Characterization and differentiation of human embryonic stem cells.

Cell replacement therapies have been limited by the availability of sufficient quantities of cells for transplantation. Human ES (hES) cell lines have recently been generated by several laboratories. When maintained for over 1 year in vitro, they remain karyotypically and phenotypically stable and may therefore provide an excellent source material for cell therapies. Currently, data is available for 26 hES cell lines. Although limited characterization has been performed on most of these lines, there are remarkable similarities in expression of markers. hES cell lines derived in different laboratories show similar expression profiles of surface markers, including SSEA-4, Tra-1-60, and Tra-1-81. In addition, markers associated with pluripotent cells such as OCT-4 are expressed at in all cell lines tested. These cells express high levels of telomerase and appear to have indefinite growth potential. The generation of the large quantities of cells necessary for cell replacement therapies will require a cell population which is stable over long term culture. We have characterized the properties of multiple hES cell lines that have been maintained in culture for extended periods. Quantitative analyses demonstrate that all of the cell lines examined show consistent marker expression and retain a normal karyotype after long-term culture. hES cells have been differentiated into the derivatives of all three germ layers. Specifically this includes cardiomyocytes, neural cells, hepatocyte-like cells, endothelial cells and hematopoietic progenitor cells. These data demonstrating the karyotypic and phenotypic stability of hES cells and their extensive differentiative capacity indicate that they may be an appropriate source of cells for multiple regenerative medicine applications.

Enrichment of neurons and neural precursors from human embryonic stem cells.

Human embryonic stem (hES) cells proliferate and maintain their pluripotency for over a year in vitro (M. Amit, M. K. Carpenter, M. S. Inokuma, C. P. Chiu, C. P., Harris, M. A. Waknitz, J. Itskovitz-Eldor, and J. A. Thomson. 2000. Dev. Biol. 227: 271-278) and may therefore provide a cell source for cell therapies. hES cells were maintained for over 6 months in vitro (over 100 population doublings) before their ability to differentiate into the neural lineage was evaluated. Differentiation was induced by the formation of embryoid bodies that were subsequently plated onto appropriate substrates in defined medium containing mitogens. These populations contained cells that showed positive immunoreactivity to nestin, polysialylated neural cell adhesion molecule (PS-NCAM) and A2B5. After further maturation, these cells expressed additional neuron-specific antigens (such as MAP-2, synaptophysin, and various neurotransmitters). Calcium imaging demonstrated that these cells responded to neurotransmitter application. Electrophysiological analyses showed that cell membranes contained voltage-dependent channels and that action potentials were triggered by current injection. PS-NCAM and A2B5 immunoselection or culture conditions could be used to produce enriched populations (60-90%) which could be further differentiated into mature neurons. The properties of the hES-derived progenitors and neurons were found to be similar to those of cells derived from primary tissue. These data indicate that hES cells could provide a cell source for the neural progenitor cells and mature neurons for therapeutic and toxicological uses.

Feeder-free growth of undifferentiated human embryonic stem cells.

Previous studies have shown that maintenance of undifferentiated human embryonic stem (hES) cells requires culture on mouse embryonic fibroblast (MEF) feeders. Here we demonstrate a successful feeder-free hES culture system in which undifferentiated cells can be maintained for at least 130 population doublings. In this system, hES cells are cultured on Matrigel or laminin in medium conditioned by MEF. The hES cells maintained on feeders or off feeders express integrin alpha6 and beta1, which may form a laminin-specific receptor. The hES cell populations in feeder-free conditions maintained a normal karyotype, stable proliferation rate, and high telomerase activity. Similar to cells cultured on feeders, hES cells maintained under feeder-free conditions expressed OCT-4, hTERT, alkaline phosphatase, and surface markers including SSEA-4, Tra 1-60, and Tra 1-81. In addition, hES cells maintained without direct feeder contact formed teratomas in SCID/beige mice and differentiated in vitro into cells from all three germ layers. Thus, the cells retain fundamental characteristics of hES cells in this culture system and are suitable for scaleup production.

Human embryonic stem cells: culture, differentiation, and genetic modification for regenerative medicine applications.

