Richard Poulsom

Lgr5+ve Stem Cells Drive Self-Renewal in the Stomach and Build Long-Lived Gastric Units In Vitro

The study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5(+ve) cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5(+ve) cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5(+ve) cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

Hepatocytes from non-hepatic adult stem cells

Stem cells are undifferentiated long-lived cells that are capable of many rounds of division. Here we show that adult human liver cells can be derived from stem cells originating in the bone marrow or circulating outside the liver, raising the possibility that blood-system stem cells could be used clinically to generate hepatocytes for replacing damaged tissue.

Bone marrow contributes to renal parenchymal turnover and regeneration.

In order to establish whether extra‐renal cells contribute to the turnover and repair of renal tissues, this study examined kidneys of female mice that had received a male bone marrow transplant and kidney biopsies from male patients who had received kidney transplants from female donors. By using in situ hybridization to detect Y‐chromosomes it could be demonstrated that circulating stem cells frequently engraft into the kidney and differentiate into renal parenchymal cells. In the human renal grafts it was confirmed that some of the recipient‐derived cells within the kidney exhibited a tubular epithelial phenotype, by combining in situ hybridization with immunostaining for the epithelial markers CAM 5.2 and the lectin Ulex europaeus. Female mouse recipients of male bone marrow grafts showed co‐localization of Y‐chromosomes and tubular epithelial markers Ricinus communis and Lens culinaris, and a specific cytochrome P450 enzyme (CYP1A2) indicating an appropriate functional capability of clustered newly formed marrow‐derived tubular epithelial cells. Y‐chromosome‐containing cells were observed within glomeruli, with morphology and location appropriate for podocytes. Within the murine kidney, these Y‐chromosome‐positive cells were negative for the mouse macrophage marker F4/80 antigen and leukocyte common antigen, but were vimentin‐positive. The presence of bone marrow‐derived cells was noted in both histologically normal mouse kidneys and in human transplanted kidneys suffering damage from a variety of causes. These data indicate that bone marrow cells contribute to both normal turnover of renal epithelia and regeneration after damage, and it is suggested that this could be exploited therapeutically. Copyright

Cell differentiation: Hepatocytes from non-hepatic adultstem cells

Stem cells are undifferentiated long-lived cells that are capable of many rounds of division. Here we show that adult human liver cells can be derived from stem cells originating in the bone marrow or circulating outside the liver, raising the possibility that blood-system stem cells could be used clinically to generate hepatocytes for replacing damaged tissue.

Bone Marrow Contribution to Tumor-Associated Myofibroblasts and Fibroblasts

The role of myofibroblasts in tissue repair and fibrosis is well documented, but the source of these myofibroblasts is unclear. There is evidence of a circulating population of fibrocytes that can home to areas of injury and contribute to myofibroblast populations. Previously, we have shown that the bone marrow is a source of myofibroblasts for many tissues including the gut, lung, and kidney and that this phenomenon is exacerbated by injury. We now show that the bone marrow can contribute to myofibroblast and fibroblast populations in tumor stroma in a mouse model of pancreatic insulinoma. Mice transgenic for the rat insulin promoter II gene linked to the large-T antigen of SV40 (RIPTag) develop solid β-cell tumors of the pancreas. Approximately 25% of myofibroblasts in these pancreatic tumors were donor-derived, and these were concentrated toward the edge of the tumor. Thus, the development of tumor stroma is at least in part a systemic response that may ultimately yield methods of targeting new therapy.

Up-regulation of Delta-like 4 Ligand in Human Tumor Vasculature and the Role of Basal Expression in Endothelial Cell Function

The Notch signaling pathway and the delta-like 4 ligand (DLL4) play key roles in embryonic vascular development. Many of the pathways involved in embryonic vascular development also play important roles in tumor angiogenesis. In this study, we assessed the expression of DLL4 in primary renal cancer and investigated the biological function of DLL4 in primary endothelial cells. Using real-time quantitative PCR and in situ hybridization, we showed that the expression of DLL4 was up-regulated within the vasculature of clear cell-renal cell carcinoma almost 9-fold more than normal kidney and was correlated with the expression of vascular endothelial growth factor (VEGF). The expression of DLL4 in endothelial cells was up-regulated by VEGF and basic fibroblast growth factor synergistically, and by hypoxia through hypoxia-inducible factor 1alpha. Down-regulation of DLL4 expression with RNA interference led to decreased expression of HEY1 and EphrinB2, and the inhibition of endothelial cell proliferation, migration, and network formation, all of which are important processes in tumor angiogenesis. The inhibition of proliferation occurred via the induction of cell cycle arrest in G0-G1 by increased expression of p21 and decreased phosphorylation of retinoblastoma. We conclude that an optimal window of the DLL4 expression is essential for tumor angiogenesis and that selective modulation of the DLL4 expression within human tumors may represent a potential novel antiangiogenic therapy.

