Rosemary Jeffery

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 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.

Lineage tracing reveals multipotent stem cells maintain human adenomas and the pattern of clonal expansion in tumor evolution

Significance The organization of cells within human colorectal adenomas, and specifically whether the tumors are maintained by stem cells, is unclear. Furthermore, the patterns of clonal evolution leading to the development of a malignant tumor have not been determined. We performed lineage tracing in human adenomas using a combination of nuclear and mitochondrial DNA lesions and epigenetic markers. Our data identify a stem cell population within adenomas and suggest that new growth of intratumor clones occurs infrequently, not as a steady continual process as often is assumed. Our work offers a unique insight into human cancer development. The genetic and morphological development of colorectal cancer is a paradigm for tumorigenesis. However, the dynamics of clonal evolution underpinning carcinogenesis remain poorly understood. Here we identify multipotential stem cells within human colorectal adenomas and use methylation patterns of nonexpressed genes to characterize clonal evolution. Numerous individual crypts from six colonic adenomas and a hyperplastic polyp were microdissected and characterized for genetic lesions. Clones deficient in cytochrome c oxidase (CCO−) were identified by histochemical staining followed by mtDNA sequencing. Topographical maps of clone locations were constructed using a combination of these data. Multilineage differentiation within clones was demonstrated by immunofluorescence. Methylation patterns of adenomatous crypts were determined by clonal bisulphite sequencing; methylation pattern diversity was compared with a mathematical model to infer to clonal dynamics. Individual adenomatous crypts were clonal for mtDNA mutations and contained both mucin-secreting and neuroendocrine cells, demonstrating that the crypt contained a multipotent stem cell. The intracrypt methylation pattern was consistent with the crypts containing multiple competing stem cells. Adenomas were epigenetically diverse populations, suggesting that they were relatively mitotically old populations. Intratumor clones typically showed less diversity in methylation pattern than the tumor as a whole. Mathematical modeling suggested that recent clonal sweeps encompassing the whole adenoma had not occurred. Adenomatous crypts within human tumors contain actively dividing stem cells. Adenomas appeared to be relatively mitotically old populations, pocketed with occasional newly generated subclones that were the result of recent rapid clonal expansion. Relative stasis and occasional rapid subclone growth may characterize colorectal tumorigenesis.

The stem cell organisation, and the proliferative and gene expression profile of Barrett's epithelium, replicates pyloric-type gastric glands

Objective Barretts oesophagus shows appearances described as ‘intestinal metaplasia’, in structures called ‘crypts’ but do not typically display crypt architecture. Here, we investigate their relationship to gastric glands. Methods Cell proliferation and migration within Barretts glands was assessed by Ki67 and iododeoxyuridine (IdU) labelling. Expression of mucin core proteins (MUC), trefoil family factor (TFF) peptides and LGR5 mRNA was determined by immunohistochemistry or by in situ hybridisation, and clonality was elucidated using mitochondrial DNA (mtDNA) mutations combined with mucin histochemistry. Results Proliferation predominantly occurs in the middle of Barretts glands, diminishing towards the surface and the base: IdU dynamics demonstrate bidirectional migration, similar to gastric glands. Distribution of MUC5AC, TFF1, MUC6 and TFF2 in Barretts mirrors pyloric glands and is preserved in Barretts dysplasia. MUC2-positive goblet cells are localised above the neck in Barretts glands, and TFF3 is concentrated in the same region. LGR5 mRNA is detected in the middle of Barretts glands suggesting a stem cell niche in this locale, similar to that in the gastric pylorus, and distinct from gastric intestinal metaplasia. Gastric and intestinal cell lineages within Barretts glands are clonal, indicating derivation from a single stem cell. Conclusions Barretts shows the proliferative and stem cell architecture, and pattern of gene expression of pyloric gastric glands, maintained by stem cells showing gastric and intestinal differentiation: neutral drift may suggest that intestinal differentiation advances with time, a concept critical for the understanding of the origin and development of Barretts oesophagus.

