We report here that cells co-purifying with mesenchymal stem cells--termed here multipotent adult progenitor cells or MAPCs--differentiate, at the single cell level, not only into mesenchymal cells, but also cells with visceral mesoderm, neuroectoderm and endoderm characteristics in vitro. When injected into an early blastocyst, single MAPCs contribute to most, if not all, somatic cell types. On transplantation into a non-irradiated host, MAPCs engraft and differentiate to the haematopoietic lineage, in addition to the epithelium of liver, lung and gut. Engraftment in the haematopoietic system as well as the gastrointestinal tract is increased when MAPCs are transplanted in a minimally irradiated host. As MAPCs proliferate extensively without obvious senescence or loss of differentiation potential, they may be an ideal cell source for therapy of inherited or degenerative diseases.

/pdf/pluripotency-of-mesenchymal-stem-cells-derived-from-adult-200s4qlizf.pdf

Pluripotency of mesenchymal stem cells derived from adult marrow

Human and mouse embryonic stem cells (HESCs and MESCs, respectively) self-renew indefinitely while maintaining the ability to generate all three germ-layer derivatives. Despite the importance of ESCs in developmental biology and their potential impact on tissue replacement therapy, the molecular mechanism underlying ESC self-renewal is poorly understood. Here we show that activation of the canonical Wnt pathway is sufficient to maintain self-renewal of both HESCs and MESCs. Although Stat-3 signaling is involved in MESC self-renewal, stimulation of this pathway does not support self-renewal of HESCs. Instead we find that Wnt pathway activation by 6-bromoindirubin-3'-oxime (BIO), a specific pharmacological inhibitor of glycogen synthase kinase-3 (GSK-3), maintains the undifferentiated phenotype in both types of ESCs and sustains expression of the pluripotent state-specific transcription factors Oct-3/4, Rex-1 and Nanog. Wnt signaling is endogenously activated in undifferentiated MESCs and is downregulated upon differentiation. In addition, BIO-mediated Wnt activation is functionally reversible, as withdrawal of the compound leads to normal multidifferentiation programs in both HESCs and MESCs. These results suggest that the use of GSK-3-specific inhibitors such as BIO may have practical applications in regenerative medicine.

Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor

Poor survival of human embryonic stem (hES) cells after cell dissociation is an obstacle to research, hindering manipulations such as subcloning. Here we show that application of a selective Rho-associated kinase (ROCK) inhibitor1,2, Y-27632, to hES cells markedly diminishes dissociation-induced apoptosis, increases cloning efficiency (from ∼1% to ∼27%) and facilitates subcloning after gene transfer. Furthermore, dissociated hES cells treated with Y-27632 are protected from apoptosis even in serum-free suspension (SFEB) culture3 and form floating aggregates. We demonstrate that the protective ability of Y-27632 enables SFEB-cultured hES cells to survive and differentiate into Bf1+ cortical and basal telencephalic progenitors, as do SFEB-cultured mouse ES cells.

A ROCK inhibitor permits survival of dissociated human embryonic stem cells

Stem cells, which are clonogenic cells with self-renewal and multilineage differentiation properties, have the potential to replace or repair damaged tissue. We have directly isolated clonogenic human central nervous system stem cells (hCNS-SC) from fresh human fetal brain tissue, using antibodies to cell surface markers and fluorescence-activated cell sorting. These hCNS-SC are phenotypically 5F3 (CD133)+, 5E12+, CD34−, CD45−, and CD24−/lo. Single CD133+ CD34− CD45− sorted cells initiated neurosphere cultures, and the progeny of clonogenic cells could differentiate into both neurons and glial cells. Single cells from neurosphere cultures initiated from CD133+ CD34− CD45− cells were again replated as single cells and were able to reestablish neurosphere cultures, demonstrating the self-renewal potential of this highly enriched population. Upon transplantation into brains of immunodeficient neonatal mice, the sorted/expanded hCNS-SC showed potent engraftment, proliferation, migration, and neural differentiation.

https://www.pnas.org/content/pnas/97/26/14720.full.pdf

Direct isolation of human central nervous system stem cells

Hebb's rule (1949) states that learning and memory are based on modifications of synaptic strength among neurons that are simultaneously active. This implies that enhanced synaptic coincidence detection would lead to better learning and memory. If the NMDA (N-methyl-D-aspartate) receptor, a synaptic coincidence detector, acts as a graded switch for memory formation, enhanced signal detection by NMDA receptors should enhance learning and memory. Here we show that overexpression of NMDA receptor 2B (NR2B) in the forebrains of transgenic mice leads to enhanced activation of NMDA receptors, facilitating synaptic potentiation in response to stimulation at 10-100 Hz. These mice exhibit superior ability in learning and memory in various behavioural tasks, showing that NR2B is critical in gating the age-dependent threshold for plasticity and memory formation. NMDA-receptor-dependent modifications of synaptic efficacy, therefore, represent a unifying mechanism for associative learning and memory. Our results suggest that genetic enhancement of mental and cognitive attributes such as intelligence and memory in mammals is feasible.

