Am J Pathol. 2014 Dec 26. pii: S0002-9440(14)00675-0.
Katano T, Ootani A, Mizoshita T, Tanida S, Tsukamoto H, Ozeki K, Kataoka H, Joh T.
PMID: 25546442 | DOI: 10.1016/j.ajpath.2014.11.007.
Stem cells are influenced by a microenvironmental niche that includes mesenchymal cells. We established a novel long-term method for primary mouse glandular stomach culture with mesenchymal myofibroblasts to investigate gastric epithelial-mesenchymal interactions. A gastric mesenchymal myofibroblast (GMF) cell line was established from mouse glandular stomach. Glandular stomach cells from neonatal mice and GMF cells were co-cultured in a collagen gel. Cultured stomach cells yielded expanding sphere-like structures. In the GMF co-culture system, the number and size of gastrospheres were increased compared with control cultures (P = 0.009 and 0.008, respectively). Immunohistochemistry showed cells positive for human gastric mucin, HIK1083, and chromogranin A, indicating differentiation into surface mucous cells, mucous neck cells, and enteroendocrine cells, respectively. RNA in situ hybridization for Lgr5 showed Lgr5+ stem cells in the cultured gastrospheres. Lgr5+ cells were observed persistently in the epithelium of gastrospheres in the GMF co-culture system for 2 months. GMFs allowed the cultured gastric epithelium to maintain active proliferation similar to that seen in vivo. Real-time quantitative RT-PCR showed that Gas1 expression was higher in GMFs (P = 0.0445), and Hoxc8, Notch1, and Sox10 expressions were higher in intestinal mesenchymal myofibroblasts (P = 0.0003, 0.0143, and 0.0488, respectively). We show the potential role of GMFs in sustaining Lgr5+ stem cell activity and affecting normal gastric epithelial differentiation and proliferation.
Futrega, K;Shajib, M;Robey, P;Doran, M;
| DOI: 10.3390/organoids2020008
(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 105 BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 105 human umbilical cord blood CD34+ cells. (3) Results: Micro-ossicles contained more human CD45+ cells, but fewer human CD34+ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34+ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation.
Obst, J;Hall-Roberts, HL;Smith, TB;Kreuzer, M;Magno, L;Di Daniel, E;Davis, JB;Mead, E;
PMID: 34615897 | DOI: 10.1038/s41598-021-96144-7
Human genetic studies have linked rare coding variants in microglial genes, such as TREM2, and more recently PLCG2 to Alzheimer's disease (AD) pathology. The P522R variant in PLCG2 has been shown to confer protection for AD and to result in a subtle increase in enzymatic activity. PLCγ2 is a key component of intracellular signal transduction networks and induces Ca2+ signals downstream of many myeloid cell surface receptors, including TREM2. To explore the relationship between PLCγ2 and TREM2 and the role of PLCγ2 in regulating immune cell function, we generated human induced pluripotent stem cell (iPSC)- derived macrophages from isogenic lines with homozygous PLCG2 knockout (Ko). Stimulating TREM2 signalling using a polyclonal antibody revealed a complete lack of calcium flux and IP1 accumulation in PLCγ2 Ko cells, demonstrating a non-redundant role of PLCγ2 in calcium release downstream of TREM2. Loss of PLCγ2 led to broad changes in expression of several macrophage surface markers and phenotype, including reduced phagocytic activity and survival, while LPS-induced secretion of the inflammatory cytokines TNFα and IL-6 was unaffected. We identified additional deficits in PLCγ2- deficient cells that compromised cellular adhesion and migration. Thus, PLCγ2 is key in enabling divergent cellular functions and might be a promising target to increase beneficial microglial functions.
Hingert D, Barreto Henriksson H, Brisby H.
PMID: 28978269 | DOI: 10.1089/ten.TEA.2017.0087
Abstract
BACKGROUND:
Low back pain is one of the most common ailments in western countries afflicting more than 80% of the population and the main cause is considered to be degeneration of intervertebral discs (IVDs). IL-1β is a vital inflammatory cytokine found in abundance in degenerated disc environment whereas BMP-3 is believed to promote chondrogenesis through TGF-β pathway.
AIM:
The aim was to study the effects of BMP-3, IL-1β and combination (pre-treatment with IL-1β) on hMSCs encapsulated in PuraMatrix™ hydrogel (Phg) especially in the absence of TGF-β in order to investigate the proliferation, and differentiation ability of hMSCs over 28 days period.
