Bone tissue formation from human embryonic stem cells in vivo.

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.

Fluorescence-activated single cell sorting of human embryonic stem cells.

Human embryonic stem cells (hESC) are the subject of intense investigation for use in regenerative medicine, in toxicity testing, and as models for the study of human development. Automated cell sorting will enhance the isolation of homogenous pools of differentiated hESCs both for basic studies and for therapeutic applications. Sorting could also be used to deplete undifferentiated, potentially tumourigenic cells. However, hESCs are sensitive to single cell disaggregation and recover poorly when plated at clonal density. Here we report a method for successful semi-automated single cell sorting of hESCs. This method utilizes an ES-specific promoter-transgene construct and automated FACS-based single cell sorting and plating. Clonal recovery in physiologic oxygen (2%) was increased fourfold over room oxygen (21%; p < 0.01). This automated protocol will help to realize proposed hESC strategies that are hampered by low throughput and poor yields.

Renal expression of interleukin-2 receptor mRNA in patients with crescentic glomerulonephritis.

We studied paraffin sections of renal biopsies from 7 patients with crescentic glomerulonephritis (CGN) by in situ hybridisation, to detect sites of interleukin-2 receptor (IL-2R) mRNA expression. Frozen sections from a further patient with CGN were studied by immunohistochemistry with a monoclonal antibody to CD25, to detect IL-2R protein. Positive control sections were taken from biopsies of acute cellular renal transplant rejection and negative controls from biopsies of membranous glomerulonephritis. No autoradiographic signal was detected in negative controls. IL-2R mRNA expression was seen in rejected transplants and in sections from 4 to 7 patients with CGN. Expression was seen in cortical interstitial cells, renal tubular epithelial cells, cells within glomerular crescents and in one glomerulus at the margin of Bowman's capsule. Tubular cell expression of IL-2R protein was confirmed by immunohistochemistry. We have confirmed that IL-2R mRNA expression occurs locally within the kidneys in CGN and have identified expression in tubular epithelial cells. The results suggest that local activation of immunocompetent cells occurs in the kidney and may be of significance in the pathogenesis of CGN.

Expression of tissue kallikrein in human kidney.

1. We studied the distribution of human tissue kallikrein mRNA in normal and diseased kidney, using in situ hybridization, together with immunohistochemical localization of renal kallikrein protein. Materials studied were (a) normal tissue from kidneys removed because of localized renal carcinoma, (b) kidneys removed because of post-traumatic haemorrhage and (c) renal biopsy specimens from patients with membranous glomerulonephritis and nephrotic syndrome. 2. A 1.35 kb EcoRI fragment of human tissue kallikrein cDNA was labelled with [32P]dCTP using the random-primer technique, and used for in situ hybridization. A specific rabbit antibody to active human urinary kallikrein was employed for immunocytochemistry, using a peroxidase-antiperoxidase method. 3. By in situ hybridization, no tissue kallikrein gene expression was seen in the carcinoma nephrectomy specimens. Positive expression was seen in the trauma nephrectomy tissue, and in four of five nephrotic syndrome biopsies. In all kidneys, expression was confined to the renal cortex. The dominant site of gene expression was the distal tubule. Apart from one area of positive signal related to an epithelial cell of Bowman's capsule, expression was not observed in glomeruli. Expression was also seen in the walls of large- and medium-sized blood vessels. 4. By immunohistochemistry, the dominant site of immunoreactivity was the distal tubule. Dense staining was also seen in granular peripolar cells and in isolated parietal epithelial cells close to the vascular pole. Isolated immunoreactive cells were seen in the media of large- and medium-sized arteries. 5. The tissue kallikrein gene in the kidney may not be constitutively expressed, but is expressed in response to physiological or pathological stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrarenal localization of interleukin-1 beta mRNA in crescentic glomerulonephritis.

