An introduction to stem cells

1998 saw the publication of two papers describing the growth in vitro of human embryonic stem (ES) cells derived either from the inner cell mass (ICM) of the early blastocyst or the primitive gonadal regions of early aborted fetuses. Work on murine ES cells over many years had already established the amazing flexibility of ES cells, essentially able to differentiate into almost all cells that arise from the three germ layers. The realization of such pluripotentiality (see below) has, of course, resulted in the field of stem cell research going into overdrive, the establishment of many new biotechnology companies (http://www.stemcellresearchnew.com/catalog1677.html), and a genuine belief that stem cell research will deliver a revolution in terms of how we treat cardiovascular disease, neurodegenerative disease, cancer, diabetes, and the like. However, many people believe that early human embryos should be accorded the same status as any sentient being and thus their 'harvesting' for stem cells is morally unjustifiable. With this in mind, other sources of malleable stem cells have been sought. In the adult, organ formation and regeneration was thought to occur through the action of organ- or tissue-restricted stem cells (i.e. haematopoietic stem cells giving rise to all the cells of the blood, neural stem cells making neurons, astrocytes, and oligodendrocytes). However, it is now believed that stem cells from one organ system, for example the haematopoietic compartment can develop into the differentiated cells within another organ system, such as the liver, brain or kidney. Thus, certain adult stem cells may turn out be as malleable as ES cells and so also be useful in regenerative medicine. This brief overview summarizes the important attributes of tissue-based stem cells and clarifies the terms used.     

Genetic linkage analysis identifies new proximal and distal flanking markers for the X-linked agammaglobulinemia gene locus, refining its localization in Xq22

Genetic linkage analysis has been instrumental in mapping thegene for X-linked agammaglobulinemia (XLA) to the proximal longarm of the human X chromosome, to Xq22. Due to the relativerarity of this disease the localization of the gene within Xq22has remained imprecise. We have investigated twenty-nine familiesaffected by XLA and have found no recombinants with the DXS178locus in over 30 informative meioses. DXS178 is now the mostreliable and informative locus for use in pre-natal diagnosisand carrier detection of XLA. In addition, we have identifiednew closely linked proximal and distal flanking markers forXLA, DXS442 and DXS101, respectively. These loci are separatedby 2cM, considerably reducing the extent of DNA within whichthe XLA locus can be contained. This will open up the way formore directed positional cloning efforts for the isolation ofthe XLA gene.     

Sequence variation and size ranges of CAG repeats in the Machado-Joseph disease, spinocerebellar ataxia type 1 and androgen receptor genes

A subgroup of trinucleotide repeat diseases result from abnormalexpansions of CAG repeats which are translated into polyglutaminestretches. As yet there is little understanding of how the polyglutaminesfunction either normally, or when expanded. We have investigatedthese sequences in the Machado-Joseph disease, androgen receptorand spinocerebellar ataxia type 1 genes in humans and otherprimates. None of the 748 normal chromosomes that were examinedhad more than 34 uninterrupted gluta-mine codons in the Machado-Josephdisease gene. Similarly, no normal alleles with more than 39uninterrupted glutamine codons have been reported for the otherdisease genes associated with polyglutamine expansions. Sequenceanalyses of the repeats in primates revealed shorter polyglutaminestretches in some of the non-human primates at all three lociand marked diversions from the expected polyglutamines in theorang-utan Machado-Joseph gene and in the marmoset spinocerebellarataxia type 1 gene. These data suggest that conservation ofthese polyglutamine stretches may not always be necessary fornormal gene function.     

Lipophosphoglycan expression and virulence in Ricin-resistant variants of Leishmania major

Lipophosphoglycan (LPG) of Leishmania is a polymorphic molecule comprising an alkylglycerol anchor, a conserved oligosaccharide core and a species-specific polymer of oligosaccharide repeats joined by phosphodiester bonds. This molecule, together with the membrane polypeptide gp63, has been implicated as a parasite receptor for host macrophages. To examine the role of LPG in parasite infectivity glycosylation variants of Leishmania major were generated by chemical mutagenesis of a virulent cloned line V121 and variants with modified LPG selected using the galactose-specific lectin Ricinus communis II (RCA II). Twenty RCA II-resistant primary clones were generated. Analysis of LPG profile by immunoblotting using LPG-specific monoclonal and polyclonal antibodies revealed that some of the clones were LPG-deficient. Three clones that did not bind any LPG-specific antibodies but expressed normal levels of the M_r 63 000 glycoprotein (gp63), a second parasite receptor for host, were chosen for detailed studies.All three clones expressed, at least to some extent, a surface molecule which could be labeled by mild periodate oxidation and sodium borotritide and behaved like LPG by hydrophobic interaction chromatography. All clones also bound a well-characterized monoclonal antibody L157 directed to the core oligosaccharide of LPG, but did not bind another monoclonal antibody, CA7AE, to an epitope on a repeating unit shared by Leishmania donovani and L. major LPG. A third monoclonal antibody, 5E6, recognizing LPG on the surface of wild-type V121 promastigotes bound only to RCA II-resistant clone 3A2-C3 and was restricted to an internal structure. The LPG molecule that this clone expressed was a form of LPG by its chromatographic behavior and by its monosaccharide and alkylglycerol composition. Clone 3A2-C3 was the only one to infect mice in vivo and survive in macrophages in vitro, albeit at a much reduced rate compared to wild-type V121 promastigotes. The data suggest that some form of LPG may be necessary to ensure parasite infectivity.     