Human embryonic stem (hES) cells can proliferate extensively in culture and can differentiate into representatives of all three embryonic germ layers in vitro and in vivo. The undifferentiated hES cells have now been cultured for more than 50 passages in vitro, yet maintain a normal karyotype. The hES cells express a series of specific surface antigens, as well as OCT-4 and human telomerase, proteins associated with a pluripotent and immortal phenotype. On differentiation, OCT-4 and human telomerase expression decreases with the emergence of a maturing population of cells. During hES cell differentiation, modulation of the expression of many genes has been evaluated using microarray analysis. To improve the ease, reproducibility, and scalability of hES culture, methods have been developed to propagate the cells in the absence of mouse embryonic cell feeders. hES cells maintained in culture using extracellular matrix factors together with mouse embryonic cell conditioned medium proliferate indefinitely while maintaining a normal karyotype, proliferation rate, and complement of undifferentiated cell markers. hES cells cultured without feeder layers retain their capacity to differentiate into cells of all three germ layers in vitro and in teratomas. The hES cells can also be genetically modified transiently or stably using both plasmid and viral gene transfer agents. These analyses and technological developments will aid in the realization of the full potential of hES cells for both research and therapeutic applications.

Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture.

Embryonic stem (ES) cell lines derived from human blastocysts have the developmental potential to form derivatives of all three embryonic germ layers even after prolonged culture. Here we describe the clonal derivation of two human ES cell lines, H9.1 and H9.2. At the time of the clonal derivation of the H9.1 and H9.2 ES cell lines, the parental ES cell line, H9, had already been continuously cultured for 6 months. After an additional 8 months of culture, H9.1 and H9.2 ES cell lines continued to: (1) actively proliferate, (2) express high levels of telomerase, and (3) retain normal karyotypes. Telomere lengths, while somewhat variable, were maintained between 8 and 12 kb in high-passage H9.1 and H9.2 cells. High-passage H9.1 and H9.2 cells both formed teratomas in SCID-beige mice that included differentiated derivatives of all three embryonic germ layers. These results demonstrate the pluripotency of single human ES cells, the maintenance of pluripotency during an extended period of culture, and the long-term self-renewing properties of cultured human ES cells. The remarkable developmental potential, proliferative capacity, and karyotypic stability of human ES cells distinguish them from adult cells.

EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum.

Immature progenitor cells (generated by in vitro propagation) may provide a useful alternative to primary cells (from dissected embryonic tissue) for transplantation if their migratory and proliferative and differentiation properties can be controlled and directed in vivo. In this study E15 murine EGF-responsive progenitor cells were transplanted to the striatum of adult rats. Simultaneously, these animals received continuous infusion of either epidermal growth factor (EGF) or vehicle, to the lateral ventricle, for 8 days. In animals that received EGF, the transplanted progenitors migrated toward the lateral ventricle and proliferated, as evidenced by bromodeoxyuridine incorporation. Progenitor cells transplanted to rats that received vehicle infusions showed neither of these responses. In all animals, transplanted progenitors expressed an immature astrocyte or oligodendrocyte phenotype, the majority of cells being astrocytes. We conclude that EGF stimulates the migration and proliferation of murine progenitor cells in vivo, either directly or indirectly, but does not influence their phenotypic differentiation.

In vitro expansion of a multipotent population of human neural progenitor cells.

The isolation and expansion of human neural progenitor cells have important potential clinical applications, because these cells may be used as graft material in cell therapies to regenerate tissue and/or function in patients with central nervous system (CNS) disorders. This paper describes a continuously dividing multipotent population of progenitor cells in the human embryonic forebrain that can be propagated in vitro. These cells can be maintained and expanded using a serum-free defined medium containing basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF). Using these three factors, the cell cultures expand and remain multipotent for at least 1 year in vitro. This period of expansion results in a 10(7)-fold increase of this heterogeneous population of cells. Upon differentiation, they form neurons, astrocytes, and oligodendrocytes, the three main phenotypes in the CNS. Moreover, GABA-immunoreactive and tyrosine hydroxylase-immunoreactive neurons can be identified. These results demonstrate the feasibility of long-term in vitro expansion of human neural progenitor cells. The advantages of such a population of neural precursors for allogeneic transplantation include the ability to provide an expandable, well-characterized, defined cell source which can form specific neuronal or glial subtypes.

Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain.

