Claudia Rosenbaum

A New Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential

Here a new, intrinsically pluripotent, CD45-negative population from human cord blood, termed unrestricted somatic stem cells (USSCs) is described. This rare population grows adherently and can be expanded to 1015 cells without losing pluripotency. In vitro USSCs showed homogeneous differentiation into osteoblasts, chondroblasts, adipocytes, and hematopoietic and neural cells including astrocytes and neurons that express neurofilament, sodium channel protein, and various neurotransmitter phenotypes. Stereotactic implantation of USSCs into intact adult rat brain revealed that human Tau-positive cells persisted for up to 3 mo and showed migratory activity and a typical neuron-like morphology. In vivo differentiation of USSCs along mesodermal and endodermal pathways was demonstrated in animal models. Bony reconstitution was observed after transplantation of USSC-loaded calcium phosphate cylinders in nude rat femurs. Chondrogenesis occurred after transplanting cell-loaded gelfoam sponges into nude mice. Transplantation of USSCs in a noninjury model, the preimmune fetal sheep, resulted in up to 5% human hematopoietic engraftment. More than 20% albumin-producing human parenchymal hepatic cells with absence of cell fusion and substantial numbers of human cardiomyocytes in both atria and ventricles of the sheep heart were detected many months after USSC transplantation. No tumor formation was observed in any of these animals.

Unrestricted somatic stem cells from human umbilical cord blood can be differentiated into neurons with a dopaminergic phenotype.

Recently, it has been shown that human unrestricted somatic stem cells (USSCs) from umbilical cord blood represent pluripotent, neonatal, nonhematopoietic stem cells with the potential to differentiate into the neural lineage. However, molecular and functional characterization of the neural phenotype and evaluation of the degree of maturity of the resulting cells are still lacking. In this study, we addressed the question of neuronal differentiation and maturation induced by a defined composition of growth and differentiation factors (XXL medium). We demonstrated the expression of different neuronal markers and their enrichment in USSC cultures during XXL medium incubation. Furthermore, we showed enrichment of USSCs expressing tyrosine hydroxylase (TH), an enzyme specific for dopaminergic neurons and other catecholamine-producing neurons, accompanied by induction of Nurr1, a factor regulating dopaminergic neurogenesis. The functionality of USSCs has been analyzed by patch-clamp recordings and high-performance liquid chromatography (HPLC). Voltage-gated sodium-channels could be identified in laminin-predifferentiated USSCs. In addition, HPLC analysis revealed synthesis and release of the neurotransmitter dopamine by USSC-derived cells, thus correlating well with the detection of TH transcripts and protein. This study provides novel insight into the potential of unrestricted somatic stem cells from human umbilical cord blood to acquire a neuronal phenotype and function.

Improved culture methods to expand schwann cells with altered growth behaviour from CMT1A patients

A duplication of the gene for myelin protein PMP22 is by far the most common cause of the hereditary demyelinating neuropathy CMT1A. A role for PMP22 in cell growth in addition to its function as a myelin protein has been suggested because PMP22 is homologous to a gene specifically upregulated during growth arrest. Furthermore, transfected rat Schwann cells overexpressing PMP22 show reduced growth. In addition, abnormal Schwann cell differentiation has been described in nerve biopsies from CMT1A patients. To analyse whether the duplication of the PMP22 gene in CMT1A neuropathy primarily alters Schwann cell differentiation and to exclude nonspecific secondary responses, we improved human Schwann cell culturing. This allowed us long‐term passaging of human Schwann cells with unchanged phenotype, assessed by expression of different Schwann cell markers. Subsequently we established Schwann cell cultures from CMT1A nerve biopsies. We find decreased proliferation of Schwann cells from different CMT1A patients in all passages. We also demonstrate PMP22 mRNA overexpression in cultured CMT1A Schwann cells. We conclude that decreased proliferation in cultured Schwann cells that carry the CMT1A duplication indicates abnormal differentiation of CMT1A Schwann cells. The identification of an abnormal phenotype of CMT1A Schwann cells in culture could possibly lead to an in vitro disease model. GLIA 23:89–98, 1998.

Long-term culture and characterization of human neurofibroma-derived Schwann cells.

