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Biomedical subjects

John D Gearhart

Publications and source records attributed to John D Gearhart.

17 recordsLinked to original sources
PubMed2006

Stem cell profiling by nuclear magnetic resonance spectroscopy.

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.

Animals↗
PubMed2006

Human embryoid body-derived stem cells in tissue engineering-enhanced migration in co-culture with bladder smooth muscle and urothelium.

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.

Cell Movement↗
PubMed2006

Pluripotent stem cells from germ cells.

To date, stem cells have been derived from three sources of germ cells. These include embryonic germ cells (EGCs), embryonal carcinoma cells (ECCs), and multipotent germ line stem cells (GSCs). EGCs are derived from primordial germ cells that arise in the late embryonic and early fetal period of development. ECCs are derived from adult testicular tumors whereas GSCs have been derived by culturing spermatogonial stem cells from mouse neonates and adults. For each of these lines, their pluripotency has been demonstrated by their ability to differentiate into cell types derived from the three germ layers in vitro and in vivo and in chimeric animals, including germ line transmission. These germ line-derived stem cells have been generated from many species including human, mice, porcine, and chicken albeit with only slight modifications. This chapter describes general considerations regarding critical aspects of their derivation compared with their counterpart, embryonic stem cells (ESCs). Detailed protocols for EGC derivation and maintenance from human and mouse primordial germ cells (PGCs) will be presented.

Adult Stem Cells↗
PubMed2006

Embryonic germ cells: when germ cells become stem cells.

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.

Animals↗
PubMed2005

Transplanted human embryonic germ cell-derived neural stem cells replace neurons and oligodendrocytes in the forebrain of neonatal mice with excitotoxic brain damage.

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.

Animals↗
PubMed2005

Human embryoid body-derived stem cells in co-culture with bladder smooth muscle and urothelium.

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.

Animals↗
PubMed2005

Human embryoid body-derived stem cells in bladder regeneration using rodent model.

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.

Animals↗
PubMed2005

Bone morphogenetic proteins produced by cells within embryoid bodies inhibit ventral directed differentiation by Sonic Hedgehog.

Mouse embryoid bodies (EBs) differentiate into dorsal spinal cord neural progenitors in response to retinoic acid (RA). Our data demonstrate that the addition of Sonic Hedgehog (Shh) directs towards a ventral spinal cord neural tube fate, but only at extremely high concentrations. One possible explanation is the presence of dorsal directing factors. Bone morphogenetic proteins (BMPs), known to direct dorsal spinal cord neural differentiation, were expressed in RA-treated EBs. Shh more potently directed ventral differentiation when combined with the BMP inhibitor Noggin. Further, when BMP7 was added, the ability of Shh to direct ventral differentiation was further mitigated.

Animals↗
PubMed2003

Human embryonic germ cell derivatives facilitate motor recovery of rats with diffuse motor neuron injury.

We have investigated the potential of human pluripotent cells to restore function in rats paralyzed with a virus-induced motor neuronopathy. Cells derived from embryonic germ cells, termed embryoid body-derived (EBD) cells, introduced into the CSF were distributed extensively over the rostrocaudal length of the spinal cord and migrated into the spinal cord parenchyma in paralyzed, but not uninjured, animals. Some of the transplanted human cells expressed the neuroglial progenitor marker nestin, whereas others expressed immunohistochemical markers characteristic of astrocytes or mature neurons. Rare transplanted cells developed immunoreactivity to choline acetyltransferase (ChAT) and sent axons into the sciatic nerve as detected by retrograde labeling. Paralyzed animals transplanted with EBD cells partially recovered motor function 12 and 24 weeks after transplantation, whereas control animals remained paralyzed. Semi-quantitative analysis revealed that the efficiency of neuronal differentiation and extension of neurites could not account for the functional recovery. Rather, transplanted EBD cells protected host neurons from death and facilitated reafferentation of motor neuron cell bodies. In vitro, EBD cells secrete transforming growth factor-alpha (TGF-alpha) and brain-derived neurotrophic factor (BDNF). Neutralizing antibodies to TGF-alpha and to BDNF abrogated the ability of EBD-conditioned media to sustain motor neuron survival in culture, whereas neutralizing antibodies to BDNF eliminated the axonal outgrowth from spinal organotypics observed with direct coculture of EBD cells. We conclude that cells derived from human pluripotent stem cells have the capacity to restore neurologic function in animals with diffuse motor neuron disease via enhancement of host neuron survival and function.

Alphavirus Infections↗
PubMed2003

Safety issues in cell-based intervention trials.

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.

Animals↗
PubMed2002

Monoallelic expression and methylation of imprinted genes in human and mouse embryonic germ cell lineages.