Adult stem cell plasticity

Observations made in the last few years support the existence of pathways, in adult humans and rodents, that allow adult stem cells to be surprisingly flexible in their differentiation repertoires. Termed plasticity, this property allows adult stem cells, assumed, until now, to be committed to generating a fixed range of progeny, to switch, when they have been relocated, to make other specialized sets of cells appropriate to their new niche. Reprogramming of some adult stem cells can occur in vivo; the stem cells normally resident in bone marrow appear particularly flexible and are able to contribute usefully to multiple recipient organs. This process produces cells with specialized structural and metabolic adaptations commensurate with their new locations. In a few examples, the degree of support is sufficient to assist or even rescue recipient mice from genetic defects. Some studies provide evidence for the expansion of the reprogrammed cells locally, but in most it remains possible that cells arrive and redifferentiate, but are no longer stem cells. Nevertheless, the fact that appropriately differentiated cells are delivered deep within organs simply by injection of bone marrow cells should make us think differently about the way that organs regenerate and repair. Migratory pathways for stem cells in adult organisms may exist that could be exploited to effect repairs using an individuals own stem cells, perhaps after gene therapy. Logical extensions of this concept are that a transplanted organ would become affected by the genetic susceptibilities of the recipient, alleles that re‐express themselves via marrow‐derived stem cells, and that plasticity after bone marrow transplantation would also transfer different phenotypes, affecting important parameters such as susceptibility to long‐term complications of diabetes, or the ability to metabolize drugs in the liver. This article reviews some of the evidence for stem cell plasticity in rodents and man. Copyright

Bone marrow derivation of pericryptal myofibroblasts in the mouse and human small intestine and colon

Background and aims: In order to establish whether extraintestinal cells contribute to the turnover and repair of gastrointestinal tissues, we studied the colons and small intestines of female mice that had received a male bone marrow transplant, together with gastrointestinal biopsies from female patients that had developed graft versus host disease after receiving a bone marrow transplant from male donors. Methods: Using in situ hybridisation to detect Y chromosomes and immunohistochemistry, we demonstrated that cells derived from injected bone marrow frequently engrafted into the intestine and differentiated into pericryptal myofibroblasts. Results: In the human intestine, we confirmed by combining in situ hybridisation with immunostaining for smooth muscle actin that the bone marrow derived cells within the intestine exhibited a myofibroblast phenotype. In female mouse recipients of male bone marrow grafts, we observed colocalisation of Y chromosomes and clusters of newly formed marrow derived myofibroblasts. While few of these were present at seven days after bone marrow transplantation, they were numerous at 14 days, and by six weeks entire columns of pericryptal myofibroblasts could be seen running up the sides of crypts in both the small intestine and colon. These columns appeared to extend into the villi in the small intestine. Within the intestinal lamina propria, these Y chromosome positive cells were negative for the mouse macrophage marker F4/80 antigen and CD34. Conclusions: Bone marrow derived pericryptal myofibroblasts were present in the mouse intestine following irradiation and bone marrow transplant, and in the intestines of human patients suffering graft versus host disease following a bone marrow transplant. Our data indicate that bone marrow cells contribute to the regeneration of intestinal myofibroblasts and epithelium after damage, and we suggest that this could be exploited therapeutically.

Multiple Organ Engraftment by Bone‐Marrow‐Derived Myofibroblasts and Fibroblasts in Bone‐Marrow‐Transplanted Mice

Myofibroblasts are ubiquitous cells with features of both fibroblasts and smooth muscle cells. We suggest that the bone marrow can contribute to myofibroblast populations in a variety of tissues and that this is exacerbated by injury. To assess this, female mice were transplanted with male bone marrow and the male cells were tracked throughout the body and identified as myofibroblasts. Skin wounding and paracetamol administration were used to assess whether myofibroblast engraftment was modulated by damage. Following radiation injury, a proportion of myofibroblasts in the lung, stomach, esophagus, skin, kidney, and adrenal capsule were bone‐marrow derived. In the lung, there was significantly greater engraftment following paracetamol administration (17% versus 41% p < 0.005). Bone‐marrow‐derived fibroblasts were also found. We suggest that bone marrow contributes to a circulating population of cells and, in the context of injury, these cells are recruited and contribute to tissue repair.

Lrig1 controls intestinal stem-cell homeostasis by negative regulation of ErbB signalling

Maintenance of adult tissues is carried out by stem cells and is sustained throughout life in a highly ordered manner. Homeostasis within the stem-cell compartment is governed by positive- and negative-feedback regulation of instructive extrinsic and intrinsic signals. ErbB signalling is a prerequisite for maintenance of the intestinal epithelium following injury and tumour formation. As ErbB-family ligands and receptors are highly expressed within the stem-cell niche, we hypothesize that strong endogenous regulators must control the pathway in the stem-cell compartment. Here we show that Lrig1, a negative-feedback regulator of the ErbB receptor family, is highly expressed by intestinal stem cells and controls the size of the intestinal stem-cell niche by regulating the amplitude of growth-factor signalling. Intestinal stem-cell maintenance has so far been attributed to a combination of Wnt and Notch activation and Bmpr inhibition. Our findings reveal ErbB activation as a strong inductive signal for stem-cell proliferation. This has implications for our understanding of ErbB signalling in tissue development and maintenance and the progression of malignant disease.