The adult human brain harbors multipotent perivascular mesenchymal stem cells

Abstract

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.

Figure 1. The adult human brain contains perivascular cells co-expressing mesenchymal stem cell and pericyte markers.

(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.

Figure 2. Isolation and expansion of perivascular mesenchymal stem cells from the adult human brain.

(A) Progenitor cells were isolated from fresh tissue biopsies from the ventricular wall (n = 2) or the temporal neocortex (n = 2). (B) The tissue was enzymatically digested and cells were plated and expanded as adherent monolayers. Once sufficient cell numbers were reached cells were purified by FACS. (C) Phase contrast image of morphology of adherent growing progenitor cells 5 days after isolation from neocortical tissue and (D) at confluency. (E) Cells have the typical flat morphology and large nucleus with prominent nucleoli as described for perivascular cells. Scale bars 50, 10, 100 µm. (F) Exponential growth characteristics of cell lines from ventricular zone and the cortex. Progenitors exhibit similar doubling time independent of region of origin. (G) Karyotype at passage 39, showing euploid number of chromosomes following long-term expansion. Data from representative culture from ventricular zone donor line.

Figure 3. FACS sorting of brain-derived progenitor cell lines.

Human progenitor cells were positively sorted for the MSC markers CD105 and CD13, and thereafter negatively for hematopoietic (CD45) and endothelial markers (CD31). Histogram illustrates results (A, B) for the two different cortical lines and (C, D) for the two lines from the ventricular zone. 99.4–100% of the cell population expressed both, CD105 and CD13, and cultures did not contain endothelial or hematopoietic cells.

Figure 4. Brain-derived cell lines express markers for mesenchymal stem cells and pericytes but not for glial or neuronal precursors.

(A) Representative histogram of flow cytometry analysis of cortical line and (B) ventricular zone line. Progenitors from all donors and both regions highly express MSC (CD90, CD73, CD105, CD29, CD166 and CD49d) and pericyte markers (CD140b/PDGFR-β, RGS5, CD146, Nestin, α-SMA and NG2). They do not express hematopoietic (CD45), endothelial (CD31, CD34), microglial (CD14, CD11), glial or neuronal precursor cell markers (GFAP, O4) or myofibroblast markers (CD56), (green = isotype, red = respective marker).

Figure 5. Progenitor cells exhibit a pericyte phenotype in vitro.

(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.

Figure 6. Characterisation of clonal perivascular MSC progenitor lines.

(A) Single cell in vitro a few hours after plating, and culture at 7 and 21 days, respectively. Scale bar 50 µm. (B) Clonally derived MSC show the same antigen profile as detected for polyclonal lines with pericyte and MSC markers but neither neuronal nor glial precursor cell markers (O4, GFAP) (example here cortical clone; green = isotype, red = respective marker).(C) QPCR comparing clones derived from the ventricular zone (n = 2), and the cortex (n = 2) to a human neuronal progenitor cell line MesC2.10. Data show the relative higher expression of mRNA for RGS5, PDGFR-β and α-SMA in the MSC clones compared to the control. As expected, Nestin mRNA was present in perivascular MSC clones and neural progenitors at comparable amounts. NG2 mRNA was slightly higher expressed in the MSC clones compared to the control. (D) Importantly, neural progenitors markers such as CD133, SOX1, NGN2, PAX6 and Musashi were highly expressed in the positive control but not detectable in MSC clones.

Figure 7. Human brain-derived clonal MSC have mesodermal potential.

Clones derived from both, the ventricular zone (n = 4) and the cortex (n = 6) can be induced to give rise to formation of osteoblasts; (A) Alzarin S staining and (B) osteocalcin staining when cultured in osteogenic medium for 21 days. (C) The same clone forms chondrocytes if cultured as pellets (450 000 cells/pellet) in induction medium. Image shows Safranin O Red staining of cortical-derived clone. (D) Culture in adipogenic medium leads to lipid-containing adipocytes after 14 days. Staining with Oil red illustrates accumulation of typical lipids. (E) qPCR shows about 8-fold increase of adipose differentiation related protein (ADRP) in cultures exposed to adipogenic differentiation medium after 14 days compared to non-induced controls.

Figure 8. Human brain-derived clonal MSC upregulate immature neuronal markers and downregulate pericyte markers upon neuronal induction.

QPCR in neural induction cultures. (A) After 10 days of neuronal induction, we observed a 180–200 fold upregulation of mRNA of the early neuronal markers DCX and β-III-tubulin and an about 50-fold increase in mRNA for GFAP, a glial marker and marker for neural progenitors. (B) At the same time, mRNA expression for pericyte markers Nestin, α-SMA, RGS5 and PDGFR-β decreased compared to undifferentiated controls. (C) Immunofluorescence image of neuronal induction culture showing TUJ1-positive cells and α-SMA-expressing cells. (D) QPCR showing upregulation of neuronal transcription factors. There was a more than 6-fold upregulation in PAX6, tvelve-fold upregulation of NeuroD1, seventy three-fold upregulation of Tbr2 and forty two-fold upregulation of Tbr1 compared to undifferentiated cells.

Figure 9. Human brain-derived clonal mesenchymal stem cells have neuroectodermal potential.

(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.