Haematopoietic lineage-committed bone marrow cells, but not cloned cultured mesenchymal stem cells, contribute to regeneration of renal tubular epithelium after HgCl2-induced acute tubular injury

Abstract.  Objective: Our previous studies have demonstrated that endogenous bone marrow cells (BMCs) contribute to renal tubular regeneration after acute tubular injury. The aim of this study was to examine which fraction of BMCs, haematopoietic lineage marrow cells (HLMCs) or mesenchymal stem cells (MSCs), are effective. Materials and methods: Six‐week‐old female mice were lethally irradiated and were transplanted with female enhanced green fluorescent protein‐positive (GFP+), plastic non‐adherent marrow cells (as a source of HLMCs) plus cloned cultured male GFP− MSCs. Four weeks later, they were assigned into two groups: control mice with vehicle treatment and mice treated with HgCl2. Tritiated thymidine was given 1 h before animal killing which occurred at intervals over 2 weeks. Kidney sections were stained for a tubular epithelial marker, cell origin indicated by GFP immunohistochemistry or Y chromosome in situ hybridization; periodic acid‐Schiff staining was performed, and samples were subjected to autoradiography. One thousand consecutive renal tubular epithelial cells per mouse, in S phase, were scored as either female (indigenous) GFP+ (HLMC‐derived) or male (MSC‐derived). Results: Haematopoietic lineage marrow cells and MSCs stably engrafted into bone marrow and spleen, but only HLMC‐derived cells, not MSCs, were found in the renal tubules and were able to undergo DNA synthesis after acute renal injury. A few MSCs were detected in the renal interstitium, but their importance needs to be further explored. Conclusion: Haematopoietic lineage marrow cells, but not cloned cultured MSCs, can play a role not only in normal wear‐and‐tear turnover of renal tubular cells, but also in repair after tubular injury.

Trefoil Peptide Gene Expression in Gastrointestinal Epithelial Cells in Inflammatory Bowel Disease

Trefoil peptides are a growing group of proteins with interesting structural and functional properties. We have defined the pattern of trefoil peptide gene expression in the ulceration-associated cell lineage (UACL) and in the nearby mucosa in Crohns disease. In the UACL, human spasmolytic polypeptide (hSP) mRNA is expressed in the acinar and proximal duct cells, while pS2 mRNA and peptide are found in the distal duct cells and in the surface cells. In adjacent mucosa, pS2 mRNA and protein are expressed by goblet cells, with the pS2 peptide concentrated in the area of the Golgi and also in the theca. Ultrastructural immunolocalisation showed the pS2 to be co-packaged in the mucous cell granules before being secreted into the intestinal lumen. In addition, pS2 peptide was demonstrated in local neuroendocrine cells and was also co-packaged with the neuroendocrine granules. The crypts associated with the UACL also showed marked neuroendocrine cell hyperplasia. We conclude that pS2 peptide is secreted locally into the viscoelastic coat covering the intestinal mucosa which surrounds Crohns disease ulcers. In addition, it is clear that intestinal goblet cells, in addition to producing mucins, are a rich source of regulatory peptides. Moreover, pS2 is clearly co-packaged with neurosecretory granules, which are released through basal and lateral membranes so that the contained peptides can act in a paracrine manner. These findings are interpreted in terms of the epidermal growth factor/urogastrone released by the UACL, stimulating pS2 gene expression in surrounding cells.(ABSTRACT TRUNCATED AT 250 WORDS)

DUOX2 and DUOXA2 form the predominant enzyme system capable of producing the reactive oxygen species H2O2 in active ulcerative colitis and are modulated by 5-aminosalicylic acid.

Background:NADPH oxidase–derived reactive oxygen species, such as H2O2, are part of the intestinal innate immune system but may drive carcinogenesis through DNA damage. We sought to identify the predominant enzyme system capable of producing H2O2 in active ulcerative colitis and assess whether it is affected by 5-aminosalicylic acid (5-ASA). Methods:We studied human mucosal biopsies by expression arrays, quantitative real-time polymerase chain reaction for NADPH oxidase family members, in situ hybridization (DUOX2 and DUOXA2) and immunofluorescence for DUOX, 8-OHdG (DNA damage), and &ggr;H2AX (DNA damage response) and sought effects of 5-ASA on ex vivo cultured biopsies and cultured rectal cancer cells. Results:DUOX2 with maturation partner DUOXA2 forms the predominant system for H2O2 production in human colon and is upregulated in active colitis. DUOX2 in situ is exclusively epithelial, varies between and within individual crypts, and increases near inflammation. 8-OHdG and &ggr;H2AX were observed in damaged crypt epithelium. 5-ASA upregulated DUOX2 and DUOXA2 levels in the setting of active versus quiescent disease and altered DUOX2 expression in cultured biopsies. Ingenuity pathway analysis confirmed that inflammation status and 5-ASA increase expression of DUOX2 and DUOXA2. An epithelial cell model confirmed that cultured cancer cells expressed DUOX protein and produced H2O2 in response to hypoxia and 5-ASA exposure. Conclusions:Both DUOX2 and DUOXA2 expression are involved specifically in inflammation and are regulated on a crypt-by-crypt basis in ulcerative colitis tissues. Synergy between inflammation, hypoxia, and 5-ASA to increase H2O2 production could explain how 5-ASA supports innate defense, although potentially increasing the burden of DNA damage.

