Multipotent Progenitors Persist Postnatally in the Gut
We have so far been unable to identify any multipotent progenitors from the postnatal sciatic nerve (data not shown), but we have found multipotent progenitors in the postnatal gut. Postnatal day (P) 5 to P15 rat gut was dissociated into single cell suspensions and plated in culture at clonal density. Clonal density allowed individual founder cells to form spatially distinct colonies so that the developmental potential of the founder cells could be assessed based on colony composition. After 14 days in culture, we consistently observed the formation of multilineage colonies containing neurons, glia, and myofibroblasts () that resembled colonies formed by embryonic NCSCs (Stemple and Anderson, 1992
; Morrison et al., 1999
; Bixby et al., 2002
), though they tended to be somewhat smaller. Based on hemocytometer counts of trypsinized cells, multilineage colonies from E14.5 and P15 progenitors averaged 184,000 ± 69,000 and 82,000 ± 17,000 cells per colony, respectively.
Multipotent Neural Progenitors Persist in the Postnatal Gut
To begin to assess the localization of these multipotent progenitors in vivo, we stripped the plexus/outer muscle layers from the gut epithelium and dissociated these tissues separately. We found that multipotent progenitors were consistently cultured from the plexus/outer muscle layers but never from the gut epithelium. This indicated that these multipotent progenitors were localized in vivo to the submucosal plexus, myenteric plexus, or outer muscle layers of the gut. All subsequent experiments on postnatal gut progenitors were performed using cells dissociated from the plexus/outer muscle layers.
Multipotent progenitors were infrequent among the unfractionated cells obtained from the preparations dissociated P15 plexus/outer muscle layers. Only 0.7% of dissociated (but unfractionated) P15 cells survived and formed multilineage colonies in culture. Since p75 has consistently been observed to mark NCSCs in the gut and other tissues (Stemple and Anderson, 1992
; Morrison et al., 1999
; Lo and Anderson, 1995
; Bixby al., 2002
), we tested whether these multipotent progenitors were enriched within the p75+
fraction of cells. As in the E14.5 gut, we found that multipotent progenitors were enriched among cells with high levels of p75 expression (see for a flow-cytometric profile p75 expression). Although p75−
cells formed no multilineage colonies and p75med
cells gave rise few multilineage colonies, p75+
cells were highly enriched for multipotent progenitors. An average of fifty-two percent of p75+
cells survived to form colonies culture, and 66% of these colonies were multilineage (). Interestingly, although E14.5 gut NCSCs are p75+
(Bixby et al., 2002
), shows that p75+
cells were largely negative for α4
integrin by P15. By staining sections of P15 gut with an antibody against p75, we found that p75+
cells localized to the myenteric and submucosal plexi (see Supplemental Figure S1
online at http://www.neuron.org/cgi/content/full/35/4/657/DC1
Multipotent Neural Crest Progenitors Can Be Isolated as p75+ Cells from the Postnatal and Adult Gut
Prospective Identification and Isolation of Postnatal Gut NCSCs by Flow-Cytometry
By isolating the 1%–2% of cells that expressed the highest levels of p75 from the plexus/outer muscle layers of the gut, we found that we could isolate highly enriched populations of NCSCs from E14.5 through the oldest rat we examined at P110 (). Sixty to seventy percent of colonies formed by p75+ cells from P5 to P22 gut contained neurons, glia, and myofibroblasts (N + G + M; ). Additional colonies contained neurons and glia, but not myofibroblasts (N + G). It is uncertain whether these N + G colonies represent restricted progenitors or whether some multipotent progenitors sometimes failed to make myofibroblasts under these culture conditions. Only 0.2% of unfractionated cells from the P22 plexus/outer muscle layer preparations survived and formed multilineage colonies in culture (data not shown). Thus, taking into account plating efficiencies, multipotent progenitors were more than 150-fold enriched within the p75+ fraction of P22 gut cells relative to unfractionated cells.
Multipotent progenitors continued to be present within the adult (250 g rats; P65–P110) rat gut (). These multipotent progenitors were still highly enriched in the p75+ fraction of cells and largely negative for α4 integrin. However, the purity of multipotent progenitors among adult p75+ cells was much lower than in the P22 gut, with only 9% of cells surviving to form colonies in culture and 17% of colonies being multilineage. Additional work will be required to improve our ability to purify adult gut multipotent progenitors by flow-cytometry.
