Jennifer Mumaw

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.

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 neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology.

BackgroundLysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development.ResultsOur results demonstrate that Lysophosphatidic Acid (LPA) and Sphingosine-1-phosphate (S1P) receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR)- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK.ConclusionThus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors.

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.

Regulation of stem cell pluripotency and differentiation by G protein coupled receptors

Stem cell-based therapeutics have the potential to effectively treat many terminal and debilitating human diseases, but the mechanisms by which their growth and differentiation are regulated are incompletely defined. Recent data from multiple systems suggest major roles for G protein coupled receptor (GPCR) pathways in regulating stem cell function in vivo and in vitro. The goal of this review is to illustrate common ground between the growing field of stem cell therapeutics and the long-established field of G protein coupled receptor signaling. Herein, we briefly introduce basic stem cell biology and discuss how several conserved pathways regulate pluripotency and differentiation in mouse and human stem cells. We further discuss general mechanisms by which GPCR signaling may impact these pluripotency and differentiation pathways, and summarize specific examples of receptors from each of the major GPCR subfamilies that have been shown to regulate stem cell function. Finally, we discuss possible therapeutic implications of GPCR regulation of stem cell function.

Superparamagnetic iron oxide nanoparticles as a means to track mesenchymal stem cells in a large animal model of tendon injury.

The goal of this study was to establish an SPIO-based cell-tracking method in an ovine model of tendonitis and to determine if this method may be useful for further study of cellular therapies in tendonitis in vivo. Functional assays were performed on labeled and unlabeled cells to ensure that no significant changes were induced by intracellular SPIOs. Following biosafety validation, tendon lesions were mechanically (n = 4) or chemically (n = 4) induced in four sheep and scanned ex vivo at 7 and 14 days to determine the presence and distribution of intralesional cells. Ovine MSCs labeled with 50 µg SPIOs/mL remained viable, proliferate, and undergo tri-lineage differentiation (p < 0.05). Labeled ovine MSCs remained detectable in vitro in concentrated cell numbers as low as 10 000 and in volumetric distributions as low as 100 000 cells/mL. Cells remained detectable by MRI at 7 days, as confirmed by correlative histology for dually labeled SPIO+/GFP+ cells. Histological evidence at 14 days suggested that SPIO particles remained embedded in tissue, providing MRI signal, although cells were no longer present. SPIO labeling has proven to be an effective method for cell tracking for a large animal model of tendon injury for up to 7 days post-injection. The data obtained in this study justify further investigation into the effects of MSC survival and migration on overall tendon healing and tissue regeneration.

Role of astrocytes, soluble factors, cells adhesion molecules and neurotrophins in functional synapse formation: implications for human embryonic stem cell derived neurons.

Availability of human embryonic stem cells (hESCs) and its neural derivatives has opened up wide possibilities of using these cells as tools for developmental studies, drug screening and cell therapies for treating neurodegenerative diseases. However, for hESC-derived neurons to fulfill their potential they need to form functional synapses and spontaneously active neural networks. Until recently very few studies have reported hESC-derived neurons capable of forming such networks, suggesting lack of certain components in culture media to promote mature synaptogenesis. In this review we discuss the various factors that enhance functional synapse formation in primary and stem cell-derived neuronal cultures. These factors include astrocytes, astrocyte-derived factors, cell adhesion molecules and neurotrophins. We discuss the current literature on studies that have used these factors for functional differentiation of primary neural cultures, and discuss its implications for stem cell -derived neural cultures.

Rapid Heterotrophic Ossification with Cryopreserved Poly(ethylene glycol-) Microencapsulated BMP2-Expressing MSCs

Autologous bone grafting is the most effective treatment for long-bone nonunions, but it poses considerable risks to donors, necessitating the development of alternative therapeutics. Poly(ethylene glycol) (PEG) microencapsulation and BMP2 transgene delivery are being developed together to induce rapid bone formation. However, methods to make these treatments available for clinical applications are presently lacking. In this study we used mesenchymal stem cells (MSCs) due to their ease of harvest, replication potential, and immunomodulatory capabilities. MSCs were from sheep and pig due to their appeal as large animal models for bone nonunion. We demonstrated that cryopreservation of these microencapsulated MSCs did not affect their cell viability, adenoviral BMP2 production, or ability to initiate bone formation. Additionally, microspheres showed no appreciable damage from cryopreservation when examined with light and electron microscopy. These results validate the use of cryopreservation in preserving the viability and functionality of PEG-encapsulated BMP2-transduced 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).

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 βIII-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.