# Comparative Microarray Analysis of Proliferating and Differentiating Murine ENS Progenitor Cells

Authors: Peter Helmut Neckel; Roland Mohr; Ying Zhang; Bernhard Hirt; Lothar Just
Journal: Stem Cells International
 (2016)

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## Abstract

Postnatal neural progenitor cells of the enteric nervous system are a potential source for future cell replacement therapies of developmental dysplasia like Hirschsprung’s disease. However, little is known about the molecular mechanisms driving the homeostasis and differentiation of this cell pool. In this work, we conducted Affymetrix GeneChip experiments to identify differences in gene regulation between proliferation and early differentiation of enteric neural progenitors from neonatal mice. We detected a total of 1333 regulated genes that were linked to different groups of cellular mechanisms involved in cell cycle, apoptosis, neural proliferation, and differentiation. As expected, we found an augmented inhibition in the gene expression of cell cycle progression as well as an enhanced mRNA expression of neuronal and glial differentiation markers. We further found a marked inactivation of the canonical Wnt pathway after the induction of cellular differentiation. Taken together, these data demonstrate the various molecular mechanisms taking place during the proliferation and early differentiation of enteric neural progenitor cells.


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## Body

## 1. Introduction

The enteric nervous system (ENS) is a largely autonomous and highly complex neuronal network found in the gastrointestinal tract. Its two major plexuses are integrated into the layered anatomy of the gut wall and, together with central modulating influences, exert control over gastrointestinal motility, secretion, ion-homeostasis, and immunological mechanisms [1]. In order to achieve this variety of functions, the ENS is composed of a multitude of different neuronal and glial cell types and closely interacts with smooth muscle cells and myogenic pacemaker cells called interstitial cells of Cajal. Furthermore, a population of neural stem or progenitor cells in the ENS has been identified in rodents [2, 3] and humans that retain their proliferative capacity throughout adult life even into old age [4, 5]. It is therefore not surprising that the correct functioning of the ENS as well as the regulation on enteric neural progenitor cells is subjected to the influence of a myriad of transmitters, neurotrophic and growth factors, signalling molecules, and extracellular matrix components, which are not exclusively expressed by neural cell types [6]. Likewise, the control of the development of the ENS is equally complex and mutations in its genetic program can lead to fatal dysplasia like Hirschsprung’s disease (HCSR) [7, 8].HSCR is hallmarked by an aganglionic distal bowel leading to life-threatening disturbances in intestinal motility. Today’s therapeutic gold standard, the surgical resection of the affected gut segments, is nevertheless associated with problematic long-term outcomes with regard to continence [9]. In order to improve the therapeutic success, the use of autologous enteric neural stem cells was proposed [10]. This concept relies on thein vitro expansion of enteric neural stem cells derived from small biopsy materials. However, we are just beginning to understand the molecular mechanisms that underlie neural stem cell biology and how this knowledge can be used for optimizingin vitro culture conditions [11, 12].Genome-wide gene-expression analyses are a useful tool to examine the genetic programs and cellular interactions and have been widely used to identify potential markers or signalling mechanisms especially in CNS neurospheres or cancer tissues. Further, gene-expression assays have also helped to unravel genetic prepositions associated with HSCR [13, 14], though little effort has so far been put into characterizing the genetic profile of enteric neural stem cellsin vitro [15].Here, we used an Affymetrix microarray analysis to evaluate the genetic expression profile of proliferating murine enteric neural stem cells and its changes during the early differentiationin vitro.


## 2. Materials and Methods

### 2.1. Cell Culturing

Cell culturing was conducted as described previously [15]. The handling of animals was in accordance to the institutional guidelines of the University of Tuebingen, which conform to the international guidelines.Neonatal (P0) C57BL/6 mice without regard to sex were decapitated and the whole gut was removed. After removal of adherent mesentery the longitudinal and circular muscle layers containing myenteric plexus could be stripped as a whole from the small intestine. Tissue was chopped and incubated in collagenase type XI (750 U/mL; Sigma-Aldrich, Taufkirchen, Germany) and dispase II (250μg/mL; Roche Diagnostics, Mannheim, Germany) dissolved in Hanks’ balanced salt solution with Ca2+/Mg2+ (HBSS; PAA, Pasching, Austria) for 30 min at 37°C. During enzymatic dissociation the tissue was carefully triturated every 10 min with a fire polished 1 mL pipette tip. Prior to the first trituration step, cell suspension was treated with 0.05% (w/v) DNAse I (Sigma-Aldrich). After 30 min, tissue dissociation was stopped by adding fetal calf serum (FCS; PAA) to a final concentration of 10% (v/v) to the medium. Undigested larger tissue pieces were removed with a 40 μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Residual enzymes were removed during two washing steps in HBSS at 200 g. After dissociation, cells were resuspended in proliferation culture medium (Dulbecco’s modified Eagle’s medium with Ham’s F12 medium (DMEM/F12; 1 : 1; PAA)) containing N2 supplement (1 : 100; Invitrogen, Darmstadt, Germany), penicillin (100 U/mL; PAA), streptomycin (100 μg/mL; PAA), L-glutamine (2 mM; PAA), epidermal growth factor (EGF; 20 ng/mL; Sigma-Aldrich), and fibroblast growth factor (FGF; 20 ng/mL; Sigma-Aldrich). Cells were seeded into 6-well plates (BD Biosciences) in a concentration of 2.5 × 104 cells/cm2. Only once before seeding, the medium was supplemented with B27 (1 : 50; Invitrogen). EGF and FGF were added daily and culture medium was exchanged every 3 days. All cultivation steps were conducted in a humidified incubator at 37°C and 5% CO2. An overview of the following cell culture protocol is shown in Figure 1. During proliferation phase of the culture, cells formed spheroid-like bodies termed enterospheres. After 5 days of proliferation, free-floating enterospheres were picked and transferred to petri dishes (Ø 60 mm; Greiner Bio One, Frickenhausen, Germany) in 5 mL fresh proliferation medium and proliferation was continued for further 4 days.Figure 1
Time schedule of enterosphere culture. The timeline illustrates the schedule ofin vitro culture. Cells were isolated at 0 div (daysin vitro) and proliferated for 5 days. Spheres were then picked and again proliferated for 4 days. At 9 div, enterospheres were picked, washed, transferred to differentiation medium, and incubated for 2 days before gene expression analyses were carried out. The micrographs show proliferating enterospheres after 1 and 5 div. Scale bar: 200 μm.Single free-floating enterospheres (50 enterospheres/dish) were picked again, washed 3 times in Tris buffer, and transferred into new petri dishes containing either proliferation medium or differentiation medium. Differentiation medium consists of DMEM/F12 containing N2 supplement (1 : 100), penicillin (100 U/mL), streptomycin (100μg/mL), L-glutamine (2 mM), and ascorbic acid-2-phosphate (200 μM; Sigma-Aldrich).Enterospheres were proliferated or differentiated for 2 more days, thereby forming the two experimental groups “proliferation” and “differentiation.” The difference in expression between those two groups (differentiation versus proliferation) was successively compared by microarray analysis as described below.


