Steve Stice

Enhanced Survivability of Cloned Calves Derived from Roscovitine-Treated Adult Somatic Cells

Abstract Nuclear transfer to produce cattle is inefficient because 1) donor cells are not easily synchronized in the proper phase of the cell cycle, 2) the nucleus of these cells is not effectively reprogrammed, 3) the rate of attrition of late-term pregnancies is high, and 4) the health of early postnatal calves is compromised. The cyclin dependent kinase 2 inhibitor, roscovitine, was used to maximize cell cycle synchrony and to produce cells that responded more reliably to nuclear reprogramming. Roscovitine-treated adult bovine granulosa cells (82.4%) were more efficiently synchronized (P < 0.05) in the quiescent G0/G1 phase of the cell cycle than were serum-starved cells (76.7%). Although blastocyst development following nuclear transfer was elevated (P < 0.05) in the serum-starved group (21.1%) relative to the roscovitine-treated cells (11.8%), the number of cells in the blastocysts derived from roscovitine-treated cells was higher (P < 0.05) than those derived from the serum-starved group (roscovitine-treated group: 142.8 ± 6.0 cells; serum-starved group: 86.8 ± 14.5 cells). The resulting fetal and calf survival after embryo transfer was enhanced in the roscovitine-treated group (seven surviving calves from six pregnancies) compared with serum-starved controls (two calves born, one surviving beyond 60 days, from five pregnancies). Roscovitine culture can predictably synchronize the donor cell cycle and increase the nuclear reprogramming capacity of the cells, resulting in enhanced fetal and calf survival and increased cloning efficiency.

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 × 106 compound library, the identification of novel chemical “hits” for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 106 compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.

KIT ligand and bone morphogenetic protein signaling enhances human embryonic stem cell to germ-like cell differentiation

BACKGROUND Signaling mechanisms involved in early human germ cell development are largely unknown and believed to be similar to mouse germ cell development; however, there may be species specific differences. KIT ligand (KITL) and Bone morphogenetic protein 4 (BMP4) are necessary in mouse germ cell development and may play an important role in human germ cell development. METHODS KITL signaling studies were conducted by differentiating human embryonic stem cells (hESCs) on KITL wild-type, hetero- or homozygous knockout feeders for 10 days, and the effects of BMP signaling was determined by differentiation in the presence of BMP4 or its antagonist, Noggin. The formation of germ-like cells was ascertained by immunocytochemistry, flow cytometry and quantitative RT-PCR for germ cell markers. RESULTS The loss of KITL in enrichment and differentiation cultures resulted in significant down-regulation of germ cell genes and a 70.5% decrease in germ-like (DDX4+ POU5F1+) cells, indicating that KITL is involved in human germ cell development. Moreover, endogenous BMP signaling caused germ-like (DDX4+ POU5F1+) cell differentiation, and the inhibition of this pathway caused a significant decrease in germ cell gene expression and in the number of DDX4+ POU5F1+ cells. Further, we demonstrated that eliminating feeders but maintaining their secreted extracellular matrix is sufficient to sustain the increased numbers of DDX4+ POU5F1+ cells in culture. However, this resulted in decreased germ cell gene expression. CONCLUSIONS From these studies, we establish that KITL and BMP4 germ cell signaling affects in vitro formation of hESC derived germ-like cells and we suggest that they may play an important role in normal human germ cell development.

Lineage-specific promoter DNA methylation patterns segregate adult progenitor cell types.

Mesenchymal stem cells (MSCs) can differentiate into multiple mesodermal cell types in vitro; however, their differentiation capacity is influenced by their tissue of origin. To what extent epigenetic information on promoters of lineage-specification genes in human progenitors influences transcriptional activation and differentiation potential remains unclear. We produced bisulfite sequencing maps of DNA methylation in adipogenic, myogenic, and endothelial promoters in relation to gene expression and differentiation capacity, and unravel a similarity in DNA methylation profiles between MSCs isolated from human adipose tissue, bone marrow (BM), and muscle. This similarity is irrespective of promoter CpG content. Methylation patterns of MSCs are distinct from those of hematopoietic progenitor cells (HPCs), pluripotent human embryonic stem cells (hESCs), and multipotent hESC-derived mesenchymal cells (MCs). Moreover, in vitro MSC differentiation does not affect lineage-specific promoter methylation states, arguing that these methylation patterns in differentiated cells are already established at the progenitor stage. Further, we find a correlation between lineage-specific promoter hypermethylation and lack of differentiation capacity toward that lineage, but no relationship between weak promoter methylation and capacity of transcriptional activation or differentiation. Thus, only part of the restriction in differentiation capacity of tissue-specific stem cells is programmed by promoter DNA methylation: hypermethylation seems to constitute a barrier to differentiation, however, no or weak methylation has no predictive value for differentiation potential.

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.

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.

Use of human embryonic stem cell derived-mesenchymal cells for cardiac repair.

Human mesenchymal stem cells (hMSC) have proven beneficial in the repair and preservation of infarcted myocardium. Unfortunately, MSCs represent a small portion of the bone marrow and require ex vivo expansion. To further advance the clinical usefulness of cellular cardiomyoplasty, derivation of “MSC‐like” cells that can be made available “off‐the‐shelf” are desirable. Recently, human embryonic stem cell‐derived mesenchymal cells (hESC‐MC) were described. We investigated the efficacy of hESC‐MC for cardiac repair after myocardial infarction (MI) compared to hMSC. Because of increased efficacy of cell delivery, cells were embedded into collagen patches and delivered to infarcted myocardium. Culture of hMSC and hESC‐MCs in collagen patches did not induce differentiation or significant loss in viability. Transplantation of hMSC and hES‐MC patches onto infarcted myocardium of athymic nude rats prevented adverse changes in infarct wall thickness and fractional area change compared to a non‐viable patch control. Hemodynamic assessment showed that hMSCs and hES‐MC patch application improved end diastolic pressure equivalently. There were no changes in systolic function. hES‐MC and hMSC construct application enhanced neovessel formation compared to a non‐viable control, and each cell type had similar efficacy in stimulating endothelial cell growth in vitro. In summary, the use of hES‐MC provides similar efficacy for cellular cardiomyoplasty as compared to hMSC and may be considered a suitable alternative for cell therapy. Biotechnol. Bioeng. 2012;109: 274–283.

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.

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.

Induced Pluripotent Stem Cell-Derived Neural Stem Cell Therapy Enhances Recovery in an Ischemic Stroke Pig Model.

Induced pluripotent stem cell-derived neural stem cells (iNSCs) have significant potential as an autologous, multifunctional cell therapy for stroke, which is the primary cause of long term disability in the United States and the second leading cause of death worldwide. Here we show that iNSC transplantation improves recovery through neuroprotective, regenerative, and cell replacement mechanisms in a novel ischemic pig stroke model. Longitudinal multiparametric magnetic resonance imaging (MRI) following iNSC therapy demonstrated reduced changes in white matter integrity, cerebral blood perfusion, and brain metabolism in the infarcted tissue. The observed tissue level recovery strongly correlated with decreased immune response, enhanced neuronal protection, and increased neurogenesis. iNSCs differentiated into neurons and oligodendrocytes with indication of long term integration. The robust recovery response to iNSC therapy in a translational pig stroke model with increased predictive potential strongly supports that iNSCs may be the critically needed therapeutic for human stroke patients.