Neurotrophin-directed differentiation of human adult marrow stromal cells to dopaminergic-like neurons.

Marrow-isolated adult multilineage inducible (MIAMI) cells were differentiated in vitro to neuronal cells in a neurotrophin-dependent fashion. After induction, the cells revealed electrophysiological features similar to those observed in mature neurons. Primary early passage human MIAMI cells without any type of co-cultures with other cell types were used. The developmental program involved a multi-step process requiring the concerted action of brain-derived neurotrophic factor, nerve growth factor and depended on neurotrophin-3, after basic fibroblast growth factor withdrawal. MIAMI-derived neuron-like cells sequentially expressed the neuronal markers, developed a complex neurite outgrowth and arborization, and acquired electrophysiological characteristics similar to those observed in mature neurons. The young and old MIAMI-derived neuronal cells developed both inward and outward currents upon depolarization, similar to those observed in normal neurons. These results represent the earliest evidence that neurotrophin-3 can direct the differentiation of non-neural stem cells from human adult bone marrow stroma to neuron-like cells in vitro. Supplementing the aforementioned multi-step process with sonic hedgehog, fibroblast growth factor 8, and retinoic acid increased the expression of molecules involved in dopaminergic differentiation and of tyrosine hydroxylase, the rate limiting enzyme of dopamine synthesis. MIAMI cells from young and old individuals represent autologous human cell populations for the treatment of disorders of the skeletal and nervous systems and for applications in cell therapy and reparative medicine approaches.

GABAA receptor subunit mRNA expression in cultured embryonic and adult human dorsal root ganglion neurons.

Previous studies have demonstrated significant pharmacological differences between the GABA(A) receptors expressed by neurons cultured from embryonic and adult human dorsal root ganglia (DRG). GABA(A) receptors of both embryonic and adult neurons are potentiated by diazepam and low concentrations of pentobarbital, and are activated by high concentrations of pentobarbital. However, in contrast to the GABA responses of embryonic neurons, the GABA responses of adult neurons are insensitive to both bicuculline and picrotoxin. We performed RT-PCR using subunit specific primer pairs, followed by Southern blot analysis with a third specific primer, to determine the pattern of subunit mRNA expression in cultures of embryonic and adult human DRG neurons. alpha2 and beta3 mRNA were expressed in all embryonic and adult cultures, while beta2 mRNA was present in all adult cultures but none of the embryonic cultures. Transcripts expressed by at least half of both embryonic and adult cultures were alpha3, alpha5, gamma2S, gamma3, theta, and rho1. Transcripts for gamma1 and delta were expressed in most adult cultures, but only a single embryonic culture. alpha4 mRNA was expressed by a single embryonic culture and pi mRNA was expressed by a single adult culture. We found no evidence for expression of alpha1, alpha6, beta1, gamma2L or rho2 transcripts. Changes in receptor subunit composition may underlie the novel pharmacological properties of GABA(A) receptor responses in adult cells. However, post-translational modification of a known subunit or the expression of a novel subunit may also contribute to the unique pharmacology of these neurons.