Efficacy of allogeneic mesenchymal stem cell administration in a model of acute ischemic kidney injury in cats.

OBJECTIVE: To evaluate the effects of allogeneic mesenchymal stem cells (MSCs) in a model of ischemic acute kidney injury (AKI). STUDY DESIGN: Randomized controlled trial. ANIMALS: Adult, purpose-bred research cats (n=15) and a historical reference group (n=3). METHODS: Cats underwent unilateral, in vivo, warm renal ischemia, then intravenous administration of 4 million adipose-derived MSCs, bone marrow-derived MSCs, or fibroblasts (n=5/treatment) 1h after reperfusion. Serum creatinine and blood urea nitrogen concentrations were measured at baseline and days 1 and 6. Urine specific gravity, urine protein to urine creatinine ratio, and glomerular filtration rate were measured at baseline and day 6. Both kidneys were harvested on day 6; histopathology was described and scored and smooth muscle actin was quantified with histomorphometry. A 2-way ANOVA was used to compare time and treatment. Chi square analysis was used to determine the % of cats with at least International Renal Interest Society (IRIS) Grade 1 AKI. RESULTS: Time, but not treatment, had a significant effect on renal function. No difference was noted in % of cats with IRIS AKI. Significantly fewer mitotic figures were observed in ischemic kidneys that received bone-marrow derived MSCs vs. fibroblasts. No differences in smooth muscle actin staining were noted. CONCLUSIONS: This study did not support the use of allogeneic MSCs in AKI in the regimen described here. Type of renal injury, MSC dose, allogenicity, duration, and route or timing of administration could influence the efficacy MSCs.

Feline mesenchymal stem cells and supernatant inhibit reactive oxygen species production in cultured feline neutrophils.

Feline bone marrow-derived MSCs (BMMSCs), adipose-derived MSCs (AMSCs) and fibroblasts (FBs) were isolated and cultured. Tri-lineage differentiation assays and flow cytometry were used to characterize MSCs. Neutrophils (NPs) were isolated from whole blood and the NPs production of reactive oxygen reactive oxygen species (ROS) was measured. NPs were cultured alone, with MSC culture supernatant (SN), BMMSCs or AMSCs. NPs incubated with BMMSCs had significantly lower ROS production than NPs incubated with AMSCs (p=0.0006) or FB (p<0.0001); NPs ROS production significantly decreased with increasing BMMSC cell number (p=0.0023) and significantly increased with NPs were incubated with FB compared to BMMSC (p=0.0003). Both BMMSC SN and AMSC SN had statistically significantly lower ROS production than FB SN when incubated with NPs (both p<0.0001). ROS production was significantly reduced with increased fractions of SN from BMMSCs (p=0.0467) and AMSCs (p=0.0017).

Nonviral minicircle generation of induced pluripotent stem cells compatible with production of chimeric chickens.

Chickens are vitally important in numerous countries as a primary food source and a major component of economic development. Efforts have been made to produce transgenic birds through pluripotent stem cell [primordial germ cells and embryonic stem cells (ESCs)] approaches to create animals with improved traits, such as meat and egg production or even disease resistance. However, these cell types have significant limitations because they are hard to culture long term while maintaining developmental plasticity. Induced pluripotent stem cells (iPSCs) are a novel class of stem cells that have proven to be robust, leading to the successful development of transgenic mice, rats, quail, and pigs and may potentially overcome the limitations of previous pluripotent stem cell systems in chickens. In this study we generated chicken (c) iPSCs from fibroblast cells for the first time using a nonviral minicircle reprogramming approach. ciPSCs demonstrated stem cell morphology and expressed key stem cell markers, including alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1. These cells were capable of rapid growth and expressed high levels of telomerase. Late-passage ciPSCs transplanted into stage X embryos were successfully incorporated into tissues of all three germ layers, and the gonads demonstrated significant cellular plasticity. These cells provide an exciting new tool to create transgenic chickens with broad implications for agricultural and transgenic animal fields at large.

α-1,3-Galactosyltransferase knockout pig induced pluripotent stem cells: a cell source for the production of xenotransplant pigs.

