Wnt Signaling Mediates the Aging-Induced Differentiation Impairment of Intestinal Stem Cells
Stem cell aging underlies aging-associated disorders, such as steeply increased incidences of tumors and impaired regeneration capacity upon stress. However, whether and how the intestinal stem cells age remains largely unknown. Here we show that intestinal stem cells derived from 24-month-old mice hardly form typical organoids with crypt-villus structures, but rather mainly form big, rounded cysts devoid of differentiated cell types, which mimics the culturing of heterozygous APC-deficient cells from the APCmin mouse line. Further analysis showed that cultured crypts derived from aged mice exhibited reduced expression levels of differentiation genes and higher expression of Wnt target genes. Lowering the concentration of R-spondin-1 in the culture system significantly reduced formation of rounded cysts, accompanied by an increased formation of organoids from crypts derived from old mice. We are the first to uncover that intestinal stem cells derived from old mice harbor significant deficiency in differentiation that can be partially rescued through a reduction in R-spondin-1 exposure. This could be highly relevant to intestinal tumor development and the reduced regeneration potential observed in the aged population. Our study provides the first experimental evidence that an over-responsiveness to Wnt/beta-catenin signaling of aged intestinal stem cells mediates the aging-induced deficiency in differentiation, and could serve as a potential target to ameliorate aging-associated intestinal pathologies.
KeywordsAging Wnt signaling Intestinal stem cells Differentiation R-spondin-1
Aging-associated intestinal pathologies are characterized by increased incidence of malignances and poor tolerances to stress, such as radiotherapy, chemotherapy and infection, which often cause severe complications in aged people. Intestinal stem cells (ISCs) are the driving force of intestinal homeostasis and regeneration [1, 2]. Functional abnormalities of ISCs underlie multiple intestinal diseases. First, ISCs were identified as the cell of origin in intestinal tumors which often exhibit high proliferative activities [3, 4, 5, 6, 7, 8]. Second, radiation and cytotoxic therapies cause massive loss of ISCs and epithelial cells, which results in complications such as infections and diarrhea. After the acute phase of cell loss, the remaining ISCs rebuild the stem cell pool and the intestinal epithelium via proliferation and differentiation . Taken together, the maintenance of intestinal homeostasis relies on balanced self-renewal, proliferation and differentiation of ISCs, which play essential roles in intestinal tumor development and regeneration after injury. However, little is known about how the core functions of ISCs alter with time, and whether their homeostatic balance is disturbed during aging.
Wnt/beta-catenin signaling pathway is a vital force to promote self-renewal and proliferation of ISCs [10, 11, 12, 13]. Numerous studies have suggested that over-activation of Wnt signaling pathway is indispensable to intestinal tumor development [14, 15, 16, 17]. Of note, recent studies have uncovered critical roles of Wnt signaling pathway in stem cell aging in other systems, such as the hematopoietic system and the skeletal muscle system [18, 19]. Up-regulation of Wnt signaling during aging leads to impairment of muscle stem cell function and increases fibrosis . Previously, we have identified Wnt signaling as a key mediator of ISC fate decision in premature aged mice, but the function of ISCs from aged mice was not studied . Recently, it was reported that aged crypts exhibited reduced regenerative potential as a result of decreased niche (Paneth/mesenchyme) secretion of Wnt signals . However, the relative activities of proliferation and differentiation of ISCs of aged mice remain to be illuminated. It is known that cell proliferation and differentiation are intimately coupled in intestinal cells, and these processes are tightly regulated by Wnt/beta-catenin signaling pathway [22, 23, 24]. Elevated Wnt signaling activity constitutes a dominant switch between proliferation and differentiation, which is an essential event in the early stages of intestinal tumorigenesis . Whether the Wnt signaling pathway would mediate a switch between proliferation and differentiation of ISCs during aging remains largely unknown.
In this study, we uncovered an unexpected deficiency in differentiation of ISCs from aged mice, using the 3D matrigel-based culture system, an accepted ex vivo assay that is reflective of ISC function in vivo. Astonishingly, ISCs from old mice mainly form rounded cysts devoid of differentiated cell types, distinctly different from the ISCs of young mice that mainly formed typical differentiated organoids. Furthermore, reducing exposure to the Wnt agonist R-spondin-1 in the culture system partially restored the deficiency in differentiation of ISCs from old mice. Our study provides first experimental evidence that ISCs derived from old mice harbor significant deficiency in differentiation, which can be partially reversed by reducing exposure to Wnt signaling. These findings could be highly relevant to aging-associated intestinal pathologies, such as tumor formation and impaired regeneration capacity after damage. Our study gives new insight in understanding the mechanisms underlying ISC aging, and offers hope to find potential ways to retard the above-mentioned aging-associated intestinal pathologies.
Materials and Methods
C57BL/6J mice were obtained from Hunan SJA Laboratory Animal Co. Ltd. (Hunan, China) and maintained in the animal facilities of Nanchang Royo Biotech under pathogen-free conditions on a 12-h light/12-h dark cycle. All mouse experiments were approved by the Animal Experimental Ethical Inspection of Nanchang Royo Biotech Co. Ltd. (RYEI20170430–1). 2 months old mice were used as young mice, and 24 months old mice were used as old mice.
