Michael J. Shamblott

Functional antigen-presenting leucocytes derived from human embryonic stem cells in vitro

BACKGROUND Differentiated cells derived from pluripotent human embryonic stem (hES) cells offer the opportunity for new transplantation therapies. However, hES cells and their differentiated progeny express highly polymorphic MHC molecules that serve as major graft rejection antigens to the immune system of allogeneic hosts. To achieve sustained engraftment of donor cells, strategies must be developed to overcome graft rejection without broadly suppressing host immunity. One approach entails induction of donor-specific immune tolerance by establishing chimeric engraftment in hosts with haemopoietic cells derived from an existing hES cell line. We aimed to develop methods to efficiently differentiate hES cells to haemopoietic cells, including immune-modulating leucocytes, a prerequisite of the tolerance induction strategies applying to hES cell-mediated transplantation. METHODS We developed a method to generate a broad range of haemopoietic cells from hES-generated embryonic bodies in the absence of murine stromal feeder cells. Embryonic bodies were further cultured in the presence of haemopoietic cytokines. In addition to flow cytometric analyses of haemopoietic cell markers, we analysed the hES cell-derived haemopoietic cells by colony-forming assays (for erythroid and myeloid progenitor cells), cytochemical staining, and mixed leucocyte reactions to determine the functional capacity of the generated antigen-presenting cells. FINDINGS 12 independent experiments were done. When selected growth factors were added, leucocytes expressing CD45 were generated and released into culture media for 6-7 weeks. Under the condition used, both erythroid and myeloid progenitor cells were generated. About 25% of the generated leucocytes acquired MHC class II and costimulatory molecule expression. These hES-derived, MHC class II+ leucocytes resembled dendritic cells and macrophages, and they functioned as antigen-presenting cells capable of eliciting allogeneic CD4 and CD8 T-cell responses in culture. INTERPRETATION The hES cell-derived antigen-presenting cells could be used to regulate alloreactive T cells and induce immune tolerance for improvement of the transplant acceptance of hES-cell derivatives.

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 (TGFβ3). 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 TGFβ3, 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 TGFβ3, 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.

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.

Use of perfluorocarbon nanoparticles for non-invasive multimodal cell tracking of human pancreatic islets

In vivo imaging of engraftment and immunorejection of transplanted islets is critical for further clinical development, with (1)H MR imaging of superparamagnetic iron oxide-labeled cells being the current premier modality. Using perfluorocarbon nanoparticles, we present here a strategy for non-invasive imaging of cells using other modalities. To this end, human cadaveric islets were labeled with rhodamine-perfluorooctylbromide (PFOB) nanoparticles, rhodamine-perfluoropolyether (PFPE) nanoparticles or Feridex as control and tested in vitro for cell viability and c-peptide secretion for 1 week. (19)F MRI, computed tomography (CT) and ultrasound (US) imaging was performed on labeled cell phantoms and on cells following transplantation beneath the kidney capsule of mice and rabbits. PFOB and PFPE-labeling did not reduce human islet viability or glucose responsiveness as compared with unlabeled cells or SPIO-labeled cells. PFOB- and PFPE-labeled islets were effectively fluorinated for visualization by (19)F MRI. PFOB-labeled islets were acoustically reflective for detection by US imaging and became sufficiently brominated to become radiopaque allowing visualization with CT. Thus, perfluorocarbon nanoparticles are multimodal cellular contrast agents that may find applications in real-time targeted delivery and imaging of transplanted human islets or other cells in a clinically applicable manner using MRI, US or CT imaging.

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.

Craniofacial abnormalities resulting from targeted disruption of the murine Sim2 gene.

Sim2 is a member of the basic helix‐loop‐helix PAS transcription factor gene family and is evolutionarily related to the Drosophila single‐minded gene, a key regulator of central nervous system midline development. In an effort to determine the biological roles of Sim2 in mammalian development, we disrupted the murine Sim2 gene through gene targeting. Mice homozygous for the disrupted allele (Sim2 ‐/‐) exhibit a cleft of the secondary palate and malformations of the tongue and pterygoid processes of the sphenoid bone. These craniofacial malformations are the most probable cause of aerophagia (air swallowing with subsequent accumulation of air in the gastrointestinal tract) and postnatal death exhibited by Sim2 ‐/‐ mice. The developing palates of the Sim2 ‐/‐ mice are hypocellular, and at embryonic day 14.5 contain excess extracellular matrix component hyaluronan (HA) compared with heterozygotes and homozygous wild‐type littermates. HA plays an important role in the regulation and mechanics of palate development. Its premature accumulation in Sim2 ‐/‐ animal palates suggests a regulatory role for Sim2 in HA synthesis and in the establishment of craniofacial architecture.

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. Magn Reson Med, 2006.

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.

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. Copyright

Engraftment of human embryonic stem cell derived cardiomyocytes improves conduction in an arrhythmogenic in vitro model

In this study, we characterized the electrophysiological benefits of engrafting human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in a model of arrhythmogenic cardiac tissue. Using transforming growth factor-β treated monolayers of neonatal rat ventricular cells (NRVCs), which retain several key aspects of the healing infarct such as an excess of contractile myofibroblasts and slowed, heterogeneous conduction, we assessed the ability of hESC-CMs to improve conduction and prevent arrhythmias. Cells from beating embryoid bodies (hESC-CMs) can form functional monolayers which beat spontaneously and can be electrically stimulated, with mean action potential duration of 275 ± 36 ms and conduction velocity (CV) of 10.6 ± 4.2 cm/s (n = 3). These cells, or cells from non-beating embryoid bodies (hEBCs) were added to anisotropic, NRVC monolayers. Immunostaining demonstrated hESC-CM survival and engraftment, and dye transfer assays confirmed functional coupling between hESC-CMs and NRVCs. Conduction velocities significantly increased in anisotropic NRVC monolayers after engraftment of hESC-CMs (13.4 ± 0.9 cm/s, n = 35 vs. 30.1 ± 3.2 cm/s, n = 20 in the longitudinal direction and 4.3 ± 0.3 cm/s vs. 9.3 ± 0.9 cm/s in the transverse direction), but decreased to even lower values after engraftment of non-cardiac hEBCs (to 10.6 ± 1.3 cm/s and 3.1 ± 0.5 cm/s, n = 11, respectively). Furthermore, reentrant wave vulnerability in NRVC monolayers decreased by 20% after engraftment of hESC-CMs, but did not change with engraftment of hEBCs. Finally, the culture of hESC-CMs in transwell inserts, which prevents juxtacrine interactions, or engraftment with connexin43-silenced hESC-CMs provided no functional improvement to NRVC monolayers. These results demonstrate that hESC-CMs can reverse the slowing of conduction velocity, reduce the incidence of reentry, and augment impaired electrical propagation via gap junction coupling to host cardiomyocytes in this arrhythmogenic in vitro model.