Neural progenitor cells obtained from the embryonic human forebrain were expanded up to 10(7)-fold in culture in the presence of epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory growth factor. When transplanted into neurogenic regions in the adult rat brain, the subventricular zone, and hippocampus, the in vitro propagated cells migrated specifically along the routes normally taken by the endogenous neuronal precursors: along the rostral migratory stream to the olfactory bulb and within the subgranular zone in the dentate gyrus, and exhibited site-specific neuronal differentiation in the granular and periglomerular layers of the bulb and in the dentate granular cell layer. The cells exhibited substantial migration also within the non-neurogenic region, the striatum, in a seemingly nondirected manner up to approximately 1-1.5 mm from the graft core, and showed differentiation into both neuronal and glial phenotypes. Only cells with glial-like features migrated over longer distances within the mature striatum, whereas the cells expressing neuronal phenotypes remained close to the implantation site. The ability of the human neural progenitors to respond in vivo to guidance cues and signals that can direct their differentiation along multiple phenotypic pathways suggests that they can provide a powerful and virtually unlimited source of cells for experimental and clinical transplantation.

Incorporation and glial differentiation of mouse EGF-responsive neural progenitor cells after transplantation into the embryonic rat brain.

In vitro, epidermal growth factor (EGF)-responsive neural progenitor cells exhibit multipotent properties and can differentiate into both neurons and glia. Using an in utero xenotransplantation approach we examined the developmental potential of EGF-responsive cells derived from E14 mouse ganglionic eminences, cortical primordium, and ventral mesencephalon, after injection into the E15 rat forebrain ventricle. Cell cultures were established from control mice or from mice carrying the lacZ transgene under control of the promoters for nestin, glial fibrillary acidic protein (GFAP), or myelin basic protein (MBP). The grafted cells, visualized with mouse-specific markers or staining for the reporter gene product, displayed widespread incorporation into distinct forebrain and midbrain structures and differentiated predominantly into glial cells. The patterns of incorporation of cells from all three regions were very similar without preference for the homotopic brain areas. These results suggest that EGF-responsive progenitor cells can respond to host derived environmental cues, differentiate into cells with glial-like features, and become integrated in the developing recipient brain.

Generation and transplantation of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice.

EGF-responsive neural stem cells isolated from murine striatum have the capacity to differentiate into both neurons and glia in vitro. Genetic modification of these cells is hindered by a number of problems such as gene stability and transfection efficiency. To circumvent these problems we generated transgenic mice in which the human GFAP promoter directs the expression of human NGF. Neural stem cells isolated from the forebrain of these transgenic animals proliferate and form clusters, which appear identical to stem cells generated from control animals. Upon differentiation in vitro, the transgenic stem cell-derived astrocytes express and secrete bioactive hNGF. Undifferentiated GFAP-hNGF or control stem cells were transplanted into the striatum of adult rats. One and 3 weeks after transplantation, hNGF was detected immunocytochemically in an halo around the transplant sites. In GFAP-hNGF-grafted animals, intrinsic striatal neurons proximal to the graft appear to have taken up hNGF secreted by the grafted cells. Ipsilateral to implants of GFAP-hNGF-secreting cells, choline acetyltransferase-immunoreactive neurons within the striatum were hypertrophied relative to the contralateral side or control-grafted animals. Further, GFAP-hNGF-grafted rats displayed a robust sprouting of p75 neurotrophin receptor-positive fibers emanating from the underlying basal forebrain. These studies indicate that EGF-responsive stem cells which secrete hNGF under the direction of the GFAP promoter display in vitro and in vivo properties similar to that seen following other methods of NGF delivery and this source of cells may provide an excellent avenue for delivery of neurotrophins such as NGF to the central nervous system.

Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: trophic and tropic effects in a rodent model of Huntington's disease.

The present study examined whether implants of epidermal growth factor (EGF)-responsive stems cells derived from transgenic mice in which the glial fibrillary acid protein (GFAP) promoter directs the expression of human nerve growth factor (hNGF) could prevent the degeneration of striatal neurons in a rodent model of Huntington's disease (HD). Rats received intrastriatal transplants of GFAP-hNGF stem cells or control stem cells followed 9 days later by an intrastriatal injection of quinolinic acid (QA). Nissl stains revealed large striatal lesions in rats receiving control grafts, which, on average, encompassed 12.78 mm3. The size of the lesion was significantly reduced (1.92 mm3) in rats receiving lesions and GFAP-hNGF transplants. Rats receiving QA lesions and GFAP-hNGF-secreting grafts stem cell grafts displayed a sparing of striatal neurons immunoreactive (ir) for glutamic acid decarboxylase, choline acetyltransferase, and neurons histochemically positive for nicotinamide adenosine diphosphate. Intrastriatal GFAP-hNGF-secreting implants also induced a robust sprouting of cholinergic fibers from subjacent basal forebrain neurons. The lesioned striatum in control-grafted animals displayed numerous p75 neurotrophin-ir (p75NTR) astrocytes, which enveloped host vasculature. In rats receiving GFAP-hNGF-secreting stem cell grafts, the astroglial staining pattern was absent. By using a mouse-specific probe, stem cells were identified in all animals. These data indicate that cellular delivery of hNGF by genetic modification of stem cells can prevent the degeneration of vulnerable striatal neural populations, including those destined to die in a rodent model of HD, and supports the emerging concept that this technology may be a valuable therapeutic strategy for patients suffering from this disease.