Multipotent Neural Crest Progenitors Are Present in Adult Gut
Although we did not detect neuron only colonies from any of the postnatal gut p75+ cells after 14 days of culture (), we wanted to be sure that this population was not substantially contaminated by committed neuronal progenitors or by immature neurons. To this end, we sorted P15 p75+ cells into culture and examined the colonies 4 days later. 2.3% of colonies contained a single neuron, and 0.6% of colonies contained two neurons. No neuron only colonies contained more than two neurons. We also sorted P15 p75+ cells into culture and stained these cells 17 hr later for the early neuronal marker neuron-specific (class III) β-tubulin (TuJ1 antibody). On average, 3.9% ± 5% of P15 gut p75+ cells expressed β-tubulin. This suggests that around 4% of P15 p75+ cells are immature neurons or committed neuronal progenitors with the ability to divide once in culture.
Postnatal Gut Multipotent Neural Crest Progenitors Self-Renew in Culture
The self-renewal capacity of the gut multipotent progenitors was assayed by depositing single p75+
cells into individual wells of 96-well plates by flow-cytometry and then culturing for 8 days under standard conditions (Morrison et al., 1999
). Multipotent colonies were then trypsinized and subcloned into secondary cultures at clonal density as previously described (Morrison et al., 1999
). Almost all multipotent primary colonies gave rise to multipotent daughter colonies (20/20 at E14.5, 11/11 at P5, 16/18 at P15, and 6/6 at P22). Since the multipotent progenitors from postnatal gut were p75+
, formed colonies similar to embryonic NCSC colonies, appeared to localize to the myenteric or submucosal plexi, and had the capacity to self-renew, we conclude that they are postnatal NCSCs.
Although the postnatal gut NCSCs consistently self-renewed in culture, the extent of self-renewal quantitatively declined with increasing age. Each E14.5 gut NCSC produced an average of 730 multipotent daughter colonies (845 total daughter colonies) subclonable after 8 days in culture, while P22 gut NCSCs produced an average of only 70 multipotent daughter colonies (360 total daughter colonies) under the same conditions (p = 0.002; ). NCSC self-renewal did not decline uniformly over time as some NCSCs at P15 and P22 self-renewed at a rate comparable to what was observed among E14.5 NCSCs. For example, one P15 NCSC gave rise to 370 multipotent daughter cells, while a P22 NCSC gave rise to 335 multipotent daughter cells.
Postnatal Gut NCSCs Self-Renew in Culture But the Extent of Self-Renewal Declines Significantly with Increasing Age
Because of the impurity of multipotent progenitors in the adult p75+ population, we cultured single adult gut cells for 15 days prior to subcloning in an attempt to distinguish multipotent colonies from restricted colonies based on size. Some of the large adult colonies gave rise to multipotent daughter cells while others did not. It is not yet clear whether this heterogeneity reflects the subcloning of restricted progenitor colonies or whether some multipotent adult progenitors may fail to detectably self-renew in this assay. Nonetheless, at least some of the adult multipotent progenitors self-renewed, producing an average of 35 multipotent subclones per founder cell ().
The 8–15 day culture assay represents one way of quantitating self-renewal potential, but it does not estimate the maximum self-renewal capacity of NCSCs. By culturing the postnatal gut NCSCs for longer periods of time, they give rise to larger numbers of multipotent daughter cells.
Postnatal Gut NCSCs Are Less Mitotically Active than E14 Gut NCSCs
In the hematopoietic system and the adult CNS, stem cells go from being highly mitotic during fetal development (Morrison et al., 1995a
; Cai et al., 1997
) to being relatively quiescent in adults (Morshead et al., 1994
; Cheshier et al., 1999
; Johansson et al., 1999
). Since this is the first identification of NCSCs in the postnatal PNS, we used our ability to prospectively identify the gut NCSCs to ask whether their cell-cycle distribution changed perinatally. The cell-cycle distribution of uncultured NCSCs was assayed at isolation by flow-cytometry, using Hoechst 33342 staining to determine the DNA content of individual cells (). E14.5 gut p75+
NCSCs included 37% ± 7% of cells in S/G2/M phases of the cell cycle (>2N DNA content), as compared to only 26% ± 2% of P5 p75+
cells (data not shown) and 10% ± 0.5% of P15 p75± cells (; p < 0.01). When BrdU was administered for a 20 hr period in vivo prior to isolation of NCSCs, 87% ± 7% of E14.5 gut p75+
cells incorporated BrdU while only 13.1% ± 2.5% of P15 gut p75+
cells incorporated BrdU. Thus, while nearly all E14.5 gut NCSCs divided at least once in a 20 hr period in vivo, only 13% of P15 gut p75+
cells divided in the same period. Either P15 gut NCSCs have a much longer cell-cycle time than E14.5 gut NCSCs, or a significant percentage of gut NCSCs are quiescent at P15.
