Reversal of dopaminergic degeneration in a parkinsonian rat following micrografting of human bone marrow-derived neural progenitors.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by the selective loss of dopaminergic (DA) neurons in the midbrain. Various types of stem cells that have potential to differentiate into DA neurons are being investigated as cellular therapies for PD. Stem cells also secrete growth factors and therefore also may have therapeutic effects in promoting the health of diseased DA neurons in the PD brain. To address this possibility in an experimental model of PD, bone marrow-derived neuroprogenitor-like cells were generated from bone marrow procured from healthy human adult volunteers and their potential to elicit recovery of damaged DA axons was studied in a partial lesion rat model of PD. Following collection of bone marrow, mesenchymal stem cells (MSC) were isolated and then genetically modified to create SB623 cells by transient transfection with the intracellular domain of the Notch1 gene (NICD), a modification that upregulates expression of certain neuroprogenitor markers. Ten deposits of 0.5 microl of SB623 cell suspension adjusted from 6,000 to 21,000 cells/microl in PBS or PBS alone were stereotaxically placed in the striatum 1 week after the nigrostriatal projection had been partially lesioned in adult F344 rats by injection of 6-hydroxydopamine (6-OHDA) into the striatum. At 3 weeks, a small number of grafted SB623 cells survived in the lesioned striatum as visualized by expression of the human specific nuclear matrix protein (hNuMA). In rats that received SB623 cells, but not in control rats, dense tyrosine hydroxylase immunoreactive (TH-ir) fibers were observed around the grafts. These fibers appeared to be rejuvenated host DA axons because no TH-ir in soma of surviving SB623 cells or coexpression of TH and hNuMA-ir were observed. In addition, dense serotonin immunoreactive (5-HT-ir) fibers were observed around grafted SB623 cells and these fibers also appeared to be of the host origin. Also, in some SB623 grafted rats that were sacrificed within 2 h of dl-amphetamine injection, hot spots of c-Fos-positive nuclei that coincided with rejuvenated dense TH fibers around the grafted SB623 cells were observed, suggesting increased availability of DA in these locations. Our observations suggest that NICD-transfected MSC hold potential as a readily available autologous or allogenic cellular therapy for ameliorating the degeneration of DA and 5-HT neurons in PD patients.

The transcription factor Nurr1 in human NT2 cells and hNT neurons.

Human, neuronally committed hNT or NT2-N cells, originally derived from the Ntera2/D1 (NT2) clone after exposure to retinoic acid (RA), represent a potentially important source of cells to treat neurodegenerative diseases. Our previous in vitro experiments showed that hNT cells possess immunocytochemically detectable markers typical of dopaminergic (DA) ventral mesencephalic (VM) neurons, including tyrosine hydroxylase (TH), dopamine transporter (DAT), dopamine receptor (D2), and aldehyde dehydrogenase (AHD-2). In the current study, we sought to examine whether Nurr1, an orphan receptor of the nuclear receptor superfamily shown to be essential for the development, differentiation and survival of midbrain DA neurons, would be expressed in 3, 4, or 5 week RA-induced hNT neurons and their NT2 precursors. Our immunocytochemical analyses indicate that NT2 cells as well as hNT neurons independent of the length of RA-driven differentiation were Nurr1-immunoreactive. RT-PCR analysis confirmed the expression of Nurr1-specific mRNA in both NT2 precursors and the hNT neurons. Furthermore, immunocytochemical co-expression of Nurr1 and TH was detected in hNT neurons. The findings of this study suggest that Nurr1 may be important during the development of hNT neurons and involved in their differentiation into the dopaminergic phenotype.

Nicotinic acetylcholine receptors on NT2 precursor cells and hNT (NT2-N) neurons.

This is the first report, to our knowledge, of prominent, natural expression of nAChR alpha4, alpha6 and alpha9 subunits in a human, neuronally-committed cell line. We performed studies with specific reference to the expression of nicotinic acetylcholine receptors (nAChR) to further characterize a human, postmitotic, transplantable, with a neuronal phenotype, cell line called hNT (also called NT2-N). hNT cells acquire a distinctive neuronal phenotype upon differentiation from their NT2 precursors. Immunocytochemical studies showed that NT2 cells were strongly immunopositive for alpha4 or alpha7 subunits, moderately immunopositive for alpha3/alpha5 subunits, and weakly immunopositive for beta2 or beta4 subunits, whereas hNT neurons showed positive, strong-to-moderate immunostaining for all of these nAChR subunits. Reverse transcription-polymerase chain reaction (RT-PCR) mRNA analyses indicated that levels of alpha7 subunit messages were similar in both NT2 and hNT cells, whereas alpha2, alpha10, and beta3 subunit transcripts were not detected. Levels of alpha3, alpha5, and beta4 subunit messages were lower in hNT neurons than in NT2 precursors. However, alpha4 and beta2 subunit messages were present in NT2 precursors but were greatly induced in hNT neurons. Levels of alpha6 and alpha9 subunit messages, not detectable in NT2 precursors, rose to high levels in hNT neurons. hNT cell nAChR subunit message levels were comparable to (alpha4, alpha5, beta4) or higher than (alpha6, alpha9, beta2) levels in adult human brain. NT2 and hNT cells may provide an excellent model for studies of neurogenesis, roles played by nAChR in differentiation and neurodegeneration, and effects of neuronal differentiation on nAChR expression.