Exogenous bone marrow cells do not rescue non-irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimaerism and cell proliferation in bone marrow and spleen

Abstract.  Objective: Various studies have shown that bone marrow stem cells can rescue mice from acute renal tubular damage under a conditioning advantage (irradiation or cisplatin treatment) favouring donor cell engraftment and regeneration; however, it is not known whether bone marrow cells (BMCs) can contribute to repair of acute tubular damage in the absence of a selection pressure for the donor cells. The aim of this study was to examine this possibility. Materials and methods: Ten‐week‐old female mice were assigned into control non‐irradiated animals having only vehicle treatment, HgCl2‐treated non‐irradiated mice, HgCl2‐treated non‐irradiated mice infused with male BMCs 1 day after HgCl2, and vehicle‐treated mice with male BMCs. Tritiated thymidine was given 1 h before animal killing. Results: Donor BMCs could not alleviate non‐irradiated mice from acute tubular damage caused by HgCl2, deduced by no reduction in serum urea nitrogen combined with negligible cell engraftment. However, donor BMCs could home to the bone marrow and spleen and display proliferative activity. This is the first report to show that despite no preparative myeloablation of recipients, engrafted donor BMCs can synthesize DNA in the bone marrow and spleen. Conclusions: Exogenous BMCs do not rescue non‐irradiated mice from acute renal tubular damage caused by HgCl2, despite establishment of chimerism and cell proliferation in bone marrow and spleen.

Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous, not bone marrow-derived cells

To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony‐stimulating factor (P‐GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P‐GCSF+, EGF+, P‐GCSF+EGF+, HgCl2, HgCl2+P‐GCSF+, HgCl2+EGF+ and HgCl2+P‐GCSF+EGF+. Following HgCl2, injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF‐treated groups were resistant to this acute kidney injury. A four‐in‐one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S‐phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P‐GCSF. Only 0.5% proximal tubular cells were seen in S‐phase in the undamaged group kidneys; this increased to 7–8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S‐phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM‐derived cells were less responsive. P‐GCSF did not influence damage or regeneration.

Remodelling of microRNAs in colorectal cancer by hypoxia alters metabolism profiles and 5-fluorouracil resistance

Abstract Solid tumours have oxygen gradients and areas of near and almost total anoxia. Hypoxia reduces sensitivity to 5-fluorouracil (5-FU)-chemotherapy for colorectal cancer (CRC). MicroRNAs (miRNAs) are hypoxia sensors and were altered consistently in six CRC cell lines (colon cancer: DLD-1, HCT116 and HT29; rectal cancer: HT55, SW837 and VACO4S) maintained in hypoxia (1 and 0.2% oxygen) compared with normoxia (20.9%). CRC cell lines also showed altered amino acid metabolism in hypoxia and hypoxia-responsive miRNAs were predicted to target genes in four metabolism pathways: beta-alanine; valine, leucine, iso-leucine; aminoacyl-tRNA; and alanine, aspartate, glutamate. MiR-210 was increased in hypoxic areas of CRC tissues and hypoxia-responsive miR-21 and miR-30d, but not miR-210, were significantly increased in 5-FU resistant CRCs. Treatment with miR-21 and miR-30d antagonists sensitized hypoxic CRC cells to 5-FU. Our data highlight the complexity and tumour heterogeneity caused by hypoxia. MiR-210 as a hypoxic biomarker, and the targeting of miR-21 and miR-30d and/or the amino acid metabolism pathways may offer translational opportunities.