### 2.2. Affymetrix Microarray Analysis

Affymetrix microarray analysis was conducted similar to previously published data in three independent experiments, each with cell cultures prepared from 2 pups from the same litter [15]. In each experiment, free-floating enterospheres were picked as described above in order to diminish the fraction of adhesive fibroblasts and smooth muscle cells.Total RNA of enterospheres of both groups was extracted using the RNeasy Micro Kit (Qiagen). RNA quality was evaluated on Agilent 2100 Bioanalyzer with RNA integrity numbers (RIN) of the samples in this study being in the range from 8 to 10. RIN numbers higher than 8 are considered optimal for downstream application [16].Double-stranded cDNA was synthesized from 100 ng of total RNA, subsequently linearly amplified, and biotinylated using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. 15μg of labeled and fragmented cDNA was hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). After hybridization, the arrays were stained and washed in a Fluidics Station 450 (Affymetrix) with the recommended washing procedure. Biotinylated cDNA bound to target molecules was detected with streptavidin-coupled phycoerythrin, biotinylated anti-streptavidin IgG antibodies and again streptavidin-coupled phycoerythrin according to the protocol. Arrays were scanned using the GCS3000 GeneChip Scanner (Affymetrix) and AGCC 3.0 software. Scanned images were subjected to visual inspection to check for hybridization artifacts and proper grid alignment and analyzed with Expression Console 1.0 (Affymetrix) to generate report files for quality control.Normalization of raw data was performed by the Partek Software 6.6, applying an RMA (Robust Multichip Average) algorithm. Significance was calculated using at-test without multiple testing correction (Partek), selecting all transcripts with a minimum change in expression level of 1.5-fold together with a p value less than 0.05.


## 2.1. Cell Culturing

Cell culturing was conducted as described previously [15]. The handling of animals was in accordance to the institutional guidelines of the University of Tuebingen, which conform to the international guidelines.Neonatal (P0) C57BL/6 mice without regard to sex were decapitated and the whole gut was removed. After removal of adherent mesentery the longitudinal and circular muscle layers containing myenteric plexus could be stripped as a whole from the small intestine. Tissue was chopped and incubated in collagenase type XI (750 U/mL; Sigma-Aldrich, Taufkirchen, Germany) and dispase II (250μg/mL; Roche Diagnostics, Mannheim, Germany) dissolved in Hanks’ balanced salt solution with Ca2+/Mg2+ (HBSS; PAA, Pasching, Austria) for 30 min at 37°C. During enzymatic dissociation the tissue was carefully triturated every 10 min with a fire polished 1 mL pipette tip. Prior to the first trituration step, cell suspension was treated with 0.05% (w/v) DNAse I (Sigma-Aldrich). After 30 min, tissue dissociation was stopped by adding fetal calf serum (FCS; PAA) to a final concentration of 10% (v/v) to the medium. Undigested larger tissue pieces were removed with a 40 μm cell strainer (BD Biosciences, Franklin Lakes, NJ, USA). Residual enzymes were removed during two washing steps in HBSS at 200 g. After dissociation, cells were resuspended in proliferation culture medium (Dulbecco’s modified Eagle’s medium with Ham’s F12 medium (DMEM/F12; 1 : 1; PAA)) containing N2 supplement (1 : 100; Invitrogen, Darmstadt, Germany), penicillin (100 U/mL; PAA), streptomycin (100 μg/mL; PAA), L-glutamine (2 mM; PAA), epidermal growth factor (EGF; 20 ng/mL; Sigma-Aldrich), and fibroblast growth factor (FGF; 20 ng/mL; Sigma-Aldrich). Cells were seeded into 6-well plates (BD Biosciences) in a concentration of 2.5 × 104 cells/cm2. Only once before seeding, the medium was supplemented with B27 (1 : 50; Invitrogen). EGF and FGF were added daily and culture medium was exchanged every 3 days. All cultivation steps were conducted in a humidified incubator at 37°C and 5% CO2. An overview of the following cell culture protocol is shown in Figure 1. During proliferation phase of the culture, cells formed spheroid-like bodies termed enterospheres. After 5 days of proliferation, free-floating enterospheres were picked and transferred to petri dishes (Ø 60 mm; Greiner Bio One, Frickenhausen, Germany) in 5 mL fresh proliferation medium and proliferation was continued for further 4 days.Figure 1
Time schedule of enterosphere culture. The timeline illustrates the schedule ofin vitro culture. Cells were isolated at 0 div (daysin vitro) and proliferated for 5 days. Spheres were then picked and again proliferated for 4 days. At 9 div, enterospheres were picked, washed, transferred to differentiation medium, and incubated for 2 days before gene expression analyses were carried out. The micrographs show proliferating enterospheres after 1 and 5 div. Scale bar: 200 μm.Single free-floating enterospheres (50 enterospheres/dish) were picked again, washed 3 times in Tris buffer, and transferred into new petri dishes containing either proliferation medium or differentiation medium. Differentiation medium consists of DMEM/F12 containing N2 supplement (1 : 100), penicillin (100 U/mL), streptomycin (100μg/mL), L-glutamine (2 mM), and ascorbic acid-2-phosphate (200 μM; Sigma-Aldrich).Enterospheres were proliferated or differentiated for 2 more days, thereby forming the two experimental groups “proliferation” and “differentiation.” The difference in expression between those two groups (differentiation versus proliferation) was successively compared by microarray analysis as described below.


