Alterations of niche to stem cell communication in the aging intestine

Aging is a biological process where tissue functions deteriorate leading to morbidity. Gastrointestinal tract is a key system for organismal health converting external energy sources to a useful form for the body. With age, intestinal function declines in humans as the absorptive capacity decreases and recovery from damage slows down. Absorptive epithelium is constantly renewed by actively proliferating intestinal stem cells (ISC) that are intermingled between specialized secretory cells named Paneth cells. These cells are located at the bottom of intestinal crypts that forms a specialized microenvironment, the stem cell niche, participating to the maintenance of ISCs and their function. What is the role of the niche in age related changes of ISC function remains poorly characterized. In this dissertation age-associated changes in the communication between ISCs and the neighboring Paneth cells are analyzed and the outcome for regenerative capacity and homeostatic maintenance of the epithelium is studied. Under homeostasis, old epithelium is functional, but ISCs are biased to differentiate towards the secretory lineage. Using in vitro organoid culture, we show that reduced regenerative function of old epithelium is due to defects in both ISCs and the supporting Paneth cells. Aged Paneth cells produce excess amount of Notum, a secreted Wnt-inhibitor, that reduces the canonical Wnt-activity in the neighboring ISCs. Moreover, old crypts have enlarged morphology reducing the physical curvature of the niche, and potentially increasing the size of ISCs in vivo. Alterations in size and shape of old ISCs can contribute to decreased capacity to receive signals from the niche. Supplementation of Wnt-ligands, genetic ablation of Notum or culture on bioengineered topologically young scaffolds improved the regenerative growth of old epithelium in vitro. Moreover, inhibition of Notum in vivo with a small molecule ABC99 enhanced recovery of old intestine from 5-Fluorouracil induced chemotherapeutic insult. These findings demonstrate that Notum inhibition could be used as a prophylactic treatment to reduce harmful side-effects in elderly patients undergoing chemotherapy. In addition, cellular shape as a facilitator of cell-cell communication in the ISC niche suggest that modulation of the tissue topology could result in enhanced stem cell function.Ikääntyminen on biologinen prosessi, missä kudosten toiminnan heikentyminen johtaa lopulta yksilön kuolemaan. Suolisto on merkittävä osa kehon toiminnan ylläpitämisessä vastaten energian talteen ottamisesta ravinnosta. Iän myötä, ihmisen suoliston toimintakyky heikkenee ja etenkin ravinnon hyödyntäminen ja vaurioiden korjaaminen hidastuu. Suoliston sisäreunaa vuoraa yksikerroksinen epiteeli, minkä tehtävä on kuljettaa energiaa sulatetusta ravinnosta muiden kudosten käyttöön ja samalla estää haitallisten patogeenien pääsy kehoon. Epiteeli uusiutuu jatkuvasti, kun suoliston kantasolut tuottavat uutta solukkoa korvaamaan kuluneen epiteelin. Kantasolut sijaitsevat nk. Lieberkühnin kryptien pohjalla erityisessä mikroympäristössä, nk. kantasolulokerossa, missä ne ovat välittömässä kosketuksessa erilaistuneiden Panethin solujen kanssa. Panethin solut ohjaavat kantasolujen toimintaa ja muodostavat täten merkittävän osan kantasolulokerosta. Kuinka paljon ikääntyminen vaikuttaa suoliston kantasolujen ja niiden mikroympäristön väliseen vuorovaikutukseen ja sitä kautta kudoksen toimintaan on tunnettu huonosti. Tässä väitöskirjassa selvitetään minkälaisia muutoksia ikääntyminen aiheuttaa suoliston epiteelin ylläpitoon, uusiutumiskykyyn sekä Panethin ja kantasolujen väliseen vuorovaikutukseen. Normaalitilassa, vanhan epiteeli on toiminnallinen, mutta kantasoluilla on suurempi taipumus muodostaa sekretorisen linjan solukkoa. Käyttämällä organotyyppisiä soluviljelymalleja, havaittiin, että vanhan epiteelin uusiutumiskyky on heikompi johtuen sekä kanta- että niitä tukevien Panethin solujen toiminnan muutoksesta. Vanhat Panethin solut tuottavat ylimäärin Wnt molekyylejä inaktivoivaa entsyymiä nimeltään Notum. Wnt molekyylit ovat elintärkeitä kantasoluille ja lisääntynyt Notumin määrä vanhassa mikroympäristössä heikentää kantasolujen toiminnallisuutta. Tämän lisäksi, vanhan mikroympäristön muoto ja kantasolujen koko on muuttunut, mikä voi madaltaa naapuruston viestien vastaanottokykyä. Antamalla vanhoille kantasoluille ylimäärin Wnt molekyylejä tai estämällä Notum entsyymin toiminta geenisaksilla, voitiin lisätä kudoksen uusiutumiskykyä soluviljelymallissa. Lisäksi, pakottamalla vanha epiteeli nuoren kaltaiseen muotoon kyettiin parantamaan uuden epiteelin muodostumista. Pienmolekyyli, jolla estetään Notumin toiminta, edisti vanhan kudoksen paranemista eläinmallissa, missä normaali kudos oli vaurioitunut syöpälääkkeen, 5-Fluorourasiili, saamisen jälkeen. Yhdessä nämä tulokset havainnollistavat, että Notumin toiminnan estämistä voitaisiin tulevaisuudessa käyttää ennaltaehkäisevästi vähentämään syöpähoitojen sivuvaikutuksia etenkin vanhoilla potilailla. Lisäksi, osoitus solujen muodon merkityksestä niiden väliseen vuorovaikutukseen kantasolulokerossa viittaa mahdollisuuteen parantaa kantasolujen toimintaa muokkaamalla kudoksen fysikaalista muotoa