METHOD:
100µL of hMSCs cell suspension was encapsulated between two layers of 100 µL hydrogels forming a sandwich-like structure. The encapsulated hMSCs were cultured in two sets of media, chondrogenic (C) and non-chondrogenic (nC) media along with addition of BMP-3 (10ng/mL) and IL-1β (10ng/mL). To study the combined effects of BMP-3 and IL-1β, the encapsulated hMSCs were first pre-treated with relevant media containing IL-1β for 24 hours, and then the media was replaced by media containing BMP-3 for the remaining experimental time period. IL-1β pre-treatment was carried out in both C and nC media. The samples were collected at day 7, 14, and 28.
RESULTS:
Proliferation and differentiation of hMSCs into chondrocyte-like cells was observed in all samples. Proteoglycans accumulation was observed in pre-treatment samples in C media. The protein and gene expression of Sox-9 and COL2A1 respectively, showed the occurrence of chondrogenesis in all samples.
CONCLUSION:
High cell viability, proliferation and differentiation was achieved in this in vitro model confirming that BMP-3 alone in the absence of TGF-β could drive hMSCs into chondrogenic lineage. Pre-treatment with IL-1β followed by BMP-3 stimulation resulted in high proteoglycans accumulation compared to stimulation with growth factors or cytokine alone. This suggests that pre-treatment with a pro-inflammatory cytokine before driving them into a chondrogeneic lineage might be of importance also in vivo.
Vange P, Bruland T, Beisvag V, Erlandsen SE, Flatberg A, Doseth B, Sandvik AK, Bakke I.
PMID: 26178168 | DOI: 10.1002/path.4591.
The oxyntic proliferative isthmus zone contains the main stem/progenitor cells that provide for physiological renewal of the distinct mature cell lineages in the oxyntic epithelium of the stomach. These cells are also proposed to be the potential cells-of-origin of gastric cancer although little is known about their molecular characteristics, and specific biological markers are lacking. In this study, we developed a method for serial section navigated laser microdissection to isolate cells from the proliferative isthmus zone of rat gastric oxyntic mucosa for genome-wide microarray gene expression analysis. Enrichment analysis showed a distinct gene expression profile for the isthmus zone, with genes regulating intracellular processes such as the cell cycle and ribosomal activity. The profile was also related to stem cell transcriptional networks and stomach neoplasia. Genes expressed uniquely in the isthmus zone were associated with E2F transcription factor 1 (E2F1), which participates in self-renewal of stem cells and in gastric carcinogenesis. One of the unique genes was Aspm (Asp (Abnormal Spindle) Homolog, Microcephaly Associated (Drosophila)). Here we show ASPM in single scattered epithelial cells located in the proliferative isthmus zone of rat, mouse and human oxyntic mucosa, which do not seem to be actively dividing. The ASPM-expressing cells are mainly mature but cell marker-deficient, except for a limited overlap with cells with neuroendocrine and tuft cell features. Further, both ASPM and E2F1 were expressed in human gastric cancer cell lines, and increased and correlated in human gastric adenocarcinomas compared to non-tumour mucosa as shown by expression profile analyses and immunohistochemistry. The association between ASPM and the transcription factor E2F1 in gastric tissue is relevant due to their common involvement in crucial cell-fate regulatory mechanisms. Our results thus introduce ASPM as a novel possible oxyntic stem/progenitor cell marker that may be involved in both normal gastric physiology and gastric carcinogenesis.
McGill Science Undergraduate Research Journal
Niu, Z;Capolicchio, T;
| DOI: 10.26443/msurj.v18i1.194
Adult hippocampal neurogenesis (AHN) is a well-studied phenomenon that involves the derivation of new neurons from neural progenitor cells in the dentate gyrus region of the hippocampus, an area responsible for cognitive functions such as learning and memory storage. Moreover, the hippocampus is known to be implicated in neurological conditions such as Alzheimer's disease. Although AHN has been extensively observed in animal models for twenty years, its existence and persistence in humans have been widely debated in academia, heavily based on post-mortem immunohistochemical markers. Using the search engines PubMed and Google Scholar for “Adult Human Neurogenesis,” 143 articles that were most relevant to the history of AHN discovery, detection in rodents, immunohistochemical studies on post-mortem human sections, and therapeutic development targeting AHN were reviewed. This review article highlights the current understanding of AHN in rodents and humans, its implications in neurodegenerative diseases and therapeutics, and the inconsistencies and methodological variabilities encountered in studying AHN in humans. Furthermore, the correlation between AHN and diseases such as mood disorders and Alzheimer's disease is still not well established, with conflicting findings reported. Standardization of transcriptomic methodologies and increased availability of post-mortem human brain samples are crucial in advancing AHN research. This review article attempts to discover the fascinating and controversial world of adult human neurogenesis and its potential implications in treating neurological disorders. Apart from the discussion on AHN existence, tackling devastating diseases with this supplemental knowledge can lead to therapeutic advancements which greatly rely on understanding not only the presence of AHN but the mechanisms mediating its availability.
