The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain

Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells.

The majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates

"NeuroStem Chip": a novel highly specialized tool to study neural differentiation pathways in human stem cells.

BACKGROUND: Human stem cells are viewed as a possible source of neurons for a cell-based therapy of neurodegenerative disorders, such as Parkinson's disease. Several protocols that generate different types of neurons from human stem cells (hSCs) have been developed. Nevertheless, the cellular mechanisms that underlie the development of neurons in vitro as they are subjected to the specific differentiation protocols are often poorly understood. RESULTS: We have designed a focused DNA (oligonucleotide-based) large-scale microarray platform (named "NeuroStem Chip") and used it to study gene expression patterns in hSCs as they differentiate into neurons. We have selected genes that are relevant to cells (i) being stem cells, (ii) becoming neurons, and (iii) being neurons. The NeuroStem Chip has over 1,300 pre-selected gene targets and multiple controls spotted in quadruplicates (approximately 46,000 spots total). In this study, we present the NeuroStem Chip in detail and describe the special advantages it offers to the fields of experimental neurology and stem cell biology. To illustrate the utility of NeuroStem Chip platform, we have characterized an undifferentiated population of pluripotent human embryonic stem cells (hESCs, cell line SA02). In addition, we have performed a comparative gene expression analysis of those cells versus a heterogeneous population of hESC-derived cells committed towards neuronal/dopaminergic differentiation pathway by co-culturing with PA6 stromal cells for 16 days and containing a few tyrosine hydroxylase-positive dopaminergic neurons. CONCLUSION: We characterized the gene expression profiles of undifferentiated and dopaminergic lineage-committed hESC-derived cells using a highly focused custom microarray platform (NeuroStem Chip) that can become an important research tool in human stem cell biology. We propose that the areas of application for NeuroStem microarray platform could be the following: (i) characterization of the expression of established, pre-selected gene targets in hSC lines, including newly derived ones, (ii) longitudinal quality control for maintained hSC populations, (iii) following gene expression changes during differentiation under defined cell culture conditions, and (iv) confirming the success of differentiation into specific neuronal subtypes

Critical issues of clinical human embryonic stem cell therapy for brain repair.

Embryonic stem cells (ESCs) provide hope as a potential regenerative therapy for neurological conditions such as Parkinson's disease and spinal cord injury. Currently, ESC-based nervous system repair faces several problems. One major hurdle is related to problems in generating large and defined populations of the desired types of neurons from human ESCs (hESCs). Moreover, survival of grafted hESC-derived cells has varied and functional recovery in recipient animals has often been disappointing. Importantly, in clinical trials, adverse effects after surgery, including tumors or vigorous immune reactions, must be avoided. Here we highlight attempts to overcome these hurdles with hESCs intended for central nervous system repair. We focus on hESC-derived dopamine-producing neurons that can be grafted in Parkinson's disease and identify critical experiments that need to be conducted before clinical trials can occur

The adult human brain contains perivascular cells co-expressing mesenchymal stem cell and pericyte markers.

<p>(A) Confocal image showing neocortical brain section with intracerebral capillary with endothelial cells on the luminal side of the blood vessel (vWF, red) surrounded by α-SMA-positive cells (green). Scale bar 100 µm. (B) Capillaries are lined with pericytes expressing PDGFR-β/CD140b (red) whereby a proportion double labeled for the MSC markers CD 105 (green) and (C) CD13 (green). Scale bar 25 µm (B) and 17 µm (C). (D) PDGFR-β-positive cells at vessel branching points stain for Ki67 (green). Scale bar 10 µm.</p

Progenitor cells exhibit a pericyte phenotype in vitro.

<p>(A) Immunofluorescence image showing expression of PDGFR-β/CD140b, (B) the pericyte-specific marker RGS5 and (C) expression of NG2 but absence of the oligodendroglial marker O4. Cells also co-express (D) Kir6.1, a marker specific for cerebral pericytes (d′) and α-SMA (d″, e″), and (E) Nestin (e′) as previously described for pericytes. Nuclei are stained with DAPI. Scale bars 50 µm.</p

Human brain-derived clonal mesenchymal stem cells have neuroectodermal potential.

<p>(A) Image shows GFAP-staining, (B) S100β-staining and (C) O4-staining after differentiation in glial induction medium. (D) If cells were exposed to neuronal induction protocol, cells stain positive for doublecortin (DCX). Ten days after neuronal induction, differentiating cells express HUC/D (E), neuron-specific enolase (NSE) (F) and the early neuronal marker β-III-tubulin (TUJ1) (G, H, h′, I, i′). Few cells express synaptophysin (G) and the pan-neuronal marker Map2 (H, h″). A few TUJ1-positive cells express GABAA-receptor (I, i″). Scale bars 50 µm. (J) Patch clamp recording of membrane currents using the cell-attached mode, in undifferentiated control cells. Note the absence of single-channel activity. (K) In contrast, single channel activity recorded in a cell differentiated in neural induction medium for 7 days. (L) Absence of electrical responses in a depolarized control cell. (M) Action currents, reflected as the first derivative of the membrane current, recorded in a depolarized differentiated cell. Several action currents are magnified in (m′). Data shown are representative of 5 experiments in differentiated and undifferentiated cells.</p