## 2.2. Affymetrix Microarray Analysis

Affymetrix microarray analysis was conducted similar to previously published data in three independent experiments, each with cell cultures prepared from 2 pups from the same litter [15]. In each experiment, free-floating enterospheres were picked as described above in order to diminish the fraction of adhesive fibroblasts and smooth muscle cells.Total RNA of enterospheres of both groups was extracted using the RNeasy Micro Kit (Qiagen). RNA quality was evaluated on Agilent 2100 Bioanalyzer with RNA integrity numbers (RIN) of the samples in this study being in the range from 8 to 10. RIN numbers higher than 8 are considered optimal for downstream application [16].Double-stranded cDNA was synthesized from 100 ng of total RNA, subsequently linearly amplified, and biotinylated using the GeneChip WT cDNA Synthesis and Amplification Kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. 15μg of labeled and fragmented cDNA was hybridized to GeneChip Mouse Gene 1.0 ST arrays (Affymetrix). After hybridization, the arrays were stained and washed in a Fluidics Station 450 (Affymetrix) with the recommended washing procedure. Biotinylated cDNA bound to target molecules was detected with streptavidin-coupled phycoerythrin, biotinylated anti-streptavidin IgG antibodies and again streptavidin-coupled phycoerythrin according to the protocol. Arrays were scanned using the GCS3000 GeneChip Scanner (Affymetrix) and AGCC 3.0 software. Scanned images were subjected to visual inspection to check for hybridization artifacts and proper grid alignment and analyzed with Expression Console 1.0 (Affymetrix) to generate report files for quality control.Normalization of raw data was performed by the Partek Software 6.6, applying an RMA (Robust Multichip Average) algorithm. Significance was calculated using at-test without multiple testing correction (Partek), selecting all transcripts with a minimum change in expression level of 1.5-fold together with a p value less than 0.05.