The shortage of human organs and tissues for transplant has led to significant interest in xenotransplantation of pig tissues for human patients. However, transplantation of pig organs results in an acute immune rejection, leading to death of the organ within minutes. The α-1,3-galactosyltransferase (GALT) gene has been knocked out in pigs to reduce rejection, yet additional genes need to be modified to ultimately make pig tissue immunocompatible with humans. The development of pig induced pluripotent stem cells (piPSCs) from GALT knockout (GALT-KO) tissue would provide an excellent cell source for complex genetic manipulations (e.g., gene targeting) that often require highly robust and proliferative cells. In this report, we generated GALT-KO piPSCs by the overexpression of POU5F1, SOX2, NANOG, LIN28, KLF-4, and C-MYC reprogramming genes. piPSCs showed classical stem cell morphology and characteristics, expressing integrated reprogramming genes in addition to the pluripotent markers AP, SSEA1, and SSEA4. GALT-KO piPSCs were highly proliferative and possessed doubling times and telomerase activity similar to human embryonic stem cells. These results demonstrated successful reprogramming of GALT-KO fibroblasts into GALT-KO piPSCs. GALT-KO piPSCs are potentially an excellent immortal cell source for the generation of pigs with complex genetic modifications for xenotransplantation, somatic cell nuclear transfer, or chimera formation.

SSEA4-positive pig induced pluripotent stem cells are primed for differentiation into neural cells.

Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However, further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still, the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study, we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming ßIII-TUB/MAP2+ neurons, GFAP+ astrocytes, and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors, piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons, astrocytes, and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans.

Avian-induced pluripotent stem cells derived using human reprogramming factors.

Avian species are important model animals for developmental biology and disease research. However, unlike in mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function, clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the generation of avian induced pluripotent stem cells (iPSCs), the first nonmammalian iPSCs, which were clonally isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accomplished using human pluripotency genes rather than avian genes, indicating that the process in which mammalian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes, and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that were only previously available in mammals.

Human haploid cells differentiated from meiotic competent clonal germ cell lines that originated from embryonic stem cells.

Early germ-like cells (GLCs) derived from human embryonic stem cells (hESCs) have presented new opportunities to study germ cell differentiation in vitro. However, differentiation conditions that facilitate the formation of haploid cells from the derived GLCs have eluded the field. The inability to propagate GLCs in culture is a further limitation, resulting in inconsistent rederivations of GLCs from hESCs with relatively few GLCs in these heterogeneous populations. Here we found in vitro conditions that enrich for DDX4/POU5F1+ GLCs (∼60%) and that has enabled continual propagation for >50 passages without loss of phenotype. Clonal isolation of single GLCs from these mixed cultures generated 3 GLC (>90% DDX4/POU5F1+) and 2 hESC (<0.1% DDX4+) lines that could be continually expanded without loss of phenotype. Differentiation of clonal GLC lines in serum resulted in expression of postmeiotic markers and >11% were haploid, ∼5-fold higher than previous studies. The robust clonal meiotic competent and incompetent GLC lines will be used to understand the factors controlling human germ cell meiosis and postmeiotic maturation.

Porcine induced pluripotent stem cells produce chimeric offspring.

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.

Neural differentiation of human embryonic stem cells at the ultrastructural level.

Neurodegerative disorders affect millions of people worldwide. Neural cells derived from human embryonic stem cells (hESC) have the potential for cell therapies and/or compound screening for treating affected individuals. While both protein and gene expression indicative of a neural phenotype has been exhibited in these differentiated cells, ultrastuctural studies thus far have been lacking. The objective of this study was to correlate hESC to neural differentiation culture conditions with ultrastructural changes observed in the treated cells. We demonstrate here that in basic culture conditions without growth factors or serum we obtain neural morphology. The addition of brain-derived neurotrophic factor (BDNF) and serum to cultures resulted in more robust neural differentiation. In addition to providing cues such as cell survival or lineage specification, additional factors also altered the intracellular structures and cell morphologies. Even though the addition of BDNF and serum did not increase synaptic formation, altered cellular structures such as abundant polyribosomes and more developed endoplasmic reticulum indicate a potential increase in protein production.