05-446 Sigma-AldrichAnti-p75NTR (Neurotrophin Receptor) Antibody, clone ME20.4

The ability to generate lung and airway epithelial cells from human pluripotent stem cells (hPSCs) would have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. We have established, based on developmental paradigms, a highly efficient method for directed differentiation of hPSCs into lung and airway epithelial cells. Long-term differentiation of hPSCs in vivo and in vitro yielded basal, goblet, Clara, ciliated, type I and type II alveolar epithelial cells. The type II alveolar epithelial cells were capable of surfactant protein-B uptake and stimulated surfactant release, providing evidence of specific function. Inhibiting or removing retinoic acid, Wnt and BMP-agonists to signaling pathways critical for early lung development in the mouse-recapitulated defects in corresponding genetic mouse knockouts. As this protocol generates most cell types of the respiratory system, it may be useful for deriving patient-specific therapeutic cells.

The ability to regulate neurogenesis in the adult dentate gyrus will require further identification and characterization of the receptors regulating this process. In vitro and in vivo studies have demonstrated that neurotrophins and the p75 neurotrophin receptor (p75NTR) can promote neurogenesis; therefore we tested the hypothesis that p75NTR is expressed by adult dentate gyrus progenitor cells and is required for their proliferation and differentiation.In a first series of studies focusing on proliferation, mice received a single BrdU injection and were sacrificed 2, 10 and 48 hours later. Proliferating, BrdU-positive cells were found to express p75NTR. In a second series of studies, BrdU was administered by six daily injections and mice were sacrificed 1 day later. Dentate gyrus sections demonstrated a large proportion of BrdU/p75NTR co-expressing cells expressing either the NeuN neuronal or GFAP glial marker, indicating that p75NTR expression persists at least until early stages of maturation. In p75NTR (-/-) mice, there was a 59% decrease in the number of BrdU-positive cells, with decreases in the number of BrdU cells co-labeled with NeuN, GFAP or neither marker of 35%, 60% and 64%, respectively.These findings demonstrate that p75NTR is expressed by adult dentate progenitor cells and point to p75NTR as an important receptor promoting the proliferation and/or early maturation of not only neural, but also glial and other cell types.

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague-Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons.

The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreER(T2) transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay.

In neuroblastoma, high levels of mRNA for p140trkA and p75LNGFR neurotrophin receptors are predictive of favorable outcome. Their evaluation by Northern blot, however, requires substantial amounts of tissue and this prevents their routine evaluation as well as the possibility for multicenter studies to be easily carried out. In an attempt to overcome these limitations, the feasibility and reliability of determining both neurotrophin receptors on cryostat sections by immunohistochemistry were assessed, and these findings were compared to those obtained from Northern blot analysis. Primary tumor samples from 28 untreated patients at all stages were evaluated by using H10 anti-p140trkA and ME20.4 anti-p75LNGFR mAbs. Although weak, positive immunostaining was found in 9 of 28 tumors for p140trkA and in 5 of 28 tumors for p75LNGFR. As compared to Northern blot, the concordance rate was 79% (22 of 28 cases) for p140trkA (p < 0.05) and 71% (20 of 28 cases) for p75LNGFR (p < 0.05). No case negative for Northern blot was found to be positive with immunohistochemistry. Since only high mRNA levels for both receptors have been shown to be clinically relevant, their immunohistochemical detection, although less sensitive than Northern blot, can be just as sufficient and reliable as a prognostic tool, and possibly with a better cost-benefit ratio.

The localization of low-affinity nerve growth factor receptor (LNGFR) in the human urinary bladder was examined immunohistochemically using the mouse monoclonal antibody (ME20-4) against human LNGFR. LNGFR immunoreactivity was present in the human urinary bladder. The distribution of LNGFR-positive fibers was more abundant in the mucosa than in the muscle layer. Results also showed that some LNGFR-positive fiber bundles contained tyrosine hydroxylase immunoreactivity. Electron microscopic examination revealed that LNGFR immunoreactivity was located on the surface of Schwann cells, and frequently on the interface of axons and Schwann cells.

Human bone marrow stromal cells have been examined with an immuno-electron microscopy technique in order to better define their structure and function in normal hematopoiesis. Bone marrow fragments from normal donors, after mild permeabilization and glutaraldehyde prefixation were labeled with the Me20.4 Mab, which recognizes the low affinity nerve growth factor (NGFR) and was recently described as specifically identifying fibroblastic-like bone marrow stromal cells. Five nm gold immuno-conjugates served as markers. NGFR+ cells were showing either a star-shape, with long and convoluted dendritic projections, and branching with each other to form a complex system of lacunae upon which hematopoietic cells were arranged. Other NGFR+ cells had an elongated spindle-like morphology. NGFR+ dendrites were seen in close contact with each other and with the different hematopoietic cells, although definite junctions were never noticed. NGFR+ dendrites were also observed surrounding mature plasma cells, in close apposition with adipocytes or surrounding bone marrow sinusoids. These findings may give some clues about the function of the bone marrow stromal cells, which are known to be involved in the homing and recirculation of hemopoietic cells; in addition, the presence and distribution of NGFR in the bone marrow stroma may support the recent evidence of a co-stimulatory effect of NGF in early hematopoiesis.