## 3. Results

In this study, we investigated the changes of the genetic expression profile that occur during the transition from proliferating to differentiating enteric neural progenitor cellsin vitro. Therefore, we generated enterospheres by 9 dayin vitro cultures, which then could be picked and either proliferated or differentiated for two more days (Figure 1). mRNA was subsequently extracted and gene expression of these two groups was analysed by Affymetrix microarray analysis.Analysis of mRNA expression was performed on a GeneChip Mouse Gene 1.0 ST array that determines the expression profile of 28.853 genes. Each gene was interrogated by a median of 27 probes that are spread along the full gene.In total, the gene chip detected 1454 transcripts to be at least 1.5-fold differentially expressed between proliferating and differentiating enterospheres. 1333 of these transcripts code for already identified proteins. 541 genes were found to be upregulated and 792 genes were found to be downregulated in comparison to proliferating enterospheres (see Supplementary Table1 of the Supplementary Material available online at http://dx.doi.org/10.1155/2016/9695827).We used the ingenuity pathway analysis software (IPA) and data mining with the science literature search enginehttp://www.ncbi.nlm.nih.gov/pubmed/ to divide the genes into different groups according to their function during cellular development. The largest functional group contained 171 genes related to cell cycle and apoptosis (Table 1, Supplementary Table 2). Here, we identified especially different cyclin proteins and cell division cycle proteins that were mainly downregulated. Further, we found several genes that are linked to neural development as well as genes regulating neural stem cell proliferation and differentiation. Furthermore, we also detected neuronal and glial differentiation markers and numerous genes involved in synapse formation (Table 2). It is noteworthy that we also identified a group of genes that are known to be involved in the differentiation of smooth muscle cells (Table 3) as well as in extracellular matrix components (Table 4). Additionally, we found regulated genes related to canonical Wnt signalling indicating a deactivation of this pathway during ENS progenitor cell differentiation (Figure 2, Table 5).Table 1
Selected genes related to cell cycle.
Gene
Encoded protein
Fold change
Cell cycle
AURKA
Aurora kinase A
−2.712
STOP
AURKB
Aurora kinase B
−4.146
STOP
CCNA2
Cyclin A2
−4.652
STOP
CCNB1
Cyclin B1
−5.752−5.820 −5.857
STOP
CCNB2
Cyclin B2
−3.392
STOP
CCND1
Cyclin D1
−2.476
STOP
CCND3
Cyclin D3
−1.539
STOP
CCNE1
Cyclin E1
−1.777
STOP
CCNE2
Cyclin E2
−2.847
STOP
CCNF
Cyclin F
−3.211
STOP
CDC6
Cell division cycle 6
−1.936
STOP
CDC20
Cell division cycle 20
−3.113
STOP
CDC25B
Cell division cycle 25B
−1.636
STOP
CDC25C
Cell division cycle 25C
−2.414
STOP
CDC45
Cell division cycle 45
−1.769
STOP
CDCA2
Cell division cycle associated 2
−3.461
STOP
CDCA3
Cell division cycle associated 3
−3.003
STOP
CDCA5
Cell division cycle associated 5
−3.053
STOP
CDCA7L
Cell division cycle associated 7-like
−4.123
STOP
CDCA8
Cell division cycle associated 8
−3.467
STOP
CDK1
Cyclin-dependent kinase 1
−3.227
STOP
CDK15
Cyclin-dependent kinase 15
1.618
GO
CDK19
Cyclin-dependent kinase 19
1.619
GO
CDK5R1
Cyclin-dependent kinase 5, regulatory subunit 1 (p35)
1.597
—
CENPA
Centromere protein A
−1.895
STOP
CENPE
Centromere protein E, 312 kDa
−4.140
STOP
CENPF
Centromere protein F, 350/400 kDa
−3.927
STOP
CENPI
Centromere protein I
−2.899
STOP
CENPK
Centromere protein K
−2.813
STOP
CENPL
Centromere protein L
−1.864
STOP
CENPM
Centromere protein M
−3.407
STOP
CENPN
Centromere protein N
−2.465
STOP
CENPU
Centromere protein U
−1.624
STOP
SKA1
Spindle and kinetochore associated complex subunit 1
−1.532
STOP
SKA2
Spindle and kinetochore associated complex subunit 2
−1.582
STOP
SKA3
Spindle and kinetochore associated complex subunit 3
−3.490
STOP
SKP2
S-phase kinase-associated protein 2, E3 ubiquitin protein ligase
−1.845
STOP
SPC25
SPC25, NDC80 kinetochore complex component
−4.148
STOPTable 2
Neural differentiation/development.
Gene
Encoded protein
Fold change
Neural stem cells
ABCG2
ATP-binding cassette, subfamily G (WHITE), member 2 (junior blood group)
−1.526
ASPM
asp (abnormal spindle) homolog, microcephaly associated  (Drosophila)
−4.911
CDT1
Chromatin licensing and DNA replication factor 1
−1.528
EGFL7
EGF-like-domain, multiple 7
3.132
EPHA2
EPH receptor A2
−1.529
ETV4
ets variant 4
−1.934
ETV5
ets variant 5
−2.844
−2.651
FABP7
Fatty acid binding protein 7, brain
−2.095
Neural differentiation
ATOH8
Atonal homolog 8 (Drosophila)
1.932
AXL
AXL receptor tyrosine kinase
2.015
CRIM1
Cysteine-rich transmembrane BMP regulator 1 (chordin-like)
1.999
CRLF1