Neurofibromas are benign tumors arising from the peripheral nerve sheath and are a typical finding in neurofibromatosis type 1 (NF1). Schwann cells are the predominant cell type in neurofibromas and thus are supposed to play a major role in the pathogenesis of these tumors. It is not known, however, if NF1 mutations in Schwann cells result in an altered phenotype that subsequently leads to tumor formation. To characterize the biological properties of neurofibroma‐derived Schwann cells we developed cell culture techniques that enabled us to isolate Schwann cells from neurofibromas and grow them in vitro for several weeks without significant fibroblast contamination. Neurofibroma‐derived Schwann cells were characterized by altered morphology, heterogeneous growth behavior, and increased expression of the P0 antigen while several other features of normal human Schwann cells were retained. We conclude that neurofibroma‐derived Schwann cells exhibit a distinct phenotype in vitro but that the observed abnormalities by themselves are insufficient to explain neurofibroma formation. Application of our improved culture conditions makes neurofibroma‐derived Schwann cells readily available for further studies to define their role in tumorigenesis in neurofibromatosis type 1. J. Neurosci. Res. 61:524–532, 2000.

Combinatorial code of growth factors and neuropeptides define neuroendocrine differentiation in PC12 cells.

Adrenal chromaffin cells constitute one of the first cell types to have been defined as a neuroendocrine cell type. Since they produce dopamine, these cells have been proposed for the treatment of neuronal deficits in human Parkinsons disease. However, the factors involved in the development of chromaffin cells are still poorly understood. Based on recent insights from stem cell research, we decided to study the role of extracellular matrices, growth factors and neuropeptides on the neuroendocrine differentiation in a serum-free medium of PC12 cells. Employing immunohistochemistry, quantitative PCR and HPLC analysis, neuroendocrine differentiation was determined by evaluating neurite outgrowth, catecholamine biosynthesis and release as well as neuropeptide and vesicular protein mRNA expression. The combination of bFGF, NGF and PACAP could prevent the inhibition of neurite process development induced by dexamethasone in PC12 cells cultured on ECM. Whereas glucocorticoids were essential in the regulation of enzymes of catecholamine biosynthesis and metabolism, growth factors and PACAP were more efficient in inducing neuropeptide and chromogranin B expression as well as release of dopamine and 3-methoxytyramine. Therefore, in addition to glucocorticoids, chromaffin cells need a gradient of matrix, growth factors, and neuropeptides to develop the full functional phenotype of a neuroendocrine cell.

Neuronal network properties of human teratocarcinoma cell line-derived neurons

Understanding the structural and functional development of neurons in networks has a high impact to estimate the potentials for restorative therapies. Neurons derived from the human NT2 cell line (hNT) formed networks with a clustered neuritic architecture in vitro, whereas primary dissociated embryonic rat cortical neurons (Cx) displayed a more homogenous cell assembly. Spontaneous spikes of both cell types were recorded on microelectrode arrays within 2 weeks after seeding, but hNT showed a mostly uncorrelated firing pattern in contrast to Cx with highly synchronized bursting. hNT neurons were less sensitive to TTX (IC50 = 5.7 +/- 0.1 nM vs. IC50 = 1.1 +/- 0.2 nM), magnesium (IC50 = 1.83 +/- 0.01 mM vs. IC50 = 0.161 +/- 0.023 mM), and APV (IC50 > 100 microM vs. IC50 = 18 microM). We conclude that embryonic cortical neurons and hNT neurons have different network properties. This should be carefully considered before hNT neurons are used in therapeutic approaches, e g., central nervous system (CNS) grafting.

Voltage-dependent membrane currents of cultured human neurofibromatosis type 2 Schwann cells.

Previous experimental observations indicate that inhibition of voltage‐dependent K+ currents suppresses proliferation of normal Schwann cells. In the present study we tested the opposite relationship, i.e., whether Schwann cells from tumors with abnormally high rates of proliferation would have an increase in membrane K+ currents. Whole‐cell membrane currents were studied in cultured cells from schwannomas of two neurofibromatosis type 2 (NF2) patients (n = 53), one patient with a sporadic schwannoma (n = 22), and two control subjects (n = 41). Five different types of voltage‐dependent membrane currents were found in all of the Schwann cells tested. Membrane depolarization activated outward K+ and Cl− currents; quinidine was found to block the K+ current (IC50 ≈ 1 μM), and NPPB reduced the Cl− current. Ba2+‐sensitive inward rectifier K+ currents, fast Na+ currents, and a transient, inactivating K+ current were less frequently observed. On average, NF2 cells were found to have statistically significant higher membrane potential and larger non‐inactivating K+ outward current as compared to controls. Electrophysiological parameters of Schwann cells from a sporadic schwannoma showed a tendency for larger outward currents; however, the difference did not reach statistical significance. Together the data support the suggestion of a possible link between K+ outward current and proliferation of Schwann cells. GLIA 24:313–322, 1998.

