm), and intestinal epithelium (endoderm).</p

Pluripotency of Monkey ESCs grown under the MT-fCFA culture condition.

<p>A. Immunocytochemical analyses show that CMK6_SFF cells (P33) have a characteristic expression pattern of typical pluripotency factors, Nanog, Oct4, and Sox2 as well as that of cell surface markers, SSEA-4, TRA-1-60, and TRA-1-81, indicating their undifferentiated and pluripotent state. <i>Scale bar</i> = 100 µm. B. Flow cytometric analysis of Nanog, Oct4, and Sox2 co-expressing CMK6_SFF cells (P35) under the MT-fCFA culture condition. Cells were co-stained with Alexa Fluor 647-conjugated anti-Nanog, Alexa Fluor 488-conjugated anti-Oct4, and PE-conjugated anti-Sox2 or the corresponding isotype controls. C. CMK6_SFF (P37) and CMK970 (P31) cells are positive for alkaline phosphatase activity. ALP, alkaline phosphatase. <i>Scale bar</i> = 100 µm.</p

Growth curves of CMK6_SFF and CMK970 cells after thawing.

<p>A. Growth curves of CMK6_SFF and CMK970 cells after thawing were constructed using the IncuCyte ZOOM System, where the growth curves were built from confluence measurements acquired at 30-min intervals. Values from each time point were averaged across 50 separate regions. Error bars represent 1 SD about the mean for 50 independent regions. B. Representative phase images of CMK6_SFF and CMK970 cells at different time points after thawing.</p