To improve our ability to identify hematopoietic stem cells (HSCs) and their localization in vivo, we compared the gene expression profiles of highly purified HSCs and non-self-renewing multipotent hematopoietic progenitors (MPPs). Cell surface receptors of the SLAM family, including CD150, CD244, and CD48, were differentially expressed among functionally distinct progenitors. HSCs were highly purified as CD150(+)CD244(-)CD48(-) cells while MPPs were CD244(+)CD150(-)CD48(-) and most restricted progenitors were CD48(+)CD244(+)CD150(-). The primitiveness of hematopoietic progenitors could thus be predicted based on the combination of SLAM family members they expressed. This is the first family of receptors whose combinatorial expression precisely distinguishes stem and progenitor cells. The ability to purify HSCs based on a simple combination of SLAM receptors allowed us to identify HSCs in tissue sections. Many HSCs were associated with sinusoidal endothelium in spleen and bone marrow, though some HSCs were associated with endosteum. HSCs thus occupy multiple niches, including sinusoidal endothelium in diverse tissues.

Stem cells persist throughout life by self-renewing in numerous tissues including the central and peripheral nervous systems. This raises the issue of whether there is a conserved mechanism to effect self-renewing divisions. Deficiency in the polycomb family transcriptional repressor Bmi-1 leads to progressive postnatal growth retardation and neurological defects. Here we show that Bmi-1 is required for the self-renewal of stem cells in the peripheral and central nervous systems but not for their survival or differentiation. The reduced self-renewal of Bmi-1-deficient neural stem cells leads to their postnatal depletion. In the absence of Bmi-1, the cyclin-dependent kinase inhibitor gene p16Ink4a is upregulated in neural stem cells, reducing the rate of proliferation. p16Ink4a deficiency partially reverses the self-renewal defect in Bmi-1-/- neural stem cells. This conserved requirement for Bmi-1 to promote self-renewal and to repress p16Ink4a expression suggests that a common mechanism regulates the self-renewal and postnatal persistence of diverse types of stem cell. Restricted neural progenitors from the gut and forebrain proliferate normally in the absence of Bmi-1. Thus, Bmi-1 dependence distinguishes stem cell self-renewal from restricted progenitor proliferation in these tissues.

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.

The c-ret proto-oncogene encodes a transmembrane tyrosine kinase that contains a cadherin-like structure in the extracellular domain (9,10,19,22,23). Its expression was detected at high levels in the peripheral nervous systems such as the enteric and autonomic nervous systems as well as in the excretory system during embryogenesis (1a, 15, 25). In addition, it is expressed preferentially in human tumors such as neuroblastoma, pheochromocytoma, and thyroid medullary carcinoma (8,18,24). Since the peripheral nervous systems and tumors mentioned above derive from neural crest cells, the physiological function of the c-ret proto-oncogene appears related to their normal growth and differentiation.Recent studies revealed that germ line mutations in the c-ret proto-oncogene are associated with the development of four different neural crest disorders (neurocristopathies): multiple endocrine neoplasia (MEN) 2A and 2B, familial medullary thyroid carcinoma, and Hirschsprung's disease (2,4,5,7,13,16). MEN 2A and MEN 2B are autosomal dominant cancer syndromes characterized by the development of medullary thyroid carcinoma and pheochromocytoma. MEN 2B is distinguished from MEN 2A by a more complex phenotype including mucosal neuroma, hyperganglionosis of the gastrointestinal tract, and marfanoid habitus. MEN 2A and familial medullary thyroid carcinoma mutations always involve cysteine residues present in the extracellular domain of the c-ret proto-oncogene (4, 12, 13). These cysteine residues are conserved in both human and mouse c-ret proto-oncogenes, suggesting that they are important for normal conformation of the c-Ret protein (9,22,23). On the other hand, a single point mutation in exon 16 of the tyrosine kinase domain has been found in 95% of patients with MEN 2B (6). This difference of the mutation sites may account for different phenotypes of MEN 2A and MEN 2B. Alternatively, it is possible that the diverse phenotypes observed in MEN 2A and MEN 2B are due to mutations in other modifier genes.Hirschsprung's disease is a developmental disorder of the enteric nervous system, inherited in an autosomal dominant manner with incomplete penetrance and variant expressivity. Several mutations have been found in different domains of the c-ret proto-oncogene, including the extracellular and tyrosine kinase domains (5, 16). Since mice homozygous for c-ret disruption showed phenotypes similar to Hirschsprung's disease (20), it is likely that the abnormalities observed in Hirschsprung's disease are caused by inactivation of the c-Ret function. On the other hand, MEN 2A and MEN 2B mutations might represent gain-of-function mutations.To elucidate the mechanism of development of MEN 2A syndrome, we introduced MEN 2A mutations in the extracellular domain of the c-ret proto-oncogene and analyzed their functions. Biochemical analysis of the Ret protein with MEN 2A mutations indicated that it is activated by ligand-independent dimerization on the cell surface. In addition, we showed that a mutation in a putative Ca 2ϩ -binding site of the ...