Cytokine receptor-like factor 1
2.382
DAB1
Dab, reelin signal transducer, homolog 1 (Drosophila)
−2.297
ELK3
ELK3, ETS-domain protein (SRF accessory protein 2)
−1.613
ESCO2
Establishment of sister chromatid cohesion N-acetyltransferase 2
−4.767
GAP43
Growth associated protein 43
1.613
GLDN
Gliomedin
5.809
HMOX1
Heme oxygenase (decycling) 1
1.884
KLF9
Kruppel-like factor 9
1.592
Lmo3
LIM domain only 3
1.542
MAP6
Microtubule-associated protein 6
1.874
MYRF
Myelin regulatory factor
2.527
NEUROD4
Neuronal differentiation 4
2.036
OLIG1
Oligodendrocyte transcription factor 1
2.660
Pvr
Poliovirus receptor
1.768
RGS4
Regulator of G-protein signaling 4
1.955
S1PR1
Sphingosine-1-phosphate receptor 1
5.073
SOCS2
Suppressor of cytokine signaling 2
2.052
2.335
WIPF1
WAS/WASL interacting protein family, member 1
1.587
Neural differentiation markers
CALB2
Calbindin 2
1.616
CNP
2′,3′-Cyclic nucleotide 3′-phosphodiesterase
1.732
GFAP
Glial fibrillary acidic protein
2.239
MBP
Myelin basic protein
1.768
Mturn
Maturin, neural progenitor differentiation regulator homolog  (Xenopus)
1.853
OMG
Oligodendrocyte myelin glycoprotein
−1.822
OPALIN
Oligodendrocytic myelin paranodal and inner loop protein
39.246
PLP1
Proteolipid protein 1
1.630
S100B
S100 calcium binding protein B
−1.675
TUBB2A
Tubulin, beta 2A class IIa
1.608
TUBB2B
Tubulin, beta 2B class IIb
1.535
TUBB3
Tubulin, beta 3 class III
1.976
Synapse and neurotransmitters
ABAT
4-Aminobutyrate aminotransferase
−1.512
ADRA1D
Adrenoceptor alpha 1D
1.803
ADRA2A
Adrenoceptor alpha 2A
2.900
ADRA2B
Adrenoceptor alpha 2B
−2.093
CHRM2
Cholinergic receptor, muscarinic 2
1.635
CHRM3
Cholinergic receptor, muscarinic 3
−1.715
CHRNA7
Cholinergic receptor, nicotinic, alpha 7 (neuronal)
1.772
COMT
Catechol-O-methyltransferase
1.515
DDC
DOPA decarboxylase (aromatic L-amino acid decarboxylase)
1.711
DNM3
Dynamin 3
2.643
EPHA5
EPH receptor A5
2.076
GRIA3
Glutamate receptor, ionotropic, AMPA 3
−1.528
GRIA4
Glutamate receptor, ionotropic, AMPA 4
−1.997
GRIK2
Glutamate receptor, ionotropic, kainate 2
−1.565
GRM5
Glutamate receptor, metabotropic 5
−1.600
HTR1B
5-Hydroxytryptamine (serotonin) receptor 1B, G-protein-coupled
−2.377
HTR2B
5-Hydroxytryptamine (serotonin) receptor 2B, G-protein-coupled
2.205
LRRTM2
Leucine-rich repeat transmembrane neuronal 2
3.665
LRRTM3
Leucine-rich repeat transmembrane neuronal 3
2.210
NTM
Neurotrimin
1.820
PENK
Proenkephalin
3.478
PRR7
Proline rich 7 (synaptic)
1.788
SLC10A4
Solute carrier family 10, member 4
1.824
1.867
SLITRK2
SLIT and NTRK-like family, member 2
−2.414
SLITRK6
SLIT and NTRK-like family, member 6
1.672
STON2
Stonin 2
4.054
STXBP3
Syntaxin-binding protein 3
1.730
Stxbp3b
Syntaxin-binding protein 3B
1.637
SV2C
Synaptic vesicle glycoprotein 2C
1.929
SYT6
Synaptotagmin VI
2.571
Neurite outgrowth
ATF3
Activating transcription factor 3
2.579
DOK4
Docking protein 4
4.937
FEZ2
Fasciculation and elongation protein zeta 2 (zygin II)
1.547
NAV2
Neuron navigator 2
1.647
NRCAM
Neuronal cell adhesion molecule
2.496
PLXNB3
Plexin B3
1.739
RGMA
Repulsive guidance molecule family member a
1.552
RNF165
Ring finger protein 165
−1.548
ROBO2
Roundabout, axon guidance receptor, homolog 2 (Drosophila)
−2.211
SEMA3B
Sema domain, immunoglobulin domain (Ig), short basic domain, secreted (semaphorin) 3B
3.692
SEMA3E
Sema domain, immunoglobulin domain (Ig), short basic domain, secreted (semaphorin) 3E
2.877
SEMA4F
Sema domain, immunoglobulin domain (Ig), transmembrane domain  (TM) and short cytoplasmic domain, (semaphorin) 4F
4.891
SEMA6A
Sema domain, transmembrane domain (TM), and cytoplasmic domain (semaphorin) 6A
−1.707
SRGAP1
SLIT-ROBO Rho GTPase activating protein 1
1.524
UNC5B
unc-5 homolog B (C. elegans)
−1.927
Growth factors
ARTN
Artemin
2.423
FGF2
Fibroblast growth factor 2 (basic)
2.264
FGF5
Fibroblast growth factor 5
7.704
GDF10
Growth differentiation factor 10
−2.361
GDF11
Growth differentiation factor 11
1.604
GDNF
Glial cell derived neurotrophic factor
4.325
GFRA3
GDNF family receptor alpha 3
1.707
MET
MET protooncogene, receptor tyrosine kinase
6.680
NGFR
Nerve growth factor receptor
1.728
NTRK3
Neurotrophic tyrosine kinase, receptor, type 3
−1.575
SNX16
Sorting nexin 16
1.641
SPHK1
Sphingosine kinase 1
1.704
SPRY1
Sprouty homolog 1, antagonist of FGF signaling (Drosophila)
−1.647Table 3
Differentiation of smooth muscle cells/ICCs.
Gene
Encoded protein
Fold change
Smooth muscle cells
ACTA2
Actin, alpha 2, smooth muscle, aorta
1.693
ACTG2
Actin, gamma 2, smooth muscle, enteric
2.336
ACTN1
Actinin, alpha 1
−1.724
AEBP1
AE binding protein 1
2.702
AFAP1
Actin filament associated protein 1
1.638
ARID5B
AT-rich interactive domain 5B (MRF1-like)
1.521
Cald1
Caldesmon 1
−1.535
CNN1
Calponin 1, basic, smooth muscle
1.652
ENG
Endoglin
−1.552
ENPP1