Postnatal Gut NCSCs Are Significantly Less Mitotically Active Than Embryonic Gut NCSCs
NCSCs from the P15 Gut Differentiate into Neurons and Glia In Vivo
To test the ability of the P15 gut NCSCs to generate neurons and glia in vivo, freshly isolated, uncultured p75+
cells were injected into two hindlimb bud somites of eight stage 17–18 chick embryos (). After 72 hr, embryos were fixed, sectioned, and processed for in situ hybridization using probes against rat SCG10
to identify neurons (Anderson and Axel, 1985
) and rat P0
to identify glia (Lemke et al., 1988
). Chick neurons were identified by hybridizing with a chick SCG10
probe in some sections. Of eight chicks analyzed, four showed engraftment with both neurons and glia, two showed engraftment of glia only, and two were not detectably engrafted. The failure of all eight chicks to detectably engraft with both neurons and glia may be due to differences between postnatal gut NCSCs and embryonic gut NCSCs or the relatively small numbers of cells injected (~310/somite; see Experimental Procedures). Neuronal engraftment was found in sympathetic ganglia (2.2 cells/positive section; ), and glia were present in peripheral nerves (17.5 cells/positive section; ). Thus, uncultured postnatal gut NCSCs migrated to embryonic neural crest structures and formed neurons and glia.
P15 Gut NCSCs Give Rise to Neurons and Glia In Vivo
Postnatal Gut NCSCs Differentiate into Neurons that Express a Variety of Neurotransmitters
We performed a clonal analysis of gut NCSCs to test whether they generate cells expressing a variety of neurotransmitters. We stained NCSC colonies that had been cultured for 14 days at clonal density using commercially available antibodies against Vasoactive Intestinal Peptide (VIP), Neuropeptide Y (NPY), and neuronal Nitric Oxide Synthase (nNOS) (). Each of these antibodies specifically stained neurons based on analyses of adult rat gut sections and multilineage colonies in culture that were double labeled with these antibodies and antibodies against neuronal markers (data not shown). VIP, NPY, and Nitric Oxide are all expressed by subsets of enteric neurons in vivo (Pham et al., 1991
; Jarvinen et al., 1999
). Nearly all E14.5, P15, and adult gut NCSC colonies contained neurons expressing VIP, NPY, and nNOS (). Based on these experiments, adult gut NCSCs retain the ability to generate neurons that express a variety of neurotransmitters normally found in the ENS.
p75+ NCSCs Give Rise to Neurons in Culture that Express a Variety of Neurotransmitters Normally Found in the Enteric Nervous System
Gut NCSCs Undergo Developmental Restrictions in Neuronal Subtype Potential
Neurons with different neurotransmitter phenotypes are born at different intervals of gut development (Pham et al., 1991
). Serotonergic progenitors last divide between E8 and E14.5 in mouse, while progenitors of NPY-expressing neurons last divide between E10 and P7. Since serotonergic differentiation is completed earliest during embryonic gut development, we examined whether gut NCSCs undergo restrictions between embryonic and postnatal stages in their potential to generate serotonergic neurons in culture.
Serotonergic neurons in the ENS can be identified by their expression of Tryptophan Hydroxylase, the initial and rate-limiting enzyme in the serotonin synthesis pathway (Gershon et al., 1977
). We cultured E14.5 p75+
cells or P5 or P15 p75+
cells at clonal density for 14 days in standard medium and then stained with an antibody specific for Tryptophan Hydroxylase (Belin et al., 1991
). Tryptophan Hydroxylase+ neurons were present at low density in most multilineage colonies formed by E14.5 gut NCSCs (66% ± 3%; ) but only in a few multilineage colonies formed by P5 gut NCSCs (9% ± 5%; ). No P15 progenitors formed Tryptophan Hydroxylase+ neurons in culture (0 ± 0; ). Between E14.5 and P15, NCSCs lost the neuability to make serotonergic neurons based on this in vitro assay.