LERK-7: a ligand of the Eph-related kinases is developmentally regulated in the brain.

The eph family is the largest subfamily of receptor tyrosine kinases (RTKs). Members of this subfamily display specific expression in the developing and adult brain. Recently, cDNAs encoding membrane bound ligands for these receptors have been identified which we have termed LERKs (ligand for eph-related kinases). We report here the isolation of LERK-7 from a human fetal brain cDNA library. LERK-7 encodes a protein of 228 amino acids and is anchored to the membrane by glycosyl-phosphatidylinositol (GPI) linkage. When transfected into CV1/EBNA cells, LERK-7 binds soluble forms of both hek and elk. In addition, a soluble form of LERK-7 will induce phosphorylation of eck expressed in a human duodenum adenocarcinoma cell line. LERK-7 expressed multiple transcripts (7.5-kb, 6.0-kb, and 3.5-kb) with the highest levels in human adult brain, heart, spleen, and ovary and human fetal brain, lung, and kidney. Similar to the other ligands in this family, LERK-7 is developmentally regulated in the brain. LERK-7 is identical to the recently described AL-1.

Altered neuronal and microglial responses to excitotoxic and ischemic brain injury in mice lacking TNF receptors.

Brain injury, as occurs in stroke or head trauma, induces a dramatic increase in levels of tumor necrosis factor-alpha (TNF), but its role in brain injury response is unknown. We generated mice genetically deficient in TNF receptors (TNFR-KO) to determine the role of TNF in brain cell injury responses. Damage to neurons caused by focal cerebral ischemia and epileptic seizures was exacerbated in TNFR-KO mice, indicating that TNF serves a neuroprotective function. Oxidative stress was increased and levels of an antioxidant enzyme reduced in brain cells of TNFR-KO mice, indicating that TNF protects neurons by stimulating antioxidant pathways. Injury-induced microglial activation was suppressed in TNFR-KO mice, demonstrating a key role for TNF in injury-induced immune response. Drugs that target TNF signaling pathways may prove beneficial in treating stroke and traumatic brain injury.

Ligands for EPH-related tyrosine kinase receptors are developmentally regulated in the CNS.

Elk is a member of the eph family of receptor-like tyrosine kinases. Although its function is unknown, elk is postulated to play a role in nervous system development. Using Northern analysis, we examined the developmental regulation of RNAs encoding elk, and several ligands for the eph family of RTKs, the LERKs. Expression of elk, LERK-1, and LERK-2 RNAs is high in all regions examined in the embryonic and postnatal rat brain and decreases to low levels with age. One exception is the adult olfactory bulb which continues to express a moderate level of LERK-2. In contrast, moderate LERK-4 expression was limited to the developing hippocampus and cerebral cortex. These data indicate that elk and some of the LERKs may play a role in nervous system development, maintenance, and/or regeneration.

LERK-2, a binding protein for the receptor-tyrosine kinase ELK, is evolutionarily conserved and expressed in a developmentally regulated pattern.

We have isolated and characterized cDNA clones that encode the rat homologue of a binding protein, LERK-2, for the receptor tyrosine kinase, elk. The cDNAs contain an open reading frame of 1527 nucleotides capable of encoding a protein 345 amino acid residues in length. The nucleotide sequence of the present clones is > 90% identical to the previously identified human LERK-2 cDNA, and the predicted proteins encoded by the rat and human clones are identical at 95% of amino acid residues. Recombinant proteins expressed from the rat cDNAs bind to elk with high affinity, similar to recombinant human LERK-2 and an endogenously-expressed rat elk-binding protein. Expression of the rat LERK-2 mRNA was detected in embryonic brain, kidney, lung, skeletal muscle, thymus, liver, and heart, and diminished in the early post-natal period. Significant LERK-2 mRNA expression in the young adult rat was restricted to the lung, kidney, heart and testes.

CNS white matter can be altered to support neuronal outgrowth.