Suoliston kantasolut

English summaryPeer reviewe

Laminin alpha 5 regulates mammary gland remodeling through luminal cell differentiation and Wnt4-mediated epithelial crosstalk

Epithelial attachment to the basement membrane (BM) is essential for mammary gland development, yet the exact roles of specific BM components remain unclear. Here, we show that Laminin alpha 5 (Lama5) expression specifically in the luminal epithelial cells is necessary for normal mammary gland growth during puberty, and for alveologenesis during pregnancy. Lama5 loss in the keratin 8-expressing cells results in reduced frequency and differentiation of hormone receptor expressing (HR+) luminal cells. Consequently, Wnt4-mediated crosstalk between HR+ luminal cells and basal epithelial cells is compromised during gland remodeling, and results in defective epithelial growth. The effects of Lama5 deletion on gland growth and branching can be rescued by Wnt4 supplementation in the in vitro model of branching morphogenesis. Our results reveal a surprising role for BM-protein expression in the luminal mammary epithelial cells, and highlight the function of Lama5 in mammary gland remodeling and luminal differentiation.Peer reviewe

Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness

By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties.National Science Foundation (U.S.). Long-Term Ecological Research Program (DEB-8811884)National Science Foundation (U.S.). Long-Term Ecological Research Program (DEB-9411972)National Science Foundation (U.S.). Long-Term Ecological Research Program (DEB-0080382)National Science Foundation (U.S.). Long-Term Ecological Research Program (DEB-0620652)National Science Foundation (U.S.). Long-Term Ecological Research Program (DEB-1234162)National Science Foundation (U.S.). (Biocomplexity Coupled Biogeocemhical Cycles. DEB-0322057)National Science Foundation (U.S.). Long-Term Research in Environmental Biology (DEB-0716587)National Science Foundation (U.S.). Long-Term Research in Environmental Biology (DEB-1242531)National Science Foundation (U.S.). Long-Term Research in Ecosystem Sciences (DEB-1120064)United States. Dept. of Energy. Program for Ecoysystem Research (DE-FG02-96ER62291)United States. Dept. of Energy. Office of Biological and Environmental Research. National Institute for Climatic Change Research (Grant DE-FC02-06ER64158

Asymmetric apportioning of aged mitochondria between daughter cells is required for stemness

By dividing asymmetrically, stem cells can generate two daughter cells with distinct fates. However, evidence is limited in mammalian systems for the selective apportioning of subcellular contents between daughters. We followed the fates of old and young organelles during the division of human mammary stemlike cells and found that such cells apportion aged mitochondria asymmetrically between daughter cells. Daughter cells that received fewer old mitochondria maintained stem cell traits. Inhibition of mitochondrial fission disrupted both the age-dependent subcellular localization and segregation of mitochondria and caused loss of stem cell properties in the progeny cells. Hence, mechanisms exist for mammalian stemlike cells to asymmetrically sort aged and young mitochondria, and these are important for maintaining stemness properties.Peer reviewe

Cellular shape reinforces niche to stem cell signaling in the small intestine

Peer reviewe

WNT2 activation through proximal germline deletion predisposes to small intestinal neuroendocrine tumors and intestinal adenocarcinomas

Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described.We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas.To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture.We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.Peer reviewe

Notum produced by Paneth cells attenuates regeneration of aged intestinal epithelium

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche(1,2). Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor alpha (PPAR-alpha)(3), and lowered PPAR-a activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.Peer reviewe