Figure 1: Wild-type neural crest stem cells (NCSCs) engraft and form neurons as efficiently in the aganglionic region of endothelin receptor B (Ednrb)-deficient gut as in wild-type gut, demonstrating that the Ednrb-deficient hindgut is permissive for the survival and differentiation of neural progenitors. This suggests a possible new strategy for treating Hirschsprung disease, a birth defect that is sometimes caused by mutations in Ednrb and that is associated with a failure to form enteric ganglia in the hindgut. Ednrb is required to modulate the response of NCSCs to migratory cues; loss of Ednrb leads to a failure of these cells to migrate into the hindgut, despite the fact that normal numbers of NCSCs are maintained throughout development in the proximal gut. The migration defect can be bypassed by transplanting NCSCs into the aganglionic region of the Ednrb-deficient embryonic gut.
A: transplanted alkaline phosphatase–expressing neural crest cells (purple) that have engrafted in the hindgut wall of an Ednrb-deficient rat. B: transplanted alkaline phosphatase–expressing neural crest cells (green) that have engrafted and formed neurons (red) in the hindgut wall of an Ednrb-deficient rat. This work demonstrates the ability to gain new insights into the etiology of disease as well as the ability to identify new potential therapeutic approaches by studying the regulation of stem cell function.
From the Morrison lab. See also Iwashita, T., Kruger, G.M., Pardal, R., Kiel, M.J., and Morrison, S.J. 2003. Science 301:972–976; and Kruger, G.M., Mosher, J.T., Tsai, Y.H., Yeager, K.J., Iwashita, T., Gariepy, C.E., and Morrison, S.J. 2003. Neuron 40:917–929.
Figure 2: Bmi-1-deficient neural stem cells exhibit a reduced rate of proliferation. Cells within Bmi1–/– stem cell colonies divide at a reduced rate, leading to reduced colony size, a reduced rate of BrdU incorporation into cells, and reduced stem cell self-renewal. BrdU (black stain) is a thymidine analog that is incorporated into DNA when cells divide and therefore marks cells that have recently divided. Bmi-1 is necessary for adult stem cells from the hematopoietic and nervous systems to self-renew normally. In the absence of Bmi-1, these stem cells are formed in normal numbers, but begin expressing senescence pathways postnatally and do not persist into adulthood.
From Molofsky, A.V., Pardal, R., Iwashita, T., Park, I.K., Clarke, M.F., and Morrison, S.J. 2003. Nature 425:962–967. © 2003 Nature Publishing Group.
Figure 3: The identification of neural crest stem cells (NCSCs ) in the adult gut (enteric nervous system), with p75 (neurotrophin receptor) staining (blue) in a transverse section of the postnatal rat gut. NCSCs were isolated from the adult gut by flow-cytometric purification of the cells that expressed the highest levels of p75. In this section, the cells that stained brightly for p75 localized to the myenteric and submucosal plexi of the enteric nervous system. Prior to this work it was thought that NCSCs terminally differentiated during fetal development and did not persist in the adult peripheral nervous system.
Cover image, Neuron, August 15, 2002. © 2002, with permission from Elsevier. See also Kruger, G.M., Mosher, J.T., Bixby, S., Joseph, N., Iwashita, T., and Morrison, S.J. 2002. Neuron 35:657–669.
Figure 4: The soluble Notch ligand, Delta-Fc, causes glial lineage determination by neural crest stem cells within 1 day in culture. In this experiment we compared the ability of Delta-Fc to cause glial lineage determination relative to Neuregulin-1, another factor that promotes glial lineage determination. Cultures of purified neural crest stem cells (NCSCs) at clonal density were supplemented either with Delta-Fc (A–C) or with Neuregulin-1 plus the control Fc protein (D–F). After only 24 hours, the cultures were washed into standard medium supplemented with bone morphogenetic protein-2 (BMP2; 50 ng/ml) and grown for 4 more days to test neuronal potential. BMP2 instructs NCSCs to undergo neuronal differentiation. Thus this experiment tests whether transient exposure to either Delta-Fc or Neuregulin-1 causes glial lineage determination quickly enough to prevent NCSCs from undergoing neuronal differentiation in response to BMP2 during the subsequent 4-day culture period.
Panels A and D show phase-contrast images, panels B and E show bright-field images of staining with the neuronal marker peripherin, and panels C and F show superimposed epifluorescence images of staining with the glial marker glial fibrillary acidic protein (GFAP, red) and the myofibroblast marker smooth muscle actin (SMA, green, negative). The control colony preincubated in Neuregulin-1 plus Fc (D–F) contained mostly neurons and neuronal precursors induced by the BMP2 treatment, as judged by the peripherin staining and the lack of GFAP and SMA staining. In contrast, the colony preincubated in Delta-Fc (A–C) contained only glia (red) despite the BMP2 treatment, as indicated by the GFAP staining and the lack of peripherin and SMA staining. Thus Delta-Fc acted more rapidly than Neuregulin-1 to instruct gliogenesis.
From Morrison, S.J., Perez, S.E., Qiao, Z., Verdi, J.M., Hicks, C., Weinmaster, G., and Anderson, D.J. Cell 2000. 101:499–510. © 1999 from Elsevier Science.
Figure 5: Neural crest stem cells (NCSCs) were isolated by flow cytometry from the sciatic nerves of embryonic day 14 rat fetuses as cells that expressed the neurotrophin receptor p75 but failed to express the myelin protein P0. These cells were cultured at clonal density for 14 days, then stained with antibodies against neuronal (peripherin, black), glial (GFAP, red), and myofibroblast (smooth muscle actin, green) markers. Single purified NCSCs gave rise to multilineage colonies like this one, containing more than 100,000 cells after only 14 days of culture.
From Morrison, S.J., White, P.M., Zock, C., and Anderson, D.J. 1999. Cell 96:737–749. © 1999 from Elsevier Science.
