Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

To proliferate and culture these derived NEP cells, ideal conditions were obtained using neurobasal medium supplemented with B27 and basic fibroblast growth factor in 5% oxygen. NEP cells were continuously propagated for longer than 6 months without losing their multipotent cell characteristics and maintained a stable chromosome number. STEM CELLS 2006;24:125-138

Over 700 drugs have failed in stroke clinical trials, an unprecedented rate thought to be attributed in part to limited and isolated testing often solely in “young” rodent models and focusing on a single secondary injury mechanism. Here, extracellular vesicles (EVs), nanometer-sized cell signaling particles, were tested in a mouse thromboembolic (TE) stroke model. Neural stem cell (NSC) and mesenchymal stem cell (MSC) EVs derived from the same pluripotent stem cell (PSC) line were evaluated for changes in infarct volume as well as sensorimotor function. NSC EVs improved cellular, tissue, and functional outcomes in middle-aged rodents, whereas MSC EVs were less effective. Acute differences in lesion volume following NSC EV treatment were corroborated by MRI in 18-month-old aged rodents. NSC EV treatment has a positive effect on motor function in the aged rodent as indicated by beam walk, instances of foot faults, and strength evaluated by hanging wire test. Increased time with a novel object also indicated that NSC EVs improved episodic memory formation in the rodent. The therapeutic effect of NSC EVs appears to be mediated by altering the systemic immune response. These data strongly support further preclinical development of a NSC EV-based stroke therapy and warrant their testing in combination with FDA-approved stroke therapies.Electronic supplementary materialThe online version of this article (10.1007/s12975-017-0599-2) contains supplementary material, which is available to authorized users.

Human pluripotent embryonic stem (ES) cells have important potential in regenerative medicine and as models for human preimplantation development; however, debate continues over whether embryos should be destroyed to produce human ES cells. We have derived four ES cell lines on mouse embryonic fibroblast cells in medium supplemented with basic fibroblast growth factor, human recombinant leukemia inhibitory factor, and fetal bovine serum. The source of these cell lines was poor-quality embryos that in the course of routine clinical practice would have been discarded. After continuous proliferation in vitro for more than 12 months, these ES cell lines maintained their developmental potential to form trophoblast and somatic cells, including cardiac muscle and neuronal tissue.

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We have developed a method, using nuclear transplantation, to produce transgenic embryonic stem (ES)-like cells from fetal bovine fibroblasts. These cells, when reintroduced into preimplantation embryos, differentiated into derivatives from the three embryonic germ layers, ectoderm, mesoderm, and endoderm, in 5-month-old animals. Six out of seven (86%) calves born were found to be chimeric for at least one tissue. These experiments demonstrate that somatic cells can be genetically modified and then de-differentiated by nuclear transfer into ES-like cells, opening the possibility of using them in differentiation studies and human cell therapy.

Bone marrow mesenchymal stem cells (MSCs) are adult pluripotent cells that are considered to be an important resource for human cell-based therapies. Understanding the clinical potential of MSCs may require their use in preclinical large-animal models, such as pigs. The objectives of the present study were 1) to establish porcine MSC (pMSC) cultures; 2) to optimize in vitro pMSC culture conditions, 3) to investigate whether pMSCs are amenable to genetic manipulation, and 4) to determine pMSC reprogramming potential using somatic cell nuclear transfer (SCNT). The pMSCs isolated from bone marrow grew, attached to plastic with a fibroblast-like morphology, and expressed the mesenchymal surface marker THY1 but not the hematopoietic marker ITGAM. Furthermore, pMSCs underwent lipogenic, chondrogenic, and osteogenic differentiation when exposed to specific inducing conditions. The pMSCs grew well in a variety of media, and proliferative capacity was enhanced by culture under low oxygen atmosphere. Transient transduction of pMSCs and isogenic skin fibroblasts (SFs) with a human adenovirus carrying the gene for green fluorescent protein (GFP; Ad5-F35eGFP) resulted in more pMSCs expressing GFP compared with SFs. Cell lines with stable genetic modifications and extended expression of transgene were obtained when pMSCs were transfected with a plasmid containing the GFP gene. Infection of pMSC and SF cell lines by an adeno-associated virus resulted in approximately 12% transgenic cells, which formed transgenic clonal lines after propagation as single cells. The pMSCs can be expanded in vitro and used as nuclear donors to produce SCNT embryos. Thus, pMSCs are an attractive cell type for large-animal autologous and allogenic cell therapy models and for SCNT transgenesis.

Human embryonic stem cells (hESC) have the potential to produce all of the cells in the body. They are able to self-renew indefinitely, potentially making them a source for large-scale production of therapeutic cell lines. Here, we developed a monolayer differentiation culture that induces hESC (WA09 and BG01) to form epithelial sheets with mesodermal gene expression patterns (BMP4, RUNX1, and GATA4). These E-cadherin+ CD90low cells then undergo apparent epithelial-mesenchymal transition for the derivation of mesenchymal progenitor cells (hESC-derived mesenchymal cells [hES-MC]) that by flow cytometry are negative for hematopoietic (CD34, CD45, and CD133) and endothelial (CD31 and CD146) markers, but positive for markers associated with mesenchymal stem cells (CD73, CD90, CD105, and CD166). To determine their functionality, we tested their capacity to produce the three lineages associated with mesenchymal stem cells and found they could form osteogenic and chondrogenic, but not adipogenic lineages. The derived hES-MC were able to remodel and contract collagen I lattice constructs to an equivalent degree as keloid fibroblasts and were induced to express alpha-smooth muscle actin when exposed to transforming growth factor (TGF)-beta1, but not platelet derived growth factor-B (PDGF-B). These data suggest that the derived hES-MC are multipotent cells with potential uses in tissue engineering and regenerative medicine and for providing a highly reproducible cell source for adult-like progenitor cells.