Il-1 beta is a potent proinflammatory peptide, and induces expression of other cytokines which are involved in the immune response. Kidney biopsies from nine patients with crescentic GN were studied by in-situ hybridization to determine the site of expression of Il-1 beta mRNA. Biopsies from nine patients with nonproliferative renal disease were studied as negative controls, and tonsillar tissue was studied as a positive control. An Il-1 beta cDNA probe was 32P-labelled by random primers and hybridized to paraffin-embedded tissue sections after de-waxing. Il-1 beta mRNA was expressed in tonsil, but not in negative controls. Positive mRNA expression was seen in four of the nine crescentic biopsies. This was observed in interstitial cells with morphological characteristics of macrophages adjacent to tubular cells, in cells within the glomerular tuft, and in tubular epithelial cells. Il-1 beta mRNA is expressed in renal tissue in crescentic GN. Tubular and interstitial expression of Il-1 beta mRNA appears of equal prominence to glomerular expression. Intrarenal cytokine synthesis may be involved in the pathogenesis of crescentic glomerulonephritis.

Detection of cytomegalovirus antigens in phagocytosed serum complexes from a patient with rheumatoid arthritis, vasculitis, peripheral neuropathy, cutaneous ulceration, and digital gangrene.

A patient with rheumatoid arthritis, vasculitis, peripheral neuropathy, cutaneous ulceration, and digital gangrene was studied. Circulating immune complexes were detected by C1q binding although serum complement levels were within the normal range. Immunofluorescent staining of buffy coat cells with specific antisera showed the presence of IgG and IgM in phagocytosed inclusions but complement C3 was not detected. A monoclonal antibody specific for cytomegalovirus detected antigens in phagocytosed inclusions on one occasion. These results may suggest that cytomegalovirus antigens are a hitherto unidentified component of serum complexes in patients with rheumatoid arthritis and may contribute to the pathogenesis of the vasculitic complications of rheumatoid arthritis by participating in immune complex formation.

In situ hybridisation.

In situ hybridisation of mRNA in tissues or cell preparations is a powerful technique for studying gene expression. When combined with cell phenotyping with monoclonal antibodies it gives insights into the cellular basis of disease in vivo. The technique has also been used widely to identify foreign nucleic acids--for example, bacterial or viral, in host cells. The major disadvantages of this approach in the past have been that it was technically demanding, time consuming, and provided qualitative rather than quantitative results. Now, with the use of non-radioactive probes and improved imaging systems, the full potential of this form of molecular analysis is increasingly accessible and should generate rapid advances in many fields.

Do polyclonal rheumatoid factors carry an 'internal image' of cytomegalovirus, Epstein-Barr virus and nuclear antigens?

Rabbit antisera have been prepared against isolated rheumatoid factors (RF's). It was considered that RFs are anti-idiotype antibodies and that the anti-RF antisera had anti-anti-idiotype specificity. Furthermore, it was considered that the RF's carry an "internal image" of the putative "antigen X" and that the rabbit antisera would have specificity for this "antigen-X". Reactions of the rabbit antisera with the early and late antigens of CMV, EBV antigens and nuclear antigens suggest that there may be an "internal image" of these antigens in rheumatoid factor molecules and that they all may be related to the immunogenesis of RF.

Immunological studies of the placenta in systemic lupus erythematosus.

An immunological study was made of the placentae from 5 mothers with lupus erythematosus. 3 of the 5 mothers had anti-DNA antibodies in their sera at the time of delivery and in one of these anti-DNA antibodies were detected in the cord blood. This patient had active renal disease and serological evidence suggestive of circulating immune complexes in her blood at the time of delivery. Immunofluorescence studies showed granular deposition of immunoglobulin and C3 on the trophoblast basement membrane similar to that previously described on the glomerular basement membrane in systemic lupus erythematosus. Anti-DNA antibodies were eluted from the placenta in this case. We suggest that immune complex deposition on the trophoblast basement membrane in patients with active systemic lupus erythematosus may play a part in the increased fetal mortality in this disease.