E14.5, But Not P15, Gut NCSCs Generate Serotonergic Neurons in Culture
In addition to examining the ability of postnatal gut NCSCs to make subtypes of neurons that normally exist in the adult gut, we also wanted to examine their ability to make subtypes of neurons that differentiate only in other regions of the PNS. To this end, we examined the ability of gut NCSCs to make noradrenergic neurons. Although some gut neural crest progenitors transiently express a noradrenergic phenotype prior to E15 (Tyrosine Hydroxylase+ [TH+] and Dopamine-β-hydroxylase + [DβH+]), these cells continue to proliferate and TH can no longer be detected in the gut after E15 (Baetge et al., 1990
NCSCs were isolated from rats ranging in age from E14.5 to P22 and cultured under conditions that promote noradrenergic differentiation (Morrison et al., 2000a
). We double labeled these cultures with antibodies against Peripherin (to identify neurons) and either Tyrosine Hydroxylase (TH) or Dopamine-β-hydroxylase (DβH). All TH+
cells coexpressed Peripherin, but only a minority of Peripherin-positive neurons expressed TH or DβH (). Double labeling experiments with antibodies against TH and DβH demonstrated that all of the TH+
cells examined coexpressed DβH, but DβH+
cells often did not express TH. The percentage of DβH positive cells also expressing TH declined with age. At E14.5, an average of 51.7% ± 14.5% of DβH positive cells also expressed TH but at P15, only 23.4% ± 21.4% of DBH+
cells did (p < 0.05). This is consistent with the fact that gut neural crest progenitors can give rise to both noradrenergic neurons (TH+
) as well as DβH+
neurons that are peptidergic, but not catecholaminergic (Baetge et al., 1990
Postnatal Gut NCSCs Decline in Their Ability to Form Tyrosine Hydroxylase-Expressing Neurons with Increasing Age
Nearly all E14.5 gut NCSC colonies contained neurons that expressed TH (87% ± 3%) and/or DβH (85% ± 12%), but the percentage of gut NCSCs that formed such neurons declined with increasing stem cell age (). By P15, only 20% of NCSCs formed TH+
neuability rons, and 38% formed DβH+
neurons (both differences were statistically significant; p < 0.01). In addition to the reduction in the proportion of NCSCs that were able to form such cells, there was also a reduction in the number of such cells per colony (; Supplemental Figure S1
). While E14.5 gut NCSC colonies contained average of 444 ± 285 DβH+ cells and 307 ± 244 TH+ cells, P15 gut NCSC colonies averaged only 14 ± DβH+
cells and 2 ± 1 TH+
cells. This reduction in the number of noradrenegic neurons per colony cannot explained by a proportionate decline in the total number of neurons per colony as we estimated that E14.5 gut NCSC colonies averaged 41,000 ± 25,000 neurons/colony, while P15 gut NCSCs averaged 16,000 ± 13,000 neurons per colony. Thus the frequency of noradrenergic neurons declined much more precipitously than the total neurons per colony over this developmental interval.
The Ability of Gut NCSCs to Generate Sympathoadrenal Neurons Declines Significantly during Development
Enteric neural crest progenitors have been observed transiently express TH and DβH in early embryonic development (before E15) prior to differentiating. To be certain that these NCSC cultures did not contain proliferating TH or DβH-positive progenitors, we added BrdU for the final 24 hr of culture prior to immunohistochemical staining. Consistent with their neuronal morphology, double labeling experiments with TH and BrdU indicated that the vast majority of TH+ cells were not proliferative: while 22% of all cells in colonies containing TH+ cells were BrdU+, only 1.9% of the cells expressing TH had incorporated BrdU.
