PMC

hMNP-transplanted animals performed significantly better than controls on the balance beam, demonstrating fewer foot faults, a greater average fault distance, and an earlier onset of functional recovery as determined by repeated measures ANOVA (p<0.05); * denotes Student's t-test p<0.05.

Human nuclear antigen-positive cells (a) double stained with Isl-1 (b; red), p75 (c; p75 in red, human nuclei in green), or ChAT (d; ChAT in blue, human nuclei in brown), consistent with a MN lineage of mixed maturation state. (e) Some human cells in the ventral horns were surrounded by synaptophysin positive processes, suggesting integration with host tissue (synaptophysin in red, human nuclei in green). (f) Many human cells extended Tuj1 positive processes, and in some animals, ectopic motor tracts were present in the dorsal and ventral white matter (Tuj1 in red, human nuclei in green). Bar = 100 µm for (a) and (b), 50 µm for (c) and (e), 10 µm for (d), 200 µm for (f).

(a) Electrophysiological activity was assessed in MNs current clamped in the whole cell configuration. (b) Injection of 20 pA of current elicited action potential trains, typical of mature MNs. The presence of glutamate receptors was evidenced using symmetrical solutions. (c) At 0 mV, the addition of glutamate mediated a small outward current, likely due to the presence of KOH in the internal solution. (d) At −70 mV, the addition of glutamate mediated a large inward current, as K⁺ and Ca²⁺ ions flowed through open glutamate receptors. Bar = 33 µm.

(a) At day 7 of the 28 day protocol, cultures contained yellow solid core neurospheres in the absence of single floating cells. (b) At day 25, morphologically homogeneous cells exhibited processes, and (c) expressed the MN lineage marker Olig 1/2 (red), and (d) Tuj1 (green); GFAP+ astrocytes (red) were rare within these Tuj1+ cultures. Nuclear counterstain for (c) and (d) is blue. (e) At day 28, the majority of Tuj1+ cells (green) double stained for the MN lineage marker HB9 (red). (f) After 3 weeks of subsequent growth, cells displayed a mature, branched morphology consistent with mature MNs, and expressed the mature MN markers (g) SMI-32 and (h) ChAT. Bar = 1000 µm for (a), 200 µm for (b) and (g), 100 µm for (c) and (d), 50 µm for (e), 33 µm for (f), and 150 µm for (h).

(a) Qualitative PCR analyses indicated that hMNPs express NT-3, NT-4, NGF, and VEGF. Neurite length was 58% longer (b) in cortical neuron cultures exposed to hMNP-CM for 7 days (c) as compared to cortical neuron cultures exposed to MN differentiation media (d). The neurofilament optical density was 45% greater (e) in the axonal chamber of microfluidic culture platforms in axotomized cortical neuron cultures exposed to hMNP-CM for 7 days (f) as compared to axotomized cortical neuron cultures exposed to MN differentiation media (g). (h) Neurite length was significantly attenuated in cortical neuron cultures exposed to hMNP-CM that contained function-blocking antibodies to MN growth factors. Immunofluorescent staining for MAP-2 in cultures exposed to control media (i) or hMNP-CM (j), in the presence of LPS. (k) Quantification of the number of MAP-2 positive neurons in the presence of control media or hMNP-CM, with and without LPS exposure. The number of neurons in cultures lacking LPS was not significantly different (p<0.05) in cultures exposed to control media or MNP CM, however, the number of neurons in cultures with LPS was significantly higher (p<0.05) in cultures exposed to hMNP-CM as compared to those exposed to control media. Bar = 50 µm.

(a) hMNP transplantation enhanced sprouting of endogenous serotonergic (5-HT) projections. hMNP-transplanted animals consistently contained aberrant projections throughout the dorsal gray matter (top panels, arrows) and dense innervation of the ventral horns (bottom panels) at 2 mm cranial to the injury site (b) At 2 mm and 3 mm cranial to the injury epicenter, and 1 mm caudal to the injury epicenter, 5-HT immunoreactivity was significantly greater than that observed in control animals. At 1 mm cranial to the injury epicenter, and 2 mm and 3 mm caudal to the injury epicenter, 5-HT immunoreactivity was not significantly different than in control animals. (c) NeuN immunostaining demonstrated that hMNP transplantation enhanced survival of endogenous (human nuclear antigen-negative) neurons 2 mm cranial to the injury site. (d) Quantification of enhanced neuronal survival in hMNP-transplanted animals cranial and caudal to the injury site. (e) hMNP transplantation attenuated phosphorylation of stress-associated protein kinase (SAPK). (f) Densitometric quantification of SAPK normalized to actin controls showed that 1 and 4 days following transplantation, phosphorylation of SAPK decreased in hMNP-transplanted animals relative to controls; no significant differences were observed between groups at 7 and 10 days. Bar = 200 µm for (a), 100 µm for (c).