Ectonucleotide pyrophosphatase/phosphodiesterase 1
−1.522
ENPP2
Ectonucleotide pyrophosphatase/phosphodiesterase 2
2.959
ENTPD1
Ectonucleoside triphosphate diphosphohydrolase 1
1.636
FOSL2
FOS-like antigen 2
2.566
GAMT
Guanidinoacetate N-methyltransferase
1.725
MYO1E
Myosin IE
1.569
MYO5A
Myosin VA (heavy chain 12, myoxin)
1.680
MYO7B
Myosin VIIB
1.710
MYO18A
Myosin XVIIIA
1.994
MYPN
Myopalladin
1.570
NEB
Nebulin
1.569
Nebl
Nebulette
2.378
NUP210
Nucleoporin 210 kDa
−1.838
RBM24
RNA binding motif protein 24
1.548
SMTN
Smoothelin
−1.778
SSPN
Sarcospan
1.603
TAGLN
Transgelin
2.706
ICC
GUCY1A3
Guanylate cyclase 1, soluble, alpha 3
−1.876
GUCY1B3
Guanylate cyclase 1, soluble, beta 3
−2.008
KIT
v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog
−1.798
KITLG
KIT ligand
−1.541Table 4
ECM.
Gene
Encoded protein
Fold change
CHSY3
Chondroitin sulfate synthase 3
−1.645
COL6A5
Collagen, type VI, alpha 5
1.527
COL12A1
Collagen, type XII, alpha 1
−1.973
COL14A1
Collagen, type XIV, alpha 1
6.135
COL16A1
Collagen, type XVI, alpha 1
1.666
COL18A1
Collagen, type XVIII, alpha 1
1.595
COL27A1
Collagen, type XXVII, alpha 1
1.522
COLGALT2
Collagen beta(1-O)galactosyltransferase 2
−1.564
CSPG4
Chondroitin sulfate proteoglycan 4
−2.952
CSPG5
Chondroitin sulfate proteoglycan 5 (neuroglycan C)
−1.585
CYR61
Cysteine-rich, angiogenic inducer, 61
1.748
ECM1
Extracellular matrix protein 1
2.580
HSPG2
Heparan sulfate proteoglycan 2
1.923
ITGA1
Integrin, alpha 1
−1.665
ITGA4
Integrin, alpha 4 (antigen CD49D, alpha 4 subunit of VLA-4 receptor)
−2.324
ITGA7
Integrin, alpha 7
4.203
ITGA8
Integrin, alpha 8
−2.262
ITGA11
Integrin, alpha 11
1.762
ITGB3
Integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)
−5.342
ITGB4
Integrin, beta 4
1.567
KRT80
Keratin 80
2.833
LAMA4
Laminin, alpha 4
−1.537
LAMA5
Laminin, alpha 5
1.684
LOX
Lysyl oxidase
3.250
LOXL4
Lysyl oxidase-like 4
2.427
2.417
MATN2
Matrilin 2
2.570
MMP2
Matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)
1.668
MMP9
Matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)
−5.557
MMP15
Matrix metallopeptidase 15 (membrane-inserted)
−2.017
MMP16
Matrix metallopeptidase 16 (membrane-inserted)
−1.634
MMP17
Matrix metallopeptidase 17 (membrane-inserted)
1.612
MMP19
Matrix metallopeptidase 19
3.236
MMP28
Matrix metallopeptidase 28
1.956
NDST3
N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 3
−5.557
P4HA1
Prolyl 4-hydroxylase, alpha polypeptide I
−1.958
PLOD3
Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3
2.250
UGDH
UDP-glucose 6-dehydrogenase
1.529Table 5
Wnt.
Gene
Encoded protein
Fold change
Wnt signaling cascade
FZD7
Frizzled class receptor 7
−2.271
LEF1
Lymphoid enhancer-binding factor 1
−2.680
LRP5
Low density lipoprotein receptor-related protein 5
−1.571
LRRK2
Leucine-rich repeat kinase 2
1.677
TCF19
Transcription factor 19
−2.217
F7L1
Transcription factor 7-like 1 (T-cell specific, HMG-box)
−1.762
WNT5A
Wingless-type MMTV integration site family, member 5A
−2.325
WNT7B
Wingless-type MMTV integration site family, member 7B
2.942
Target gene
ARL4C
ADP-ribosylation factor-like 4C
2.179
AXIN2
Axin 2
1.744
CCND1
Cyclin D1
−2.476
CSRNP1
Cysteine-serine-rich nuclear protein 1
1.822
RACGAP1
Rac GTPase activating protein 1
−3.201
SPRY2
Sprouty homolog 2 (Drosophila)
−1.771
SPRY4
Sprouty homolog 4 (Drosophila)
−2.771
WISP1
WNT1 inducible signaling pathway protein 1
2.489
Wnt antagonists/inhibitors
APOE
Apolipoprotein E
1.704
DKK2
Dickkopf WNT signaling pathway inhibitor 2
1.731
EDIL3
EGF-like repeats and discoidin I-like domains 3
2.258
FRZB
Frizzled-related protein
1.938
HIC1
Hypermethylated in cancer 1
−1.731
JADE1
Jade family PHD finger 1
−1.656
LRP4
Low density lipoprotein receptor-related protein 4
1.979
NARF
Nuclear prelamin A recognition factor
−1.699
NEDD4L
Neural precursor cell expressed, developmentally downregulated 4-like, E3 ubiquitin protein ligase
1.588
NKD1
Naked cuticle homolog 1 (Drosophila)
3.220
NOTUM
Notum pectinacetylesterase homolog (Drosophila)
2.631
NOV
Nephroblastoma overexpressed
2.050
PRICKLE1
Prickle homolog 1 (Drosophila)
1.536
TLE3
Transducin-like enhancer of split 3
1.714
TRIB2
Tribbles pseudokinase 2
−1.637
Wnt activators
DAAM2
Dishevelled associated activator of morphogenesis 2
−1.993
PSRC1
Proline/serine-rich coiled-coil 1
−2.235
TNIK
TRAF2 and NCK interacting kinase
1.677
TRAF4
TNF receptor-associated factor 4
−1.673Figure 2
Detected regulatory influences on the canonical Wnt pathway. Scheme of the canonical Wnt pathway. Pointy arrowheads indicate an activating, blunt arrowheads, an inhibitory influence. The fold-change in expression of genes is written under the respective gene acronyms and colours indicate a general upregulation (red) or downregulation (green). For detailed explanation of the signalling cascade and regulated genes, see text.