Reduced Neuronal Subtype Potential May Result from Decreased BMP Sensitivity
We investigated the mechanism by which postnatal NCSCs lose the potential to form serotonergic and noradrenergic neurons. BMPs are necessary and sufficient for the differentiation of noradrenergic neurons in vivo (Reissman et al., 1996
; Schneider et al., 1999
), and BMPs promote the differentiation of at least certain types of neurons from embryonic enteric progenitors in culture (Pisano et al., 2000
). BMPs promote Mash-1 expression in NCSCs, and both serotonergic and noradrenergic differentiation are Mash-1-dependent (Guillemot et al., 1993
; Blaugrund et al., 1996
). Therefore, a possible mechanism by which NCSCs might lose the ability to generate serotonergic and noradrenergic neurons is by losing their sensitivity to the neurogenic effects of BMPs.
To test this, E14.5 and P15 gut NCSCs were cultured at clonal density with BMP4 for 24 hr or 5 days and stained for Mash-1 or Peripherin, respectively. Although BMP4 significantly increased neuronal differentiation in colonies formed by E14.5 gut p75+α4+ cells, as judged by either Mash-1 or Peripherin expression, it did not promote neuronal differentiation based on morphology or Mash-1 or Peripherin expression in colonies formed by P15 gut p75+ cells (). To ensure that neurons were not dying from a lack of trophic factor support in the P15 cell cultures, we supplemented these cultures with GDNF, NT3, and NGF, but this did not increase the number of Peripherin+ neurons that arose in the presence of BMP4 (data not shown). This suggests that the mechanism by which P15 gut NCSCs lose the ability to make serotonergic and noradrenergic neurons in culture involves a loss of responsiveness to the neurogenic effects of BMPs.
Postnatal Gut NCSCs Are Not Responsive to the Neurogenic Effects of BMP4
Postnatal Gut NCSCs Are More Responsive to Gliogenic Factors Than Embryonic Gut NCSCs
Based on an analysis of rats that were administered BrdU from P14–P16, we detected many more newborn glial cells than neurons in the gut during this period (data not shown). This suggests that gliogenesis predominates in the P15 gut just as in the E14 sciatic nerve (Bixby et al., 2002
). We thus tested whether P15 gut NCSCs exhibit increased sensitivity to gliogenic factors relative to E14 gut NCSCs. shows that postnatal gut NCSCs became increasingly responsive to the gliogenic effects of soluble Notch ligand Delta-Fc with increasing time after birth. Although few or no glial only gliocolonies were detected from E14.5 gut NCSCs treated with soluble Delta, P5 and P15 gut NCSCs did generate significantly increased numbers of glial only colonies after treatment with soluble Delta. Treatment with soluble Delta increased the formation of glia only colonies by P15 NCSCs by over 40% while increasing plating efficiency by only 10%–13%. These results are consistent with Notch promoting gliogenesis through an instructive mechanism as we previously documented (Morrison et al., 2000b
). Postnatal gut NCSCs become responsive to the gliogenic effect of Notch activation in a way that is not observed among E14.5 gut NCSCs. P15 gut NCSCs are also more responsive to the gliogenic effects of Neuregulin than E14 gut NCSCs (data not shown).
Gut NCSCs Become Increasingly Responsive to the Gliogenic Effect of Soluble Delta as Development Proceeds
Temporal Changes in the Responsiveness of Gut NCSCs to Lineage Determination Factors Affect Cell Fate Determination In Vivo
P15 gut NCSCs are less responsive to the neurogenic factor BMP4 () but are more responsive to gliogenic factors () than E14.5 gut NCSCs. If temporal changes in the responsiveness of gut NCSCs to lineage determination factors affect cell-fate determination in vivo, then P15 gut NCSCs should give rise primarily to glia upon transplantation into developing chick nerves in contrast to E14.5 gut NCSCs, which gave rise primarily to neurons (Bixby et al., 2002
). To test this, we examined the peripheral nerves of chicks injected with uncultured P15 gut NCSCs (). Six of eight chicks exhibited glia in their peripheral nerves (17.5 cells/positive section) while one of eight chicks had a single neuron in a peripheral nerve. Thus P15 gut NCSCs are biased toward adopting a glial fate in developing peripheral nerves like E14.5 sciatic nerve NCSCs, but unlike E14.5 gut NCSCs. This demonstrates that NCSCs undergo perinatal changes in their responsiveness to lineage determination factors that affect cell fate determination in vivo.