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Purpose: To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions., Methods: Limbal epithelial cells were isolated from 139 donors using 15 variations of three dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) keratinocyte growth medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and epidermal growth factor (EGF) were evaluated at 5%, 14%, and 20% O2. Human MRC-5, dermal (fetal, neonatal, or adult), and limbal stromal fibroblasts were compared. Statistical analysis was performed on the number of maximum serial weekly passages, percentage of aborted colonies, colony-forming efficiency (CFE), p63α(bright) cells, and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ , K12, and MUC1 were performed to evaluate phenotype., Results: Dispase/TrypLE was the isolation method that consistently showed the best yield, viability, and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1 mM and EGF 10 ng/mL at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells, and p63α/K12 RT-PCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2., Conclusions: The LSC function can be maintained in vitro under appropriate Xeno-free conditions.

Oligodendrocytes (OLs) are glial cells of the central nervous system, which produce myelin. Cultured OLs provide immense therapeutic opportunities for treating a variety of neurological conditions. One of the most promising sources for such therapies is human embryonic stem cells (ESCs) as well as providing a model to study human OL development. For these purposes, an investigation of proteome level changes is critical for understanding the process of OL differentiation. In this report, an iTRAQ-based quantitative proteomic approach was used to study multiple steps during OL differentiation including neural progenitor cells, glial progenitor cells and oligodendrocyte progenitor cells (OPCs) compared to undifferentiated ESCs. Using a 1% false discovery rate cutoff, ∼3145 proteins were quantitated and several demonstrated progressive stage-specific expression. Proteins such as transferrin, neural cell adhesion molecule 1, apolipoprotein E and wingless-related MMTV integration site 5A showed increased expression from the neural progenitor cell to the OPC stage. Several proteins that have demonstrated evidence or been suspected in OL maturation were also found upregulated in OPCs including fatty acid-binding protein 4, THBS1, bone morphogenetic protein 1, CRYAB, transferrin, tenascin C, COL3A1, TGFBI and EPB41L3. Thus, by providing the first extensive proteomic profiling of human ESC differentiation into OPCs, this study provides many novel proteins that are potentially involved in OL development., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Manganas et al. (Reports, 9 November 2007, p. 980) reported the discovery of a biomarker specific for neural progenitor cells detectable using magnetic resonance spectroscopy. A new algorithm was developed to extract the biomarker from noisy in vivo data. We question how this algorithm was validated, because the biomarker overlaps with peaks from nonspecific lipid signals.

Stem cells are potential agents for the treatment of myocardial infarcts among other heart diseases. Over the past decade, the scientific community has extensively used a wide variety of cells and examined their capacity to both regenerate the infarcted myocardium and improve functionally the diseased hearts. Some of the cells used include skeletal myoblasts, bone marrow-derived cells, adult cardiac resident stem cells, mesenchymal stem cells, and both mouse and human embryonic stem cells (Nat Biotechnol 2005;23:845-856). The reported cardiogenic capacity of the utilitized stem cells is assayed both in vitro through the use of differentiation paradigms and in vivo through transplantation into a variety of animal models of cardiac disease. The purpose of this review article is to summarize recent stem cell applications in cell-based cardiac therapies and their outcomes.

Embryonic germ cells (EGCs) are pluripotent stem cells derived from primordial germ cells (PGCs). PGCs are progenitors of adult gametes, which diverge from the somatic lineage between late embryonic to early fetal development. First derived in the mouse, EGCs have also been derived from human, chicken, and pig. As pluripotent stem cells, EGCs demonstrate long-term self-renewal via clonal expansion in an undifferentiated state, and differentiate in vitro to form embryoid bodies containing cells that represent all three germ layers as well as mixed cell populations of less differentiated progenitors and precursors. This is also demonstrated in vivo by their formation into experimentally induced teratocarcinomas following transplantation. Furthermore, mice, pig, and chicken EGCs have also been shown to contribute to experimentally produced chimeric animals, including germline transmission. Importantly, EGCs demonstrate normal and stable karyotypes as well as normal patterns of genomic imprinting, including X-inactivation. Transplantation studies have begun in a variety of models in hopes of defining their potential use to treat a wide variety of human conditions, including diabetes and urological and neurological disorders.

The classification of embryonic and adult stem cells, including their derivatives, is still limited, and often these cells are best defined by their functional properties. Recent gene array studies have yielded contradictory results. Also, very little is known about the metabolic properties of these exciting cells. In this study, proton (1H) NMR spectroscopy was used to identify the major low-molecular-weight metabolites in murine embryonic stem cells (ESC) and their neural stem cell (NSC) derivatives. ESC are characterized by an unusually low number of NMR-detectable metabolites, high phosphocholine (PC) content, and nondetectable glycerophosphocholine (GPC). The metabolic profiles of NSC resemble glial cells and oligodendrocyte progenitors, but with considerably higher PC, GPC, and myo-inositol content. The results suggest that NMR spectroscopy in vitro can provide markers to study the effects of differentiation on cell metabolism, and potentially to assess stem cell preparations for differentiation status., (Copyright (c) 2006 Wiley-Liss, Inc.)