## 4. Discussion

The proliferation and differentiation of enteric neural progenitor cells during embryonic and postnatal development are controlled by a complex interplay of various intrinsic and extrinsic factors. Their exact timing is crucial for proper migration and proliferation of neural crest cells and for their differentiation into the various neural cell types that compose the complex neural structures of the ENS. Although research in recent years extended our understanding of ENS development and its pathologies [13], there are still many genes and processes unknown. Particularly, factors regulating neural progenitor proliferation and differentiation in the developing and postnatal gut as well as cellular and molecular interaction systems remain largely elusive. Here, we usedin vitro cultures of enteric neural progenitor cells derived from murine tunica muscularis to scan for molecular programs and signalling pathways acting on cell proliferation and early differentiation.Our experiment aimed to elucidate gene regulations in enterospheres that occur while ENS progenitor cells leave their proliferative state and begin to differentiate into more defined and specific cell types. The results of the Affymetrix gene expression analysis showed the up- and downregulation of overall 1333 known genes that code for already identified proteins. 171 of these genes could be linked to cell proliferation (Table1, Supplementary Table 1). Amongst them we detected genes coding for proteins related to the kinetochore complex (like NSL1 [17], NUF2 [18], SKA1-3 [19], and ZWILCH [20]), cyclin proteins [21], cyclin-dependent kinases (CDK) [22], and several types of centromere proteins. The regulation of 145 of these genes strongly indicates a slowdown of cell cycle progression as it was intended by the experimental deprivation of growth factor supplementation by the end of the proliferation phase (see Section 3). Interestingly, betacellulin (BTC) was upregulated nearly 6-fold although it was reported to promote cellular proliferation in the neural stem cell niche [23]. Nonetheless, the vast majority of genes including all regulated cyclins, cell division cycle proteins, and kinetochore proteins were found to be downregulated.We also checked the regulated genes for apoptosis markers to see whether the stop in proliferation was related to cell death (Supplementary Table2). Since only 3 of 12 apoptotic genes were regulated in the direction that indicates apoptosis, it is unlikely that apoptosis played a leading role in the interruption of proliferation. Still, the effect and regulation of apoptosis during enteric sphere cultures are an important cornerstone of understanding enteric neural progenitors in culture andin vivo and require further investigation. Together, on a broad basis, this dataset provides strong evidence that this cell culture design is applicable to decreasing the proliferative rate of enteric neural progenitor cells without inducing cell death or apoptosis in an appreciable quantity.To further evaluate the proliferative conditions of cell types present in enterospheres, we focused on different cell specific markers of neural progenitors as well as neurons, glial, or smooth muscle cells. We consider this complex cellular composition of the enterospheres an advantage compared to more purified neural crest derived neurospheres as we are able to capture complex interactions and secretion mechanisms between cell types that might also play an important rolein vivo. Interestingly, we found 8 genes involved in adult central or embryonic neural stem cells homeostasis (Table 2). The majority of genes like EPHA2 [24] are regulated in a way that suggests that neural stem cells exit the proliferative cell cycle to enter differentiation programs. This idea was supported by the upregulation of numerous genes that drive neuronal and glial differentiation like NEUROD4 [25] or OLIG1 [26]. In this context, we identified several upregulated genes involved in proper myelination. As enteric and central glia cells are known to temporally express myelin-related proteins during development, it is conceivable that this regulation is part of the early glial differentiation program [27]. Moreover, also typical markers of differentiated neurons (class III beta-tubulin, CALB2 [28]) and enteric glia (GFAP [29]) were found to be upregulated. Intriguingly, S100B, a common glia cell marker, was downregulated contrasting the rest of our data. Again, this might be due to the complex differentiation program of enteric glia, in which S100B plays a role at later stages.Furthermore, the establishment of neuronal cell communication was strongly regulated. Here, we found an increased expression of genes related to synaptogenesis (LRRTM2 and 3 [30], neurotrimin [31]) and to SNARE or vesicle protein function (STXBP3, SV2C [32], and SYT6 [33]). We also identified a number of genes involved in transmitter metabolism (COMT, DDC) as well as neurotransmitter receptor like 5-HT, glutamate, and adrenergic receptors. However, the regulation of those genes was highly variable shedding light on the intricacy of synapse formation in the developing enteric nervous system. This complexity is carried on by genes related to axon sprouting and guidance like semaphorins [34] or RGMa [35].Additionally, we found that regulated genes directly involved in the differentiation of muscle cells and/or enteric pacemaker cells called interstitial cells of Cajal (Table3). Particularly interesting is the upregulation of a number of genes known to drive smooth muscle differentiation like ARID5B [36], FOSL2 [36] and genes that are expressed in differentiated smooth muscle cells in the intestine like AFAP1 [37], ENPP2 [38], and CNN1 [39] as