Previous work has demonstrated that white matter in the adult mammalian CNS inhibits cell adhesion and neurite outgrowth. This phenomenon has been investigated most recently by culturing neurons on cryostat sections of the adult CNS. Employing this same technique, we have found, in accord with others, that neurons seldom adhere to or grow on central nervous system white matter but will attach and grow on gray matter. In the experiments presented here, embryonic rat hippocampal neurons were grown on cryostat sections from the adult rat CNS, in the presence of brain derived glial cocultures. It was found that the white matter in cryostat sections can be modified by interaction with medium conditioned by brain-derived glial cells. Neurons plated on sections pretreated by such media show significant increases in both attachment and neurite outgrowth. The activity contained in glial conditioned medium is likely complex in nature. While the majority of the activity can be eliminated by heat treatment and trypsinization, neural adhesion but not neurite initiation is affected by protease treatment. Therefore, cell attachment and neurite outgrowth may be regulated by different factors in the conditioned media.

An analysis of the effects of Alzheimer's plaques on living neurons.

Although senile plaques represent a consistent neuropathological feature in Alzheimer's brains, it is not known what role plaques play in the etiology of the disease. Both growth-promoting and growth-inhibiting influences have been postulated. One of the major components in plaques, beta-amyloid, has been shown to affect neuron survival and neurite outgrowth in vitro. Because plaques consist of other components in addition to beta-amyloid, we undertook the present study to determine whether neuronal survival and neurite outgrowth are affected by the presence of a senile plaque. This was accomplished by using cryostat sections from the cerebral cortex of Alzheimer's patients as a substratum for cultured rat hippocampal neurons. Evaluation of these living neurons on Alzheimer's tissue demonstrated that senile plaques affect the amount, complexity, and direction of neurite outgrowth. In addition, neurons were more likely to extend processes away from plaques rather than toward a plaque. Although cell survival on plaques and in control regions was similar, cell survival was significantly reduced in the peri-plaque region. These observations suggest that senile plaques could have deleterious effects on neural organization in situ.

Messenger RNAs coding for receptors and channels in the cerebral cortex of adult and aged rats.

Poly(A)+ mRNAs from the cerebral cortex of aged (24 months) and young adult (3 months) rats were isolated and injected into Xenopus oocytes to express functional neurotransmitter receptors and voltage-operated channels. Electrophysiological recordings of induced membrane currents were used as a measure of the relative amounts of mRNA encoding different receptors and channels, and to study their functional properties. There were no large differences apparent between mRNAs from aged and adult rats, in marked contrast to the dramatic (1000-fold) changes in mRNA expression that occur during embryonic and postnatal development. The membrane currents induced by glutamate or acetylcholine (ACh) application were roughly one third smaller in oocytes injected with mRNA from aged cerebral cortex than in oocytes injected with mRNA from adult cerebral cortex, whereas currents induced by gamma-aminobutyric acid (GABA), kainate or serotonin (5-HT) application, and by activation of voltage-operated Na+ and Ca2+ channels were not significantly different. We did not observe any age-related differences in the properties of the receptors and channels studied.

Changes in messenger RNAs coding for neurotransmitter receptors and voltage-operated channels in the developing rat cerebral cortex.

The ontogenetic development of poly(A)+ mRNAs coding for receptors to several neurotransmitters (kainate, glutamate, acetylcholine, and serotonin) and voltage-operated channels (sodium and calcium) was studied by isolating total poly(A)+ mRNA from the brains of rats at various developmental stages and injecting it into Xenopus oocytes. The oocytes translated the foreign mRNA and incorporated functional receptor/ion channel complexes into the cell membrane. Thus, recording of induced membrane currents in voltage-clamped oocytes gave a measure of the relative amounts of the different messengers. Responses induced by kainate, glutamate, acetylcholine, and serotonin all increased with age and reached a maximum in oocytes injected with mRNA from adult cortex. Messenger RNAs for the earliest ages examined, Embryonic Days 15 and 18, expressed little or no response to kainate, glutamate, or acetylcholine, while 50-70% of the adult response was reached by Postnatal Day 10. In contrast, the serotonin-induced response was already comparatively large (16% of the adult level) in oocytes injected with mRNA from Embryonic Day 15 brain and increased postnatally to adult levels. The expression of voltage-dependent sodium and calcium channels was small in oocytes injected with mRNA from embryonic animals and increased postnatally to reach a maximum in oocytes injected with mRNA from adult animals.

Reflections and remembrances.

Two brief and very personal essays written by practicing nurses in the Rhode Island community. These commentaries reflect their remembrances of the human dimensions of dying and the role of hospice in easing the accompanying burdens of anguish and grief.