No evidence that common allelic variation in the Amyloid Precursor Protein (APP) gene confers susceptibility to Alzheimer's disease

In order to test the hypothesis that allelic variation withinthe Amyloid Precursor Protein (APP) gene influences susceptibilityto common forms of Alzheimer's disease (AD) we screened theentire coding, promoter and 3' untranslated sequences of theAPP gene for DNA variations in 30 unrelated patIents and eightcontrols with probable AD by a combination of RT-PCR, PCR andchemical cleavage mismatch analysis. Although we were unableto detect commonly occurring allelic variants, we were ableto detect a novel mutation within the APP gene in one individualwith late-onset AD. This mutation resulted in the substitutionof a tryptophan residue for an arginine residue at codon 328wIthin exon 7 which encodes the so-called protease Inhibitordomain of the 751 residue APP Isoform. However, the pathologicalsignificance of this mutation is uncertain as neither this,nor any other mutation occurring within exon 7 of the APP genewas found in any of a further 102 AD patients and 86 age-matchedcontrols. In conclusion, it is unlikely that susceptibilityto AD results from commonly occurring allelic variants of theAPP gene and it is even less probable that mutations withinexon 7 of the APP gene are important risk factors for late-onsetAD.     

Molecular analysis of de novo germline mutations in the von Hippel-Lindau disease gene

VHL disease is a dominantly inherited familial cancer syndromewith variable expression and age-dependent penetrance. The diagnosisof isolated cases is often delayed compared with familial cases,and estimates of the new mutation rate have varied more than20-fold. To investigate the frequency and origin of de novoVHL gene mutations we have analysed: (i) families with identicalmutations to determine if there is a common haplotype, and (ii)apparent new mutation cases to determine whether the clinicaldiagnosis of such cases is reliable and to define the parentalorigin of de novo VHL gene mutations. Haplotyping of 12 VHLmutations occurring in two or more families (total 42 kindreds)revealed that for most mutations there was no evidence of afounder effect. A marked bias for a paternal origin of new mutationshas been reported in other familial cancer syndromes such asneurofibromatosis type 1 (NF1), multiple endocrine neoplasia(MEN) 2B and bilateral retinoblastoma, but it is unclear whetherthis bias results from a greater susceptibility for mutagenesisduring male gametogenesis because of the larger number of celldivisions compared with that in oogenesis, or from genomic imprintingeffects. Analysis of 13 de novo VHL mutations in which the parentof origin could be established, showed no evidence for a biasfor a paternal origin (seven paternal, six maternal), and differedsignificantly from that reported in NF1, MEN2B and bilateralretinoblastoma. This result demonstrates that an increased susceptibilityto paternal allele mutation is not a universal finding in autosomalgenetic diseases and that the origin of new mutations may beinfluenced by both genomic imprinting effects and the increasednumber of cell divisions in spermatogenesis compared with oogenesis.     

Macrophage-lymphocyte association in in vitro mouse spleen cultures; the formation of B-cell colonies.

Colonies of predominantly B lymphocytes have been grown in liquid culture, without agar or specific mitogen treatment. An essential component of the colonies is a macrophage-like cell. Colonies appear to result from continued growth of B lymphocytes following a primary macrophage-lymphocyte association which frequently occurs under a variety of culture conditions. Factors promoting colony growth are foetal calf serum and 2 mercapto-ethanol, the latter acting in part through reduction of a non-dialysable sulphur-containing component of serum.     

The effect of hapten-specific suppression of IgE on antigen-induced histamine release from mouse peritoneal mast cells.

Subcutaneous injections of a mixture of dinitrophenylated ovalbumin (DNP3-OA) and dextran sulfate into Swiss-Webster mice elicited short-lived primary and long-lasting secondary IgE antibody responses to both DNP and OA. Histamine was released on in vitro challenge with antigen (OA or DNP22-BSA) of washed peritoneal mast cells (PMC) obtained from mice during a primary or a secondary IgE response. Administration of an intravenous injection of a tolerogenic conjugate of DNP8-mouse gamma-globulin, either prior to immunizationor during an ongoing IgE response, resulted in almost complete disappearance of circulating anti-DNP IgE antibody and in a very marked decrease in histamine release from PMC on challenge with DNP22-BSA. However, the IgE response to OA of these mice and the histamine release from their PMC on challenge with OA were not affected. Moreover, the PMC of mice, which had been tolerized to DNP, could be passively sensitized with serum containing DNP-specific IgE antibody for the release of histamine on DNP22-BSA challenge. The most significant finding of this study is the observation that the time course for the loss of reactivity of PMC to DNP22-BSA, after administration of the tolerogen during an ongoing secondary response, paralleled the decrease in circulating anti-GNP IgE antibody.     

Use of hybridot assay to screen for BK and JC polyomaviruses in non-immunosuppressed patients.

Urine samples from 50 patients attending a genitourinary outpatient clinic and from 13 renal allograft recipients were investigated for evidence of infection with human BK and JC polyomaviruses using cytology and a new DNA hybridot assay. Forty four per cent of samples from the renal allograft recipients were positive by cytology and 75% by DNA hybridisation, indicating that hybridot assay is more sensitive than cytological screening. BK and JC viral DNA was found in 20% of the patients attending the genitourinary clinic, showing infection with BK virus and JC virus in a group of patients with clinical conditions not normally associated with immunological deficiency-a finding that has not been reported before.