Optimization of culture conditions for the derivation and propagation of baboon (Papio anubis) induced pluripotent stem cells

<div><p>Induced pluripotent stem cells (iPSCs) offer the possibility of cell replacement therapies using patient-matched cells to treat otherwise intractable diseases and debilitations. To successfully realize this potential, several factors must be optimized including i) selection of the appropriate cell type and numbers to transplant, ii) determination of the means of transplantation and the location into which the transplanted cells should be delivered, and iii) demonstration of the safety and efficacy of the cell replacement protocol to mitigate each targeted disease state. A majority of diseases or debilitations likely to be targeted by cell-based therapeutic approaches represent complex conditions or physiologies manifest predominantly in primates including humans. Nonhuman primates afford the most clinically relevant model system for biomedical studies and testing of cell-based therapies. Baboons have 92% genomic similarity with humans overall and especially significant similarities in their immunogenetic system, rendering this species a particularly valuable model for testing procedures involving cell transplants into living individuals. To maximize the utility of the baboon model, standardized protocols must be developed for the derivation of induced pluripotent stem cells from living adults and the long-term maintenance of these cells in culture. Here we tested four commercially available culture systems (ReproFF, mTeSR1, E8 and Pluristem) for competence to maintain baboon iPSCs in a pluripotent state over multiple passages, and to support the derivation of new lines of baboon iPSCs. Of these four media only Pluristem was able to maintain baboon pluripotency as assessed by morphological characteristics, immunocytochemistry and RT-qPCR. Pluristem also facilitated the derivation of new lines of iPSCs from adult baboon somatic cells, which had previously not been accomplished. We derived multiple iPS cell lines from adult baboon peripheral blood mononuclear cells cultured in Pluristem. These were validated by expression of the pluripotency markers OCT4, NANOG, SOX2, SSEA4 and TRA181, as well as the ability to differentiate into tissues from all three germ layers when injected into immunocompromised mice. These findings further advance the utility of the baboon as an ideal preclinical model system for optimizing iPS cell-based, patient-specific replacement therapies in humans.</p></div

Pluripotency assayed by immunocytochemistry and teratoma formation.

<p>Baboon iPSCS were positive for the pluripotent transcription factors NANOG (A), OCT4 (B), SOX2 (C) and the cell surface marker SSEA4 (D). Following injection into NOD-SCID immunocompromised mice baboon iPSCs formed teratomas including tissues representative of all three germ layers. Neural rosettes of ectoderm (E, white asterisks), ossifying bone of mesoderm (F, white arrow) and ciliated lung epithelial of endoderm (G, black arrow).</p

Immunocytochemical localization of pluripotency associated factors.

<p>As evaluated by immunocytochemical staining, baboon iPSCs cultured in conditioned media and Pluristem had similar expression levels and subcellular distribution of OCT4 (A,F, Green), NANOG (B,G, Green), SOX2 (C,H, Green), TRA181 (D,I, Green) and SSEA4 (E,J, Green) Blue = DNA, Bar = 20 μm.</p

Comparison of culture conditions.

<p>Baboon iPSCs cultured on MEFs in conditioned media maintained closely packed cells with well-defined colony borders (A, F). Similar results were observed with iPSCs cultured in Pluristem (B, G). iPSCs cultured in ReproFF (C, H), mTeSR-1 (D, I) or mTeSR-E8 (E, J) showed signs of differentiation including dark areas (white arrowheads) and expanding differentiating zones (white arrows).</p

Gene network of known relationships among those genes differentially expressed in control- compared to vinclozolin lineage F3 generation germ cells.

<p>(A) E13 network, (B) E16 network. Gene node shape code: oval and circle – protein; diamond – ligand; irregular polygon – phosphatase; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Red colored nodes are up-regulated genes, blue color are down-regulated genes. Grey connecters represent general regulation, blue – expression regulation, purple – binding, green – promoter binding, orange – microRNA effect. Cell membrane, nucleus, mitochondria, endoplasmic reticulum and golgi localizations are indicated. Network was derived using Pathway Studio™ software.</p

Known relationships between genes having DMR in their promoter regions (grey nodes) and differentially expressed genes (red nodes) in control- compared to vinclozolin lineage F3 generation germ cells from: (A) E13 and (B) E16.

<p>Gene node shape code: oval and circle – protein; diamond – ligand; circle/oval on tripod platform – transcription factor; ice cream cone – receptor. Grey connecters represent general regulation, blue – expression regulation, purple – binding, green – promoter binding. Network was derived using Pathway Studio™ software.</p

Pathways enriched with F3 generation E13 and E16 germ cell gene lists.

<p>Pathways enriched with F3 generation E13 and E16 germ cell gene lists.</p

Olfactory Transduction Pathway showing olfactory receptor genes differentially expressed between F3 generation E13 PGC vinclozolin and control lineage rats.

<p>Adapted from Kyoto Encyclopedia of Genes and Genomes pathway rno04740.</p

DMR from (A) E13 PGC and (B) E16 germ cells.

<p>DMR from (A) E13 PGC and (B) E16 germ cells.</p

Tertiary Epimutations – A Novel Aspect of Epigenetic Transgenerational Inheritance Promoting Genome Instability

<div><p>Exposure to environmental factors can induce the epigenetic transgenerational inheritance of disease. Alterations to the epigenome termed “epimutations” include “primary epimutations” which are epigenetic alterations in the absence of genetic change and “secondary epimutations” which form following an initial genetic change. To determine if secondary epimutations contribute to transgenerational transmission of disease following in utero exposure to the endocrine disruptor vinclozolin, we exposed pregnant female rats carrying the <i>lacI</i> mutation-reporter transgene to vinclozolin and assessed the frequency of mutations in kidney tissue and sperm recovered from F1 and F3 generation progeny. Our results confirm that vinclozolin induces primary epimutations rather than secondary epimutations, but also suggest that some primary epimutations can predispose a subsequent accelerated accumulation of genetic mutations in F3 generation descendants that have the potential to contribute to transgenerational phenotypes. We therefore propose the existence of “tertiary epimutations” which are initial primary epimutations that promote genome instability leading to an accelerated accumulation of genetic mutations.</p></div