Genetic enhancement of learning and memory in mice

Parkinson's disease is a widespread condition caused by the loss of midbrain neurons that synthesize the neurotransmitter dopamine. Cells derived from the fetal midbrain can modify the course of the disease, but they are an inadequate source of dopamine-synthesizing neurons because their ability to generate these neurons is unstable. In contrast, embryonic stem (ES) cells proliferate extensively and can generate dopamine neurons. If ES cells are to become the basis for cell therapies, we must develop methods of enriching for the cell of interest and demonstrate that these cells show functions that will assist in treating the disease. Here we show that a highly enriched population of midbrain neural stem cells can be derived from mouse ES cells. The dopamine neurons generated by these stem cells show electrophysiological and behavioural properties expected of neurons from the midbrain. Our results encourage the use of ES cells in cell-replacement therapy for Parkinson's disease.

/pdf/dopamine-neurons-derived-from-embryonic-stem-cells-function-35euvat5ic.pdf

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease

Embryonic stem (ES) cells are clonal cell lines derived from the inner cell mass of the developing blastocyst that can proliferate extensively in vitro and are capable of adopting all the cell fates in a developing embryo. Clinical interest in the use of ES cells has been stimulated by studies showing that isolated human cells with ES properties from the inner cell mass or developing germ cells can provide a source of somatic precursors. Previous studies have defined in vitro conditions for promoting the development of specific somatic fates, specifically, hematopoietic, mesodermal, and neurectodermal. In this study, we present a method for obtaining dopaminergic (DA) and serotonergic neurons in high yield from mouse ES cells in vitro. Furthermore, we demonstrate that the ES cells can be obtained in unlimited numbers and that these neuron types are generated efficiently. We generated CNS progenitor populations from ES cells, expanded these cells and promoted their differentiation into dopaminergic and serotonergic neurons in the presence of mitogen and specific signaling molecules. The differentiation and maturation of neuronal cells was completed after mitogen withdrawal from the growth medium. This experimental system provides a powerful tool for analyzing the molecular mechanisms controlling the functions of these neurons in vitro and in vivo, and potentially for understanding and treating neurodegenerative and psychiatric diseases.

Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells

Copper/zinc superoxide dismutase (CuZn-SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene encoding CuZn-SOD resides on human chromosome 21 and is overexpressed in Down syndrome (DS) patients. Overexpression of CuZn-SOD in transgenic (Tg) mice and cultured cells creates chronic oxidative stress leading to enhanced susceptibility to degeneration and apoptotic cell death. We have now found that three lines of Tg-CuZn-SOD mice, one of which also overexpresses S100beta, a glial calcium binding protein, are deficient in spatial memory. Furthermore, hippocampal slices taken from these mice have an apparently normal synaptic physiology, but are impaired in the ability to express long-term potentiation (LTP). This effect on hippocampal LTP was abrogated by treatment of slices with the H2O2 scavenger catalase or the antioxidant N-t-butyl-phenylnitrone (BPN). It is proposed that elevated CuZnSOD causes an increase in tetanic stimulation-evoked formation of H2O2 which leads to diminished LTP and cognitive deficits in these mice.

Reversible impairment of long‐term potentiation in transgenic Cu/Zn‐SOD mice

Exposure of rat hippocampal slices to low concentrations of the muscarinic agonist carbachol (CCh) has been shown to produce a slow onset long-term potentiation (LTP) of reactivity to afferent stimulation in CA1 neurons. Although this potentiation shares a number of properties with tetanic LTP, muscarinic LTP (LTPm) is independent of activation of the NMDA receptor. We now demonstrate that low levels of hydrogen peroxide (H2O2) cause hippocampal slices to lose the ability to express LTPm. This powerful effect of H2O2 is selective in that it does not affect the reactivity of hippocampal neurons to higher concentrations of CCh. In fact, H2O2 also blocks induction of a slow onset, non-NMDA-dependent tetanic LTP (NN-LTP). The functional relevance of this action of H2O2 is exemplified by the fact that the hippocampus of aged rats, which produces higher levels of endogenous H2O2 than that of young rats, lacks LTPm and expresses a markedly reduced NN-LTP. In aged rats, the lack of LTPm contrasts with an apparently normal muscarinic suppression of the EPSP slope induced by higher concentrations of CCh. When hippocampal slices from aged animals are treated with catalase, an enzyme that breaks down H2O2, LTPm is restored, and NN-LTP is enhanced. Thus, our study proposes a unique and novel age-dependent peroxide regulation of LTPm in the brain and provides a link between the cholinergic system, aging, and memory functions.

https://www.jneurosci.org/content/jneuro/17/22/8695.full.pdf

Jonathan M. Auerbach

Papers

Dopamine neurons derived from embryonic stem cells function in an animal model of Parkinson's disease

Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells

Reversible impairment of long‐term potentiation in transgenic Cu/Zn‐SOD mice

Peroxide Modulation of Slow Onset Potentiation in Rat Hippocampus

Muscarinic receptors involved in hippocampal plasticity.