well as various myosin and actin isoforms. These data confirm the fact that cultured spheroids are composed of different cell types present in the intestinal tunica muscularis and further indicate that deprivation of growth factors induces differentiation of smooth muscle cells resembling molecular processes in the developing gut. In fact, we among others were previously able to confirm the presence of smooth muscle cells derived from enterosphere culture by BrdU-immunolabeling costudies [4]. However, it is noteworthy that a few genes related to muscular differentiation (endoglin [40], smoothelin [41], NUP210 [42], caldesmon 1 [43], and ACTN1 [44]) were downregulated contrasting the expression pattern observed in the majority of regulated genes. This hints to complex regulatory mechanisms controlling the myogenic differentiation program in which these genes are not required at all or in a different temporal sequence not mapped by our experimental design. It is further remarkable that five markers expressed in interstitial cells of Cajal (ICC) including KIT [45] were downregulated.Moreover, the regulation of 43 extracellular matrix proteins like collagens, integrins, proteoglycans, and matrix metallopeptidases points to a reconstruction of extracellular environment that has been discussed to influence neural stem cell behaviour [46] (Table 4). Taken together, these results illustrate the ongoing genetic programs during early differentiation of enterospheres.Within the dataset, it was of special interest to find particularly many regulated genes related to the canonical Wnt pathway (Table5). The involvement of canonical Wnt signalling has frequently been shown in the regulation of various stem cell niches, like intestinal epithelium or CNS derived neural stem cells. However, these studies exhibited different and partly contradicting outcomes, which strongly hint to the variable functions of canonical Wnt signals in different tissues during embryonic and postnatal development. In previous work, we found regulation of several Wnt-related genes in the context of thyroid hormone dependent differentiation of enteric neural progenitor cells indicating a potential role of the canonical Wnt pathway activation during the proliferation of this progenitor cell pool [15]. Canonical Wnt signalling has frequently been reviewed in the literature—just recently by Ring et al. [47]. In brief, secreted Wnt proteins bind to frizzled receptors (FZD) complexed with low density lipoprotein receptor-related protein 5/6 (LRP5/6) coreceptors. Thereafter, the scaffolding protein disheveled (DVL) is recruited to FZD and inhibits the β-catenin destruction complex (AXIN2, APC, and GSK-3β). Therefore, β-catenin accumulates in the cytoplasm and translocates to the nucleus where it binds to TCF/LEF transcription factors to initiate Wnt target gene expression. Interestingly, our current data strongly indicate that the canonical Wnt pathway is switched off during the first two days of enteric progenitor differentiation on several levels of the signalling cascade (Figure 2). On the one hand we identified a downregulation of activating parts of the signalling cascade itself like the receptor proteins FZD7 and LRP5 or the transcription factors TCF19, TCF7L1, and LEF1. On the other hand, inactivating elements of the pathway like parts of the β-catenin destruction complex AXIN2 and LRRK2 [48] were upregulated. We also found numerous modulators of the signalling cascade. It is of interest that the majority of those genes are reported to inhibit the signalling process extracellularly or on receptor level (Notum [49], FRZB [50], DKK2 [51], and LRP4 [52]), in the cytoplasm (NEDD4L [53], NKD1 [54], PRICKLE1 [55], NOV [56], and APOE [57]), or in the nucleus (TLE3 [58], EDIL3 [59]). Furthermore, we identified target genes of the canonical Wnt pathway that were either upregulated (e.g., AXIN2 that exerts a negative feedback on the pathway) or downregulated like the cell cycle progression genes CCND1 and SPRY4 [60]. We also found a lower expression of SPRY2 [61], a Wnt target gene and known inhibitor of GDNF signalling [62], in the differentiation group. Together with a strong upregulation of GDNF itself by 4.325-fold, this might drive enteric progenitor cells into neural differentiation [12].Taken together, it is conceivable that canonical Wnt signalling plays a role in the maintenance of the enteric progenitor pool during proliferation and is switched off at the beginning of differentiation conditions. Indeed, our previous gene expression analyses [15] as well as recently published cell culture experiments [63] and yet unpublishedin vitro analyses strongly support this hypothesis.


## 5. Conclusion

This study focused on the changes in gene expression of enteric neural progenitor cells occurring within the first two days of transition from a proliferative state to differentiationin vitro. Using microarray analysis, we found a marked inhibition of cell cycle progression in general as well as strong evidence for neural stem cells differentiation into enteric neurons and glia cells. These findings were substantiated by the upregulation of genes related to synapse formation and neural connectivity. Most interesting, we found that this transition from enteric neural progenitor proliferation to differentiation was accompanied by a considerable inactivation of the canonical Wnt signalling pathway. This, together with previous work, strongly indicates that canonical Wnt activation is one of the driving mechanisms of enteric neural progenitor proliferation and thus might play a role in the homeostasis of this cell poolin vivo andin vitro.



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*Source: 9695827-2015-11-30.xml*