International journal of molecular sciences
Raslan, AA;Oh, YJ;Jin, YR;Yoon, JK;
PMID: 35328508 | DOI: 10.3390/ijms23063089
The lungs have a remarkable ability to regenerate damaged tissues caused by acute injury. Many lung diseases, especially chronic lung diseases, are associated with a reduced or disrupted regeneration potential of the lungs. Therefore, understanding the underlying mechanisms of the regenerative capacity of the lungs offers the potential to identify novel therapeutic targets for these diseases. R-spondin2, a co-activator of WNT/β-catenin signaling, plays an important role in embryonic murine lung development. However, the role of Rspo2 in adult lung homeostasis and regeneration remains unknown. The aim of this study is to determine Rspo2 function in distal lung stem/progenitor cells and adult lung regeneration. In this study, we found that robust Rspo2 expression was detected in different epithelial cells, including airway club cells and alveolar type 2 (AT2) cells in the adult lungs. However, Rspo2 expression significantly decreased during the first week after naphthalene-induced airway injury and was restored by day 14 post-injury. In ex vivo 3D organoid culture, recombinant RSPO2 promoted the colony formation and differentiation of both club and AT2 cells through the activation of canonical WNT signaling. In contrast, Rspo2 ablation in club and AT2 cells significantly disrupted their expansion capacity in the ex vivo 3D organoid culture. Furthermore, mice lacking Rspo2 showed significant defects in airway regeneration after naphthalene-induced injury. Our results strongly suggest that RSPO2 plays a key role in the adult lung epithelial stem/progenitor cells during homeostasis and regeneration, and therefore, it may be a potential therapeutic target for chronic lung diseases with reduced regenerative capability.
Hilkens J, Timmer NC, Boer M, Ikink GJ, Schewe M, Sacchetti A, Koppens MA, Song JY, Bakker ER.
PMID: 27511199 | DOI: 10.1136/gutjnl-2016-311606
Abstract
OBJECTIVE:
The gross majority of colorectal cancer cases results from aberrant Wnt/β-catenin signalling through adenomatous polyposis coli (APC) or CTNNB1 mutations. However, a subset of human colon tumours harbour, mutually exclusive with APC and CTNNB1 mutations, gene fusions in RSPO2 or RSPO3, leading to enhanced expression of these R-spondin genes. This suggested that RSPO activation can substitute for the most common mutations as an alternative driver for intestinal cancer. Involvement of RSPO3 in tumour growth was recently shown in RSPO3-fusion-positive xenograft models. The current study determines the extent into which solely a gain in RSPO3 actually functions as a driver of intestinal cancer in a direct, causal fashion, and addresses the in vivo activities of RSPO3 in parallel.
DESIGN:
We generated a conditional Rspo3 transgenic mouse model in which the Rspo3 transgene is expressed upon Cre activity. Cre is provided by cross-breeding with Lgr5-GFP-CreERT2 mice.
RESULTS:
Upon in vivo Rspo3 expression, mice rapidly developed extensive hyperplastic, adenomatous and adenocarcinomatous lesions throughout the intestine. RSPO3 induced the expansion of Lgr5+ stem cells, Paneth cells, non-Paneth cell label-retaining cells and Lgr4+ cells, thus promoting both intestinal stem cell and niche compartments. Wnt/β-catenin signalling was modestly increased upon Rspo3 expression and mutant Kras synergised with Rspo3 in hyperplastic growth.
CONCLUSIONS:
We provide in vivo evidence that RSPO3 stimulates the crypt stem cell and niche compartments and drives rapid intestinal tumorigenesis. This establishes RSPO3 as a potent driver of intestinal cancer and proposes RSPO3 as a candidate target for therapy in patients with colorectal cancer harbouring RSPO3 fusions.
