Multilineage potential of stable human mesenchymal stem cell line derived from fetal marrow

Human bone marrow contains two major cell types, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). MSCs possess self-renewal capacity and pluripotency defined by their ability to differentiate into osteoblasts, chondrocytes, adipocytes and muscle cells. MSCs are also known to differentiate into neurons and glial cells in vitro, and in vivo following transplantation into the brain of animal models of neurological disorders including ischemia and intracerebral hemorrhage (ICH) stroke. In order to obtain sufficient number and homogeneous population of human MSCs, we have clonally isolated permanent and stable human MSC lines by transfecting primary cell cultures of fetal human bone marrow MSCs with a retroviral vector encoding v-myc gene. One of the cell lines, HM3.B10 (B10), was found to differentiate into neural cell types including neural stem cells, neurons, astrocytes and oligodendrocytes in vitro as shown by expression of genetic markers for neural stem cells (nestin and Musashi1), neurons (neurofilament protein, synapsin and MAP2), astrocytes (glial fibrillary acidic protein, GFAP) and oligodendrocytes (myelin basic protein, MBP) as determined by RT-PCR assay. In addition, B10 cells were found to differentiate into neural cell types as shown by immunocytochical demonstration of nestin (for neural stem cells), neurofilament protein and beta-tubulin III (neurons) GFAP (astrocytes), and galactocerebroside (oligodendrocytes). Following brain transplantation in mouse ICH stroke model, B10 human MSCs integrate into host brain, survive, differentiate into neurons and astrocytes and induce behavioral improvement in the ICH animals. B10 human MSC cell line is not only a useful tool for the studies of organogenesis and specifically for the neurogenesis, but also provides a valuable source of cells for cell therapy studies in animal models of stroke and other neurological disorders.     

Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours

The intermediate filament protein nestin is predominantly expressed in some stem/progenitor cells and appears to be a useful molecular tool to characterise tumours originating from precursor cells of neuroectodermal and mesenchymal lineages. Leydig cells originate in the adult testis by differentiation from stem cells and express a variety of neural and neuroendocrine markers. The possible expression of the neural stem cell marker nestin in Leydig cells and testicular tumour cells was determined by analysing the patterns of nestin expression in normal and pathological human testes by Western blot and immunohistochemical methods. In normal testis, nestin was found in some vascular endothelial cells, a subset of peritubular spindle-shaped cells and some Leydig cells; spermatogenic and Sertoli cells were unstained. In normal Leydig cells, nestin was distributed in the perinuclear cytoplasm and accumulated in the crystalloids of Reinke with ageing. In non-tumour pathologies (cryptorchidism, impaired spermatogenesis), the seminiferous tubules were immunonegative, whereas hyperplastic Leydig cells showed cytoplasmic immunolabelling. In testicular malignancies, nestin was localised in the Sertoli cells of the seminiferous tubules affected with intratubular germ cell neoplasia, in the hyperplastic Leydig cells associated with these tumours and in some components (mesenchymal and neuroepithelial cells) of teratomas; spermatocytic and non-spermatocytic seminomas were unstained. Some vascular endothelial cells were immunolabelled in all tumour samples. Thus, nestin is expressed in a population of normal and hyperplastic Leydig cells and in Sertoli cells in the presence of intratubular germ-cell neoplasia. Nestin may be a good marker for identifying components of testicular teratomas.The two first authors participated equally in this workThis work was supported by a grant from the Fondo de Investigaciones Sanitarias (FIS 02/3003 to M.V.T. Lobo)     

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.     

Expression of nestin in the podocytes of normal and diseased human kidneys

The complex cyto-architecture of the podocyte is critical for glomerular permselectivity. The present study characterizes the expression of nestin, an intermediate filament protein, in human kidneys. In normal kidneys, nestin was detected at the periphery of glomerular capillary loops. Colabeling showed nestin was expressed in WT1-positive cells. Within the podocyte, nestin immunoreactivity was present in the cell body and primary process. This was supported by immunoelectron microscopy. Nestin also colocalized with vimentin in the periphery of capillary loops but not in the mesangium. Nestin was not detected in other structures of the adult human kidney. To determine the potential role of nestin in proteinuria, nestin was examined in kidney biopsies from patients with or without proteinuria. These patients were diagnosed with IgA nephropathy with mild mesangial expansion but without proteinuria, IgA nephropathy with proteinuria, membranous nephropathy (MN), and focal segmental glomerular sclerosis (FSGS). The distribution of nestin in these biopsies was similar to that in the normal kidney. Semiquantitative analysis of immunostaining showed that glomerular nestin expression in IgA nephropathy without proteinuria was not different from normal kidney; however, nestin expression in kidneys of patients with IgA nephropathy and proteinuria, or MN and FSGS with proteinuria was significantly reduced compared with normal kidney (P < 0.01). Reduced nestin mRNA expression in the patients with IgA nephropathy with proteinuria and FSGN was also observed by quantitative real-time PCR. These studies suggest that nestin may play an important role in maintaining normal podocyte function in the human kidney.     

Expression of intermediate filament proteins and neuronal markers in the human fetal gut.

The human enteric nervous system (ENS) derives from migrating neural crest cells (NCC) and is structured into different plexuses embedded in the gastrointestinal tract wall. During development of the NCC, a rearrangement of various cytoskeletal intermediate filaments such as nestin, peripherin, or alpha-internexin takes place. Although all are related to developing neurons, nestin is also used to identify neural stem cells. Until now, information about the prenatal development of the human ENS has been very restricted, especially concerning potential stem cells. In this study the expression of nestin, peripherin, and alpha-internexin, but also of neuronal markers such as protein gene product (PGP) 9.5 and tyrosine hydroxylase, were investigated in human fetal and postnatal gut. The tissue samples were rapidly removed and subsequently processed for immunohistochemistry or immunoblotting. Nestin could be detected in all samples investigated with the exception of the 9th and the 12th week of gestation (WOG). Although the neuronal marker PGP9.5 was coexpressed with nestin at the 14th WOG, this could no longer be observed at later time points. Alpha-internexin and peripherin expression also did not appear before the 14th WOG, where they were coexpressed with PGP9.5. This study reveals that the intermediate filament markers investigated are not suitable to detect early neural crest stem cells.     

Evidence for neural progenitor cells in the human adult enteric nervous system

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.