HIV-1 protein Tat reduces the glutamate-induced intracellular Ca2+ increase in cultured cortical astrocytes

The trans‐activator protein Tat of the human immunodeficiency virus type 1 (HIV‐1) is regarded as an injurious molecule in the pathogenesis of HIV‐1 associated encephalopathy (HIVE). We investigated the effects of Tat on neuroligand‐induced intracellular Ca2+ increase in cultured astroglial cells. Rat cortical astrocytes, human glioblastoma cells and glial restricted precursor cells, from a human embryonic teratocarcinoma cell line, were incubated with recombinant Tat (100 ng/mL for 60 min) which induced a significant reduction of glutamate or ATP‐induced intracellular Ca2+ increase (‘glutamate response’, ‘ATP response’). The reduction of the glutamate response was also observed following cell incubation with cell extracts of HeLa‐T4+ cells transiently transfected with an expression plasmid coding for Tat. However, inactivation of the transcriptional trans‐activity of Tat, by using a mutant form of Tat, as well as inhibition of de novo protein synthesis by cycloheximide abolished the effect on the glutamate response. This suggests that Tat acts upon induction of a so far unknown cellular gene whoes gene product causes the reduction of glutamate responses. As the effect of Tat resembles the effect of TNFα on glutamate responses [Köller et al. (2001) Brain Res., 893, 237–243] which is locally released within the brains of HIVE patients, we also tested for synergistic effects of Tat and TNFα on the glutamate response. Low concentrations of Tat in combination with subthreshold concentrations of TNFα also elicited a marked reduction of astroglial glutamate responses. Our data suggest that Tat and TNFα, both by itself and synergistically, induce astroglial dysfunction.

Enhanced proliferation and potassium conductance of Schwann cells isolated from NF2 schwannomas can be reduced by quinidine.

Abstract Neurofibromatosis type 2 (NF2) is an autosomal dominant disease that is characterized mainly by schwannomas, as well as menigiomas and gliomas. The NF2 gene product merlin/schwannomin acts as a tumor suppressor. Schwann cells derived from NF2 schwannomas showed an enhanced proliferation rate, and electrophysological studies revealed larger K + outward currents as compared with controls. Schwann cells isolated from schwannomas of NF2 patients or multiorgan donors were treated with different concentrations of the K + current blockers quinidine, tetraethylammonium chloride, and 4-aminopyridine and K + outward currents and proliferation rates of these cells were compared. K + outward currents of both cell types can be blocked by quinidine. Importantly, treatment with quinidine reduces proliferation of NF2 Schwann cells in a concentration dependent manner but did not reduce proliferation of normal Schwann cells. Therefore, the use of quinidine or quinidine-like components would possibly provide a novel adjuvant therapeutic option for NF2 patients to slow down or freeze growth of schwannomas.

Lack of immune responses to immediate or delayed implanted allogeneic and xenogeneic Schwann cell suspensions

Previous studies have shown that Schwann cell implantation offers a potential therapeutic approach to a variety of neurodegenerative disorders and traumatic injuries. In a clinically relevant paradigm, however, the implantation of autologous Schwann cells is problematic and the use of heterogenetic Schwann cells will be required. In the present study we addressed this important issue and analysed the immunogenicity and survival of allogeneic and xenogeneic Schwann cell suspension grafts in a prelesioned CNS fiber tract, the transected postcommissural fornix of the adult Wistar rat. Cultured Schwann cells from Wistar rat or human peripheral nerve were injected either immediately or after a delay into the transection site and the spatio‐temporal pattern of leukocyte infiltration and of major histocompatibility antigen expression was characterized and semiquantified with immunocytochemical methods. Our main findings are that (1) invasive cerebral lesions induce the expression of MHC class I and II antigens, but only sparse infiltration of T‐lymphocytes, (2) both allogeneic and xenogeneic discordant Schwann cell suspension grafts, from either neonatal or adult peripheral nerve, survive without any overt signs of rejection for up to 10 weeks after implantation; and (3) delayed implantation procedures have no effect on immune responses to allogeneic Schwann cell grafts. These results demonstrate that there is no marked ongoing immune reactions to heterogenetic Schwann cell suspension grafts and that long‐term survival of cross‐species Schwann cell grafts can be achieved in the absence of any immunsuppressive treatment. Thus the conditions for functional transplantation of Schwann cells across immunological barriers seem to be favourable and will have implications for future cross‐species studies, and possibly also for clinical application. GLIA 21:299–314, 1997.