Crypt culture was performed as previously described  . Briefly, isolated crypts were resuspended in cold matrigel (BD) containing Y27632 (Abcam), hR-spondin-1 (PeproTech), mEGF (PeproTech), hNoggin (PeproTech), and penicillin/streptomycin, and plated in 24-well plates at a density of 200 crypts, 50 μl per well. The plate was incubated at 37 °C for 5 min, and then 500 μl of advanced DMEM/F 12 medium (Invitrogen) containing B27 (Invitrogen), N2 supplement (Invitrogen) and 1.25 mM N-acetylcystein was added to each well to cover the matrigel. In the culture system final concentrations of the following components were R-spondin-1 1 μg/ml, mEGF 50 ng/ml, and Noggin 100 ng/ml. Y27632 10 μM was included in the first 3 days after seeding. Final concentration of mWnt3a (R&D Systems) was 100 ng/ml. The medium containing growth factors was changed every 3 days, and crypts were passaged after 10 days of primary culture. Growth factors were added every other day and culture medium was changed every 4 days.
RNA Isolation and cDNA Synthesis
Total RNA was isolated from cultured crypts by using RNApure Tissue Kit (CWbiotech) following the manufacturer’s instructions. Reverse transcriptions were performed to synthesize first-strand DNA by using TransScript-Uni One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech).
Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was performed with an ABI 7900 Real-Time PCR system (Applied Biosystems). TransStart Tip Green qPCR SuperMix (TransGen Biotech) was used. Primer sets for the detection of single genes are listed in Supplementary Table S1. mRNA expression of genes was normalized to beta-actin in each sample.
GraphPad Prism 7.0 software was used for statistical analysis. The unpaired two-tailed Student’s t test and two-way ANOVA were used to calculate p-values for two-group datasets and four-group datasets, respectively.
Results and Discussion
ISCs from Aged Mice Exhibit Significant Deficiency in Differentiation in Culture
Reduction in R-Spondin-1 Exposure Ameliorates the Aging-Induced Deficiency in Differentiation of ISCs
We further tested the effect of up-regulation of Wnt signaling on old ISCs. Addition of Wnt3a led to increased Wnt activity in the cultured crypts as shown by up-regulated expression of Axin2 and Ascl2 (Fig. S2A), and an elevated absolute number of big cysts, though the difference in the constituent ratio was not significant (Fig.S2B-D). The overall outgrowth also had a trend to increase, though again, not significantly (Fig.S2B). These results indicate that elevated activity of Wnt signaling further blocks differentiation of cultured ISCs, which was in line with several previous studies in which addition of Wnt3a caused the typical crypt-villus architecture to change into rounded cysts devoid of differentiated cell types [26, 29, 30].
In summary, the current study revealed that ISCs derived from old mice harbor a significant deficiency in differentiation that can be partially rescued through a reduction in R-spondin-1 exposure. This could be highly relevant to intestinal tumor development and the reduced regeneration potential observed in the aged population. Our study provides the first experimental evidence that an over-responsiveness to Wnt/beta-catenin signaling of aged intestinal stem cells mediates the aging-induced deficiency in differentiation, and could serve as a potential target to ameliorate aging-associated intestinal pathologies.
Previously, Nalapareddy et al. have reported a decrease in niche Wnt signal mainly resulting from reduced secretion of Wnts by Paneth cells and the mesenchyme in aged mice, which led to lower Wnt signaling levels in ISCs in vivo and reduced their regenerative potential. They described that the number of lobes or buds per crypt from aged intestine was lower after 3 passages in culture. The addition of Wnt3a rescued the phenotype after 3 passages. Our study showed that old ISCs formed a large proportion of big cysts with no budding, and formed significantly less differentiated, big organoids. Our study stands in agreement with their study in respect to the comparison between the number of lobes or buds per crypt.
Of note, Nalapareddy et al. only used duodenal (proximal) crypts in culture, while we tested the potential of crypts derived from the whole intestine. Multiple studies have shown that distal intestine has a higher potential to form adenomas [31, 32, 33], and the formation of adenomas was closely related to impairment of differentiation. Therefore it is highly possible that the deficiency of differentiation of the old crypts was less obvious in the Nalapareddy study where only duodenal (proximal) crypts were used for experiments.
Furthermore, our study suggested that ISCs from aged mice showed a higher sensitivity to sufficient Wnt signals supplied in the culture system which reflects an intrinsic over-responsiveness to Wnt signaling. Incidences of intestinal tumors increase sharply upon aging, with aberrant expression of APC along with other components of the beta–catenin destruction complex resulting in hyperactive Wnt signaling [23, 34, 35, 36]. This has been identified as a driving force for the development of intestinal tumors, which accumulate with age [23, 34, 35, 36]. It is tempting to speculate that the increased intrinsic over-responsiveness to Wnt signals in old ISCs could result from an altered epigenetic or genetic profile, which could be therapeutically targetable and reversed. Our study reveals an intrinsic and abnormal over-responsiveness of old ISCs to Wnt signaling, which we hope will shed new insight into the field of ISC aging.
This work was supported by the National Natural Science Fund of China (NSFC-81660520, NSFC-81660244 and NSFC-81860027), and the Jiangxi Provincial Nature Science Foundation (20171ACB21060, 20181BAB205060 and 2018ACB21034).
HC performed and analyzed majority of all experiments, including crypt isolation and culture. DT participated in most of the experiments. GBG gave suggestions and helped with some of the experiments. TZ, YW, ZT and LZ helped with sample collection and helped in some crypt isolation experiments. DT and ST conceived and designed the experiments. The manuscript was written by HC, GBG and ST and commented on by all other authors.
Compliance with Ethical Standards
Conflict of Interest
The authors declare no potential conflicts of interest.
- 31.Haigis, K. M., Hoff, P. D., White, A., Shoemaker, A. R., Halberg, R. B., & Dove, W. F. (2004). Tumor regionality in the mouse intestine reflects the mechanism of loss of Apc function. Proceedings of the National Academy of Sciences of the United States of America, 101, 9769–9773.CrossRefGoogle Scholar
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