Glial cell line derived neurotrophic factor (GDNF) signals through a multicomponent receptor complex consisting of RET receptor tyrosine kinase and a member of GDNF family receptor a (GFRa). Recently, it was shown that tyrosine 1062 in RET represents a binding site for SHC adaptor proteins and is crucial for both RAS/mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3-K)/AKT signaling pathways. In the present study, we characterized how these two pathways diverge from tyrosine 1062, using human neuroblastoma and primitive neuroectodermal tumor cell lines expressing RET at high levels. In response to GDNF stimulation, SHC bound to GAB1 and GRB2 adaptor proteins as well as RET, and SHC and GAB1 were highly phosphorylated on tyrosine. The complex formation consisting of SHC, GAB1 and GRB2 was almost abolished by replacement of tyrosine 1062 in RET with phenylalanine. Tyrosine-phosphorylated GAB1 was also associated with p85 subunit of PI3-K, resulting in PI3-K and AKT activation, whereas SHC-GRB2-SOS complex was responsible for the RAS/ERK signaling pathway. These results suggested that the RAS and PI3-K pathways activated by GDNF bifurcate mainly through SHC bound to tyrosine 1062 in RET. Furthermore, using luciferase reporter-gene assays, we found that the RAS/ERK and PI3-K signaling pathways are important for activation of CREB and NF-kB in GDNF-treated cells, respectively. Oncogene (2000) 19, 4469 ± 4475.

Genes associated with Hirschsprung disease, a failure to form enteric ganglia in the hindgut, were highly up-regulated in gut neural crest stem cells relative to whole-fetus RNA. One of these genes, the glial cell line-derived neurotrophic factor (GDNF) receptor Ret, was necessary for neural crest stem cell migration in the gut. GDNF promoted the migration of neural crest stem cells in culture but did not affect their survival or proliferation. Gene expression profiling, combined with reverse genetics and analyses of stem cell function, suggests that Hirschsprung disease is caused by defects in neural crest stem cell function.Although stem cell properties have been characterized in many tissues (1), we are only beginning to understand how stem cell function is regulated at the molecular level. Gene expression profiles have been described for uncultured hematopoietic stem cells and cultured central nervous system neurospheres (2-8), but not for prospectively identified, uncultured neural stem cells. Because stem cell properties change in culture (9-11), the gene expression profile of uncultured neural stem cells might better reflect their properties in vivo.Molecular links between stem cell function and disease are of particular interest. Many diseases involve defects in neural development and may be caused by mutations that impair neural stem cell function. One potential example is Hirschsprung disease, a relatively common (1 in 5000 births) gut motility defect caused by a failure to form enteric nervous system ganglia in the hindgut. This can lead to fatal distention of the gut (megacolon). Although a number of the mutations that cause Hirschsprung disease have been identified (12), the ways in which these mutations affect neural development have been controversial, and it is unknown whether they affect gut neural crest stem cell (NCSC) function.Gut NCSCs are self-renewing and multipotent, give rise to diverse types of neurons and glia in vivo, and persist in the gut throughout adult life (13-15). Uncultured gut NCSCs can be isolated by flow cytometry by selecting freshly dissociated fetal gut cells that express the highest levels of p75 (the neurotrophin receptor) and α 4 integrin (14). These p75 + α 4 + cells represent only 1 to 2% of cells in the E14.5 (embryonic day 14.5) rat gut (14). Of the single p75 + α 4 + cells that were added to culture, 60 ± 9% survived to form colonies, and 80 ± 7% of these colonies contained neurons (peripherin), glia [glial fibrillary acidic protein (GFAP)], and myofibroblasts [smooth muscle actin (SMA)]. These colonies typically contained 1 × 10 5 to 2 × 10 5 cells after 14 days of culture. These colonies are characteristic of NCSCs (13,14,16,17). †To whom correspondence should be addressed. Email: seanjm@umich.edu. * These authors contributed equally to this work. We compared the gene expression profiles of gut NCSCs and whole-fetus RNA using oligonucleotide arrays (26,379 probe sets). Three independent 10,000-cell aliquots of freshly isolated, uncultured gut NCSCs were...

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.

Using our diagnostic criteria, we could recruit relatively many patients with similar characteristics to those of idiopathic PPFE patients in the literature. The possibility of clinical diagnosis of idiopathic PPFE should be further discussed.