Biomedical subjects

Michael J Shamblott

Publications and source records attributed to Michael J Shamblott.

9 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

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↗

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

Musculoskeletal differentiation of cells derived from human embryonic germ cells.

Stem cells have the potential to significantly improve cell and tissue regeneration therapies, but little is understood about how to control their behavior. We investigated the potential differentiation capability of cells derived from human embryonic germ (EG) cells into musculoskeletal lineages by providing a three-dimensional environment with increased cell-cell contact and growth factors. Cells were clustered into pellets to mimic the mesenchyme condensation process during limb development. LVEC cells, an embryoid body-derived (EBD) cell culture generated from EG cells, were cultured in micromass pellets for 21 days in the presence of bone morphogenetic protein 2 (BMP2) and/or transforming growth factor beta-3 (TGFbeta3). Gene expression for cartilage-, bone-, and muscle-specific matrix proteins--including collagen types I, II, III, IX, X; aggrecan; cartilage proteoglycan link protein; cartilage oligomeric protein; chondroitin sulfate-4-S; and myf5--was upregulated in the pellets treated with TGFbeta3, while mRNAs for neurofilament heavy (NFH), a neuron marker, and flk-1, a hematopoietic marker, decreased. Total collagen and proteoglycan production exhibited a time-dependent increase in the pellets treated with TGFbeta3, further confirming the expression of characteristic musculoskeletal markers. Furthermore, our results indicate the ability to select or differentiate stem cells toward a musculoskeletal lineage from a heterogenous EBD cell line.

Cartilage↗

PubMed2004

Cell therapies for type 1 diabetes mellitus.

Type 1 diabetes is caused by autoimmune destruction of pancreatic beta-cells and is characterised by absolute insulin insufficiency. The monocellular nature of this disease and endocrine action of insulin make this disease an excellent candidate for cellular therapy. Furthermore, precedent for cellular therapies has been set by successful cadaveric whole pancreas and islet transplantation. In order to expand the supply of cells to meet current and future needs, several novel cell sources have been proposed, including human beta-cells or islets expanded in culture, islet xenografts and pancreatic ductal progenitor cells. Surrogate beta-cells derived from hepatocytes, intestinal K cells or non-endodermal cell types have also been suggested. Stem cells found in bone marrow and umbilical cord blood have been used extensively to repopulate the haematopoietic system and offer the possibility of autologous transplantation. Recent studies have suggested that these stem cells may also have a broader capacity to differentiate, possibly into beta-cells. Stem cells from embryonic sources, such as human embryonic stem and embryonic germ cells, have the ability to proliferate extensively in culture and have an inherent developmental plasticity that may make them a potentially unlimited source of cells that can sense glucose and produce mature insulin. The wide range of proposed cell sources and our increasingly clear picture of pancreatic development suggest that novel cellular therapies might one day compete with non-cellular glucose sensing and insulin delivery devices.

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

Prolactin gene expression in human ovarian follicular cells.

OBJECTIVE: To investigate prolactin gene expression in human ovarian follicular cells. DESIGN: RNA was isolated from follicular cells obtained at the time of transvaginal oocyte retrieval from patients after controlled ovarian hyperstimulation. The RNA was subjected to reverse transcription and polymerase chain reaction (RT-PCR) using prolactin-specific primers. The RT-PCR products were analyzed by gel electrophoresis, followed by Southern blot analysis using prolactin-specific probes. SETTING: Department of gynecology and obstetrics research laboratory. PATIENT(S): Women undergoing controlled ovarian hyperstimulation followed by transvaginal oocyte retrieval. INTERVENTION(S): Controlled ovarian hyperstimulation followed by transvaginal oocyte retrieval. Expression of prolactin mRNA by follicular cells. RESULT(S): The presence of prolactin-specific cDNA amplified by RT-PCR from RNA isolated from human follicular cells was confirmed by Southern blot analysis. CONCLUSION(S): Human ovarian follicular cells are an extrapituitary site of prolactin gene expression.

Blotting, Southern↗

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↗

PubMedDate not supplied

Glucose-responsive insulin-producing cells from stem cells.

Recent success with immunosuppression following islet cell transplantation offers hope that a cell transplantation treatment for type 1 (juvenile) diabetes may be possible if sufficient quantities of safe and effective cells can be produced. For the treatment of type 1 diabetes, the two therapeutically essential functions are the ability to monitor blood glucose levels and the production of corresponding and sufficient levels of mature insulin to maintain glycemic control. Stem cells can replicate themselves and produce cells that take on more specialized functions. If a source of stem cells capable of yielding glucose-responsive insulin-producing (GRIP) cells can be identified, then transplantation-based treatment for type 1 diabetes may become widely available. Currently, stem cells from embryonic and adult sources are being investigated for their ability to proliferate and differentiate into cells with GRIP function. Human embryonic pluripotent stem cells, commonly referred to as embryonic stem (ES) cells and embryonic germ (EG) cells, have received significant attention owing to their broad capacity to differentiate and ability to proliferate well in culture. Their application to diabetes research is of particular promise, as it has been demonstrated that mouse ES cells are capable of producing cells able to normalize glucose levels of diabetic mice, and human ES cells can differentiate into cells capable of insulin production. Cells with GRIP function have also been derived from stem cells residing in adult organisms, here referred to as endogenous stem cell sources. Independent of source, stem cells capable of producing cells with GRIP function may provide a widely available cell transplantation treatment for type 1 diabetes.

Animals↗