Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

Abstract

BackgroundThe adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin+ Sox2+ neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium).ResultsHere we report the isolation and long term propagation of another population of Nestin+ cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges.ConclusionSmooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.

Adult Human Brain Neural Progenitor Cells (NPCs) and Fibroblast-Like Cells Have Similar Properties In Vitro but Only NPCs Differentiate into Neurons

“…Overall, these data indicate a neurovasculature origin of these FbCs. In fact, a cell type strongly resembling our FbCs has been described from the adult human spinal cord [48] . Although there are certain differences, such as their inability to form spheres under NPC culture conditions, when cultured under serum-containing conditions, these spinal cord cells behaved like our FbCs in that they proliferated as an adherent monolayer and expressed Nestin, Sox2, PDGFRβ and α-SMA.…”

“…Although there are certain differences, such as their inability to form spheres under NPC culture conditions, when cultured under serum-containing conditions, these spinal cord cells behaved like our FbCs in that they proliferated as an adherent monolayer and expressed Nestin, Sox2, PDGFRβ and α-SMA. Furthermore, they also found these antigens to be localized to the blood vessels of the spinal cord [48] . Neurovasculature contamination of brain tissue cultures is unavoidable and when taken together with the above results, it appears our brain-derived FbCs are likely to be of neurovascular origin.…”

“…Therefore, the origin(s) of FbCs were investigated based on the three most probable sources. First, mesenchymal stem/precursor cells from the blood [45] , [46] , [47] , second, the perivascular/vascular cells in the brain parenchyma [48] , [49] , and third, the fibroblast cells from the leptomeninges or the surrounding support tissue [50] , [51] , [52] , [53] , [54] . The results showed that fibroblast-related genes in the FbCs, such as P4H, Vimentin and Nestin, were expressed at comparable levels to a control fibroblast cell line, reconfirming the highly fibroblast-like characteristics of these cells.…”

The ependymal region of the adult human spinal cord differs from other species and shows ependymoma-like features

“…Only one group has been able to perform in vitro assays with human ependymal cells; they found that human ependymal neurospheres may give rise to cells expressing glial and neuronal markers (GFAP, GABA, 5-HT), but cannot be passaged (Dromard et al, 2008;Hugnot and Franzen, 2011). Moreover, when cultured in adherent conditions, these cells can be passaged, but produce cells with a molecular profile compatible with mesodermal cell types (Mamaeva et al, 2011). Together, the morphological, molecular, and functional data may question the role of adult spinal cord ependyma as a neurogenic niche in adult humans, but more functional studies are needed to fully support this claim.…”

A Molecular Predictor Reassesses Classification of Human Grade II/III Gliomas

“…In glioma, its expression correlates with high FLAIR volumes and the mesenchymal subtypes of GBM. Further accuracy in the overall survival is provided by the overexpression of genes associated with proliferation ( AURKA [ 37 ], BUB1 [ 38 ], BUB1B [ 38 ], DLG7/DLGAP5 [ 39 ], FOXM1 [ 40 ], KI67 [ 13 ], NEK2 [ 41 ], PLK1 [ 42 ]), apoptosis ( BIRC5/SURVIVIN [ 12 ]) and vasculature ( COL1A1 [ 43 ], HSPG2 [ 44 ], JAG1 [ 45 ], NKX6.1 [ 46 ], TNC [ 47 ]). Their overexpression in poor-prognosis patients is consistent with the fact that enhanced proliferation, apoptosis inhibition and angiogenesis are hallmarks of disease progression in many cancers [48].…”