Kim M, von Muenchow L, Le Meur T, Kueng B, Gapp B, Weber D, Dietrich W, Kovarik J, Rolink AG, Ksiazek I.
PMID: 29709545 | DOI: 10.1016/j.imlet.2018.04.008
Dipeptidyl peptidase 9 (DPP9) is a ubiquitously expressed intracellular prolyl peptidase implicated in immunoregulation. However, its physiological relevance in the immune system remains largely unknown. We investigated the role of DPP9 enzyme in immune system by characterizing DPP9 knock-in mice expressing a catalytically inactive S729A mutant of DPP9 enzyme (DPP9ki/ki mice). DPP9ki/ki mice show reduced number of lymphoid and myeloid cells in fetal liver and postnatal blood but their hematopoietic cells are fully functional and able to reconstitute lymphoid and myeloid lineages even in competitive mixed chimeras. These studies demonstrate that inactivation of DPP9 enzymatic activity does not lead to any perturbations in mouse hematopoiesis.
Gioftsidi, S;Relaix, F;Mourikis, P;
PMID: 35459219 | DOI: 10.1186/s13395-022-00293-w
Skeletal muscle stem cells have a central role in muscle growth and regeneration. They reside as quiescent cells in resting muscle and in response to damage they transiently amplify and fuse to produce new myofibers or self-renew to replenish the stem cell pool. A signaling pathway that is critical in the regulation of all these processes is Notch. Despite the major differences in the anatomical and cellular niches between the embryonic myotome, the adult sarcolemma/basement-membrane interphase, and the regenerating muscle, Notch signaling has evolved to support the context-specific requirements of the muscle cells. In this review, we discuss the diverse ways by which Notch signaling factors and other modifying partners are operating during the lifetime of muscle stem cells to establish an adaptive dynamic network.
Stem Cell Reports. 2015 Jun 3.
Finkbeiner SR, Hill DR, Altheim CH, Dedhia PH, Taylor MJ, Tsai YH, Chin AM, Mahe MM, Watson CL, Freeman JJ, Nattiv R, Thomson M, Klein OD, Shroyer NF, Helmrath MA, Teitelbaum DH, Dempsey PJ, Spence JR.
PMID: 26067134
Human intestinal organoids (HIOs) are a tissue culture model in which small intestine-like tissue is generated from pluripotent stem cells. By carrying out unsupervised hierarchical clustering of RNA-sequencing data, we demonstrate that HIOs most closely resemble human fetal intestine. We observed that genes involved in digestive tract development are enriched in both fetal intestine and HIOs compared to adult tissue, whereas genes related to digestive function and Paneth cell host defense are expressed at higher levels in adult intestine. Our study also revealed that the intestinal stem cell marker OLFM4 is expressed at very low levels in fetal intestine and in HIOs, but is robust in adult crypts. We validated our findings using in vivo transplantation to show that HIOs become more adult-like after transplantation. Our study emphasizes important maturation events that occur in the intestine during human development and demonstrates that HIOs can be used to model fetal-to-adult maturation.
Abstract Aims Intestinal stem cell (ISC) markers such as LGR5, ASCL2, EPHB2 and OLFM4 and their clinical implications have been extensively studied in colorectal cancers (CRCs). However, little is known about their expression in precancerous lesions of CRCs. Here, we investigated the expression and distribution of ISC markers in serrated polyps and conventional adenomas. Methods and results RT-PCR analysis revealed that all ISC markers were significantly upregulated in conventional adenomas with low grade dysplasia (CALGs) compared with other lesions. RNA in situ hybridization confirmed that CALGs exhibited strong and diffuse expression of all ISC markers, which indicate a stem cell-like phenotype. However, normal colonic mucosa hyperplastic polyps and sessile serrated adenomas harbored LGR5+ cells that were confined to the crypt base and demonstrated an organized expression of ISC markers. Notably, in traditional serrated adenomas, expression of LGR5 and ASCL2 was localized to the ectopic crypts as in the normal crypts, but expression of EPHB2 and OLFM4 was distributed in a diffuse manner, which is suggestive of a progenitor-like features. Conclusions The expression and distribution profile of ISC markers possibly provides insights into the organization of stem and progenitor-like cells in each type of precancerous lesion of CRC