Objectives: To evaluate the affinity between human stem cells and human bladder cells by analyzing their migration and proliferation patterns in co-culture. Human stem cells have great potential for tissue engineering purposes. Co-culture of stem cells with mature cells may promote differentiation., Methods: Equal numbers of green fluorescent protein-labeled human embryonic germ cell derivates (SDECs) were plated, either alone or in the presence of red fluorescence-labeled (PKH 26) human bladder smooth muscle cells (SMCs) or urothelial cells (UROs). The co-cultures shared the same media (EGM2MV). The migration patterns of the different cell lines were measured daily, using an integrated grid, for 8 days with fluorescence microscopy., Results: SDECs, grown alone, had a robust basal migration rate of between 0.3 and 0.7 mm/day compared with SMCs, which had a rate of 0.1 to 0.3 mm/day and UROs with a rate of 0.1 to 0.2 mm/day. Stem cell migration was enhanced in co-culture with SMCs or UROs to 0.5 to 1.0 mm/day. Migration of SDECs was more linear, directed toward SMCs or UROs, compared with the circumferential growth when plated alone. SMCs, more than UROs, migrated more rapidly in the presence of stem cells., Conclusions: Human stem cells showed improved migration in the presence of mature human bladder cells and were attracted to them, as shown by the altered direction of growth. Thus, co-culture of human stem cells with host SMCs can enhance seeding of matrices due to positive chemotaxis. Identifying the responsible factors may help to augment chemotaxis between desired cell types and optimize tissue regeneration.

Stem cell therapy is a hope for the treatment of some childhood neurological disorders. We examined whether human neural stem cells (hNSCs) replace lost cells in a newborn mouse model of brain damage. Excitotoxic lesions were made in neonatal mouse forebrain with the N-methyl-D-aspartate (NMDA) receptor agonist quinolinic acid (QA). QA induced apoptosis in neocortex, hippocampus, striatum, white matter, and subventricular zone. This degeneration was associated with production of cleaved caspase-3. Cells immunopositive for inducible nitric oxide synthase were present in damaged white matter and subventricular zone. Three days after injury, mice received brain parenchymal or intraventricular injections of hNSCs derived from embryonic germ (EG) cells. Human cells were prelabeled in vitro with DiD for in vivo tracking. The locations of hNSCs within the mouse brain were determined through DiD fluorescence and immunodetection of human-specific nestin and nuclear antigen 7 days after transplantation. hNSCs survived transplantation into the lesioned mouse brain, as evidenced by human cell markers and DiD fluorescence. The cells migrated away from the injection site and were found at sites of injury within the striatum, hippocampus, thalamus, and white matter tracts and at remote locations in the brain. Subsets of grafted cells expressed neuronal and glial cell markers. hNSCs restored partially the complement of striatal neurons in brain-damaged mice. We conclude that human EG cell-derived NSCs can engraft successfully into injured newborn brain, where they can survive and disseminate into the lesioned areas, differentiate into neuronal and glial cells, and replace lost neurons. (c) 2005 Wiley-Liss, Inc.

Objectives: Human pluripotent stem cells have excellent regenerative abilities, making them attractive in bladder regeneration. Porcine small intestinal submucosa (SIS) is an established matrix well-suited to the urinary tract. We evaluated the ability of human embryonic germ (hEG) cell-derived stem cells to form a composite graft on SIS, grown alone or in combination with human bladder smooth muscle cells (SMCs) or urothelial (URO) cells., Methods: The hEG cell-derived cell line SDEC had the best-fit profile for smooth muscle, as determined by its gene and protein expression. SDEC cells were seeded on SIS, either on its serosal or mucosal surface, and cultured for 7 or 14 days. Co-cultures of stem cells with URO cells or SMCs were also assessed under similar conditions. The grafts were analyzed by histologic examination for cell growth, morphology, and matrix penetration., Results: SDEC cells grew in a monolayer on SIS, with a marked increase in three-dimensional growth when co-cultured with URO cells or SMCs. Penetration of the matrix was evident when seeded on the rough surface of the SIS, progressing with time. Stratification of the cell layers occurred on the smooth side of the SIS., Conclusions: This is the first description of hEG cell-derived stem cells in co-culture with bladder cells. hEG cell-derived stem cells grow well on SIS even when seeded at low concentrations in the presence of bladder cells (URO cells or SMCs). These composite grafts will be ideal to evaluate the in vivo functional characteristics of stem cells using an animal model of bladder regeneration.

