Focal nodular hyperplasia with concomitant hepatocellular carcinoma: a case report and clonal analysis

This report describes a hepatocellular carcinoma (HCC) with concomitant focal nodular hyperplasia (FNH) in a 56 years old Chinese man. There were two well circumscribed tumours measuring 3×2.5×2 cm and 2×1.5×1.5 cm. The larger mass was grey and soft with a small area of bleeding and necrosis and an intact capsule. The smaller mass was yellow and had no capsule. Clonal analysis was carried out to clarify the relation between the HCC and the adjacent FNH. The clonal analysis was based on the methylation pattern of the polymorphic X chromosome linked androgen receptor gene (HUMARA). In FNH, after Hpa Ⅱ digestion, the allelic bands showed two well defined peaks. The intensity of the two peaks in the DNA from cirrhotic tissue did not differ significantly, consistent with a random pattern of X chromosome inactivation. However, in HCC, after Hpa Ⅱ digestion, the allelic bands differed significantly in intensity. Therefore, there was a typical polyclonal pattern of inactivation in FNH but the HCC was interpreted as being monoclonal.

EARLY DIAGNOSIS OF MYELODYSPLASTIC SYNDROMES USING CLONAL ANALYSES

Objective: To study the value of clonal analysis to the early diagnosis of myelodysplastic syndrome (MDS). Methods: Four types of clonal analyses were performed on the bone marrow samples from 50 patients suspected of MDS: (1) Conventional Cytogenetics (CC) for clonal chromosomal abnormalities; (2) BrdU-Sister Chromatid Differentiation (BrdU-SCD) for cell cycle kinetics; (3) Fluorescence in Situ Hybridization (FISH) for trisomy 8; (4) Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) for N-ras mutation. Results: The diagnosis of forty-three patients was compatible with the FAB criteria for MDS. The other seven cases didn’t meet the FAB criteria, with only one lineage of dyspoiesis or with no obvious dysplastic changes. Among these seven cases, two were morphologically diagnosed with suspicious refractory anemia, one with sideroblastic anemia, one with leukemoid reaction, one with hypercellular anemia and two with chronic aplastic anemia. Clonal analyses of the 7 patients showed that six cases had clonal karyotype abnormalities, four had prolonged cell cycle patterns, four had trisomy 8 of different proportions and one had mutation of the exon 1 of N-RAS. Thus, they were revaluated as MDS patients. Conclusion: The untypical MDS patients with one lineage dyspoiesis or without obvious dysplastic changes can be diagnosed early by combining multiple clonal analysis techniques such as CC, SCD, FISH and PCR-SSCR.

Dissection of gene function at clonal level using mosaic analysis with double markers

MADM （Mosaic Analysis with Double Markers） technology offers a genetic approach in mice to visualize and concomitantly manipulate genetically defined cells at clonal level and single cell resolution. MADM employs Cre recombinase/loxP-dependent interchromosomal mitotic recombination to reconstitute two split marker genes--green GFP and red tdTomato -- and can label sparse clones of homozygous mutant cells in one color and wild-type cells in the other color in an otherwise unlabeled background. At present, major MADM applications include lineage tracing, single cell labeling, conditional knockouts in small populations of cells and induction of uniparental chromosome disomy to assess effects of genomic imprinting. MADM can be applied universally in the mouse with the sole limitation being the specificity of the promoter controlling Cre recombinase expression. Here I review recent developments and extensions of the MADM technique and give an overview of the major discoveries and progresses enabled by the implementation of the novel genetic MADM tools.

Tbr2-expressing intermediate progenitor cells in the adult mouse hippocampus are unipotent neuronal precursors with limited amplification capacity under homeostasis

Neurogenesis persists in two locations of the adult mammalian brain, the subventricular zone of the lateral ventricles and the subgranular zone of the dentate gyrus in the hippocampus. In the adult subgranular zone, radial glial- like cells （RGLs） are multipotent stem cells that can give rise to both astrocytes and neurons. In the process of generating neurons, RGLs divide asymmetrically to give rise to one RGL and one intermediate progenitor cell （IPC）. IPCs are considered to be a population of transit amplifying cells that proliferate and eventually give rise to mature granule neurons. The properties of individual IPCs at the clonai level are not well understood. Furthermore, it is not clear whether IPCs can generate astrocytes or revert back to RGLs, besides generating neurons. Here we developed a genetic marking strategy for clonal analysis and lineage-tracing of individual Tbr2-expressing IPCs in the adult hippocampus in vivo using Tbr2-CreERT2 mice. Using this technique we identified Tbr2-CreERT2 labeled IPCs as unipotent neuronal precursors that do not generate astrocytes or RGLs under homeostasis. Additionally, we showed that these labeled IPCs rapidly generate immature neurons in a synchronous manner and do not undergo a significant amount of amplification under homeostasis, in animals subjected to an enriched environment/running, or in animals with different age. In summary, our study suggests that Tbr2-expressing IPCs in the adult mouse hippocampus are unipotent precursors and rapidly give rise to immature neurons without major amplification.

Recent updates on the biological basis of heterogeneity in bone marrow stromal cells/ skeletal stem cells

Based on studies over the last several decades,the self-renewing skeletal lineages derived from bone marrow stroma could be an ideal source for skeletal tissue engineering.However,the markers for osteogenic precursors;i.e.,bone marrow-derived skeletal stem cells(SSCs),in association with other cells of the marrow stroma(bone marrow stromal cells,BMSCs)and their heterogeneous nature both in vivo and in vitro remain to be clarified.This review aims to highlight:i)the importance of distinguishing BMSCs/SSCs from other“mesenchymal stem/stromal cells”,and ii)factors that are responsible for their heterogeneity,and how these factors impact on the differentiation potential of SSCs towards bone.The prospective role of SSC enrichment,their expansion and its impact on SSC phenotype is explored.Emphasis has also been given to emerging single cell RNA sequencing approaches in scrutinizing the unique population of SSCs within the BMSC population,along with their committed progeny.Understanding the factors involved in heterogeneity may help researchers to improvise their strategies to isolate,characterize and adopt best culture practices and source identification to develop standard operating protocols for developing reproducible stem cells grafts.However,more scientific understanding of the molecular basis of heterogeneity is warranted that may be obtained from the robust high-throughput functional transcriptomics of single cells or clonal populations.