Imprinting is an epigenetic modification leading to monoallelic expression of some genes, and disrupted imprinting is believed to be a barrier to human stem cell transplantation, based on studies that suggest that epigenetic marks are unstable in mouse embryonic germ (EG) and embryonic stem (ES) cells. However, stem cell imprinting has not previously been examined directly in humans. We found that three imprinted genes, TSSC5, H19, and SNRPN, show monoallelic expression in in vitro differentiated human EG-derived cells, and a fourth gene, IGF2, shows partially relaxed imprinting at a ratio from 4:1 to 5:1, comparable to that found in normal somatic cells. In addition, we found normal methylation of an imprinting control region (ICR) that regulates H19 and IGF2 imprinting, suggesting that imprinting may not be a significant epigenetic barrier to human EG cell transplantation. Finally, we were able to construct an in vitro mouse model of genomic imprinting, by generating EG cells from 8.5-day embryos of an interspecific cross, in which undifferentiated cells show biallelic expression and acquire preferential parental allele expression after differentiation. This model should allow experimental manipulation of epigenetic modifications of cultured EG cells that may not be possible in human stem cell studies.

Alleles↗
PubMed2002

Decreased progesterone receptor expression in the intermediate trophoblastic cells of spontaneous abortions.

OBJECTIVE: To determine whether there are differences in the expression of progesterone receptor (PR) in intermediate trophoblastic cells of pregnancies ending in either spontaneous abortion (SAB) or elective abortion. DESIGN: Immunohistochemical identification of PR in intermediate trophoblastic cells. SETTING: Academic medical center. PATIENT(S): Subjects were 86 patients who either underwent first trimester SAB or elective abortion. INTERVENTION(S): All SAB and elective abortion specimens were serially sectioned and immunohistochemically stained for PR and for melanoma cell adhesion molecule. Melanoma cell adhesion molecule immunohistochemical staining was used as a sensitive and specific marker to identify intermediate trophoblastic cells on the adjacent tissue section. MAIN OUTCOME MEASURE(S): The PR staining of intermediate trophoblastic cells by semiquantitative immunostaining score. RESULT(S): The PR expression in intermediate trophoblastic cells was significantly greater in elective abortion specimens than in SAB specimens. When controlling for estimated gestational age, the difference in PR expression was even greater. CONCLUSION(S): The quantity of PR in intermediate trophoblastic cells is significantly less in SAB when compared to elective abortion pregnancies. Although it is unknown whether this is a primary or secondary event, this information may be an important finding in attempting to characterize both the molecular etiology of implantation and the molecular pathophysiology of SAB.

Abortion, Induced↗
PubMed2002

The effects of piracetam on cognitive performance in a mouse model of Down's syndrome.

Piracetam is a nootropic agent that has been shown to improve cognitive performance in a number of animal model systems. Piracetam is reported to be used widely as a means of improving cognitive function in children with Down's syndrome (DS). In order to provide a preclinical assessment of the potential efficacy of piracetam, we examined the effects of a dose range of piracetam in the Ts65Dn mouse model of DS. Ts65Dn mice are trisomic for a region of mouse chromosome 16 with homology to human chromosome 21. Daily piracetam treatment at doses of 0, 75, 150, and 300 mg/kg ip was initiated in 6-week-old male Ts65Dn and euploid control mice. Following 4 weeks of treatment, mice were tested in the visible and hidden-platform components of the Morris water maze and were placed overnight in computerized activity chambers to assess effects on overall activity. Piracetam treatment was continued through the 4 weeks of testing. In control mice, 75 and 150 mg/kg/day piracetam improved performance in both the visible- and hidden-platform tasks. Although low doses of piracetam reduced search time in the visible-platform component in Ts65Dn mice, all piracetam doses prevented trial-related improvements in performance in Ts65Dn mice. The 300-mg/kg/day-piracetam dose was associated with a reversal of the nocturnal spontaneous hyperactivity in Ts65Dn. These data do not provide support for piracetam treatment for individuals with DS.

Animals↗
PubMed2002

The Role of E-cadherin in the Motility and Invasion of Implantation Site Intermediate Trophoblast.

During early pregnancy, intermediate (extravillous) trophoblast infiltrates the basal plate and invades the spiral arteries, a physiological process required to establish the maternal-fetal circulation. Immunostaining studies have shown that differentiation of trophoblast into this invasive subpopulation is associated with down-regulation of E-cadherin expression. To study the function of E-cadherin in trophoblast in vitro, we restored E-cadherin expression in an E-cadherin negative human implantation site intermediate trophoblastic cell line, IST-1, using a recombinant adenovirus, E-cad/Ad5 which constitutively expresses E-cadherin. In contrast to the control IST-1 cells which were individual and pleomorphic in shape, E-cad/Ad5 transduced cells were cohesive, uniform, and round. The motility and invasiveness of E-cad/Ad5 transduced IST-1 cells, as compared with the control cells, was significantly reduced. These effects were contact-dependent and were attenuated by a function-perturbing anti-E-cadherin antibody. In conclusion, our results indicate that expression of E-cadherin in IST-1 cells results in a contact-mediated inhibition of motility and invasion and suggest an important role for E-cadherin down-regulation in the intermediate trophoblast during implantation.

Adenoviridae↗