Objectives: To evaluate the capability of a human embryonic germ (hEG) cell-derived cell line (SDEC), previously characterized in our laboratory, seeded on porcine small intestinal submucosa (SIS) to regenerate the injured rat bladder., Methods: Fluorescent-labeled SDEC cells seeded on SIS for 8 days in vitro were used as bladder grafts in rats. A total of 30 congenitally athymic rats (six groups of 5 rats each), underwent partial cystectomy and replacement with plain SIS (groups 1 to 3) or cell-seeded SIS (groups 4 to 6). The rats were sacrificed after 7 (groups 1 and 4), 14 (groups 2 and 5), and 28 (groups 3 and 6) days. The bladders were analyzed by histopathologic examination and fluorescence microscopy., Results: No graft rejection or diminution in bladder capacity occurred. Plain SIS implants had multiple calcareous deposits, not seen with the cell-seeded implants. Macroscopically, at 7 days, the grafts were healed with a cellular lining on the luminal aspect in groups 4 to 6. Microscopically, the rat bladder was completely regenerated 28 days after stem cell-seeded SIS implantation. Labeled stem cells were identified throughout the graft and contributed significantly to bladder regeneration., Conclusions: The results of this study have demonstrated the successful replacement of a bladder defect in a rat model using hEG cell-derived cells seeded on SIS grafts. Longer term analysis of these bladder grafts will allow evaluation of function, cell migration, and differentiation processes of human stem cells.

We report on the deliberations of an interdisciplinary group of experts in science, law, and philosophy who convened to discuss novel ethical and policy challenges in stem cell research. In this report we discuss the ethical and policy implications of safety concerns in the transition from basic laboratory research to clinical applications of cell-based therapies derived from stem cells. Although many features of this transition from lab to clinic are common to other therapies, three aspects of stem cell biology pose unique challenges. First, tension regarding the use of human embryos may complicate the scientific development of safe and effective cell lines. Second, because human stem cells were not developed in the laboratory until 1998, few safety questions relating to human applications have been addressed in animal research. Third, preclinical and clinical testing of biologic agents, particularly those as inherently complex as mammalian cells, present formidable challenges, such as the need to develop suitable standardized assays and the difficulty of selecting appropriate patient populations for early phase trials. We recommend that scientists, policy makers, and the public discuss these issues responsibly, and further, that a national advisory committee to oversee human trials of cell therapies be established.

Objective: To discuss the current state of the science surrounding human pluripotent stem cells and to show that the derivation of such cells from donated preimplantation human embryos should be eligible for federal funding provided that certain protections are met., Design: A literature search focusing on the scientific aspects of pluripotent stem-cell research and analyses of current and past legislation and federal panel recommendations., Conclusion(s): The current federal laws regulating the permission necessary to obtain fetal tissue from elective pregnancy terminations are intended to insulate the decision to terminate a pregnancy from the potential positive influence of fetal tissue transplantation. A similar situation can be created for the derivation of cells from excess preimplantation human embryos produced by IVF programs. If, as in fetal tissue research, assurances can be made that the research will have no influence on the decision to dispose of the embryo, the derivation of pluripotent stem cells from embryo should proceed with federal funding.

Human pluripotent stem cells would be invaluable for in vitro studies of aspects of human embryogenesis. With the goal of establishing pluripotent stem cell lines, gonadal ridges and mesenteries containing primordial germ cells (PGCs, 5-9 weeks postfertilization) were cultured on mouse STO fibroblast feeder layers in the presence of human recombinant leukemia inhibitory factor, human recombinant basic fibroblast growth factor, and forskolin. Initially, single PGCs in culture were visualized by alkaline phosphatase activity staining. Over a period of 7-21 days, PGCs gave rise to large multicellular colonies resembling those of mouse pluripotent stem cells termed embryonic stem and embryonic germ (EG) cells. Throughout the culture period most cells within the colonies continued to be alkaline phosphatase-positive and tested positive against a panel of five immunological markers (SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) that have been used routinely to characterize embryonic stem and EG cells. The cultured cells have been continuously passaged and found to be karyotypically normal and stable. Both XX and XY cell cultures have been obtained. Immunohistochemical analysis of embryoid bodies collected from these cultures revealed a wide variety of differentiated cell types, including derivatives of all three embryonic germ layers. Based on their origin and demonstrated properties, these human PGC-derived cultures meet the criteria for pluripotent stem cells and most closely resemble EG cells.

Mutations in the gene encoding the stimulatory G protein of adenylyl cyclase (G alpha(s)) are present in subjects with Albright hereditary osteodystrophy, a syndrome of characteristic developmental defects and, in some patients, resistance to multiple hormones that stimulate cAMP accumulation (pseudohypoparathyroidism type Ia). As the first step in generating a model of Albright hereditary osteodystrophy, the gene encoding G alpha(s) (Gnas) was disrupted in mouse embryonic stem (ES) cells by homologous recombination. Northern blot analysis and immunoblot analysis demonstrated that steady-state levels of G alpha(s) messenger RNA and G alpha(s) protein in targeted ES cells were approximately 50% of levels in untargeted ES cells. In response to 10 microM forskolin and to various concentrations of isoproterenol (0.1-3.0 microM), cAMP accumulation was reduced in the G alpha(s) knockout ES cell lines, relative to wild-type ES cells and to five of six ES cell lines with randomly integrated targeting vector. These results support the role of G alpha(s) haploinsufficiency in reducing the ability of hormones to generate cAMP in subjects with pseudohypoparathyroidism type Ia. The targeted disruption of Gnas in mouse ES cells establishes an in vitro system for further studies of the role of G alpha(s) and cAMP coupled signal transduction in differentiation and development.