Knowledge of the prevalence of latent Mycobacterium tuberculosis infection is crucial for effective tuberculosis control, but tuberculin skin test surveys have major limitations, including poor specificity because of the broad antigenic cross-reactivity of tuberculin. The M. tuberculosis RD1 genomic segment encodes proteins, such as early secretory antigenic target (ESAT)-6, that are absent from M. bovis bacille Calmette-Guérin (BCG) and most environmental mycobacteria. We recently identified circulating ESAT-6-specific T cells as an accurate marker of M. tuberculosis infection. Here, interferon-gamma-secreting T cells specific for peptides derived from ESAT-6 and a second RD1 gene product, CFP10, were enumerated in 100 prospectively recruited healthy adults in Bombay (Mumbai), India. Eighty percent responded to >/=1 antigen, and many donors had high frequencies of T cells that were specific for certain immunodominant peptides. In contrast, of 40 mostly BCG-vaccinated, United Kingdom-resident healthy adults, none responded to either antigen. This study suggests an 80% prevalence of latent M. tuberculosis infection in urban India.
Very small embryonic-like stem cells (VSELs) are possibly lost during cord blood banking and bone marrow (BM) processing for autologus stem cell therapy mainly because of their small size. The present study was conducted on human umbilical cord blood (UCB, n=6) and discarded red blood cells (RBC) fraction obtained after separation of mononuclear cells from human BM (n=6), to test this hypothesis. The results show that VSELs, which are pluripotent stem cells with maximum regenerative potential, settle along with the RBCs during Ficoll-Hypaque density separation. These cells are very small in size (3-5 μm), have high nucleo-cytoplasmic ratio, and express nuclear Oct-4, cell surface protein SSEA-4, and other pluripotent markers such as Nanog, Sox-2, Rex-1, and Tert as indicated by immunolocalization and quantitative polymerase chain reaction (Q-PCR) studies. Interestingly, a distinct population of slightly larger, round hematopoietic stem cells (HSCs) with cytoplasmic Oct-4 were detected in the "buffy" coat, which usually gets banked or used during autologus stem cell therapy. Immunohistochemical studies on the umbilical cord tissue (UCT) sections (n=3) showed the presence of nuclear Oct-4-positive VSELs and many fibroblast-like mesenchymal stem cells (MSCs) with cytoplasmic Oct-4. These VSELs with nuclear Oct-4, detected in UCB, UCT, and discarded RBC fraction obtained after BM processing, may persist throughout life, maintain tissue homeostasis, and undergo asymmetric cell division to self-renew as well as produce larger progenitor stem cells, viz. HSCs or MSCs, which follow differentiation trajectories depending on the somatic niche. Hence, it can be concluded that the true stem cells in adult body tissues are the VSELs, whereas the HSCs and MSCs are actually progenitor stem cells that arise by asymmetric cell division of VSELs. The results of the present study may help explain low efficacy reported during adult autologous stem cell trials, wherein unknowingly progenitor stem cells are injected rather than the pluripotent stem cells with maximum regenerative potential.
Very small embryonic-like stem cells (VSELs) are immature primitive cells residing in adult and fetal tissues. This study describes enrichment strategy and molecular and phenotypic characterization of human cord blood VSELs. Flow cytometry analysis revealed that a majority of VSELs (LIN(-)/CD45(-)/CD34(+)) were present in the red blood cell (RBC) pellet after Ficoll-Hypaque centrifugation in contrast to the hematopoietic stem cells (LIN(-)/CD45(+)/CD34(+)) in the interphase layer. Thus, after lyses of RBCs, VSELs were enriched using CD133 and SSEA4 antibodies. These enriched cells were small in size (4-6 μm), spherical, exhibited telomerase activity and expressed pluripotent stem cell (OCT4A, OCT4, SSEA4, NANOG, SOX2, REX1), primordial germ cell (STELLA, FRAGILIS) as well as primitive hematopoietic (CD133, CD34) markers at protein and transcript levels. Heterogeneity was noted among VSELs based on subtle differences in expression of various markers studied. DNA analysis and cell cycle studies revealed that a majority of enriched VSELs were diploid, non-apoptotic and in G0/G1 phase, reflecting their quiescent state. VSELs also survived 5-fluorouracil treatment in vitro and treated cells entered into cell cycle. This study provides further support for the existence of pluripotent, diploid and relatively quiescent VSELs in cord blood and suggests further exploration of the subpopulations among them.
This study describes the successful derivation of two human embryonic stem (hES) cell lines using 53 frozen and 18 fresh "slow-growing" surplus embryos, obtained from collaborating in vitro fertilization clinics, on in-house-derived human feeder layers. The cell lines have been derived by whole embryo culture followed by further expansion of manually dissected inner cell mass from the surrounding trophoectodermal cells. Immunocytochemical localization of cell surface markers like SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81, staining for alkaline phosphatase and reverse transcriptase polymerase chain reaction (RT-PCR) analysis of pluripotency state markers viz. Oct-4, TERT, Nanog, Rex1, and Sox2 indicate that both cell lines possess typical features of embryonic stem cells. Both cell lines exhibit normal female karyotype after 40 passages in culture. Pluripotent nature of the cell lines was confirmed both in vitro and in vivo. Embryoid bodies, formed in suspension culture, express markers for all three lineages as indicated by RT-PCR analysis for SOX 1 (ectoderm), HAND 1 (mesoderm), AFP (endoderm), and CDX2 (trophoectoderm). Teratoma formed in vivo in severe combined immunodeficient mice revealed cells of all the three embryonic germ layers. Comparison of the STR and human leukocyte antigen profiles of these cell lines with the existing human ES cell lines indicate that they are genetically distinct. The addition of our hES cell lines contributes usefully to the globally restricted repertoire of human ES cell lines.
BackgroundPolycomb Group (PcG) proteins are chromatin modifiers involved in early embryonic development as well as in proliferation of adult stem cells and cancer cells. PcG proteins form large repressive complexes termed Polycomb Repressive Complexes (PRCs) of which PRC1 and PRC2 are well studied. Differentiation of human Embryonic Stem (hES) cells into insulin producing cells has been achieved to limited extent, but several aspects of differentiation remain unexplored. The PcG protein dynamics in human embryonic stem (hES) cells during differentiation into pancreatic lineage has not yet been reported. In the present study, the expression of RING1A, RING1B, BMI1, CBX2, SUZ12, EZH2, EED and JARID2 during differentiation of hES cells towards pancreatic lineage was examined.ResultsIn-house derived hES cell line KIND1 was used to study expression of PcG protein upon spontaneous and directed differentiation towards pancreatic lineage. qRT-PCR analysis showed expression of gene transcripts for various lineages in spontaneously differentiated KIND1 cells, but no differentiation into pancreatic lineage was observed. Directed differentiation induced KIND1 cells grown under feeder-free conditions to transition from definitive endoderm (Day 4), primitive gut tube stage (Day 8) and pancreatic progenitors (Day 12-Day 16) as evident from expression of SOX17, PDX1 and SOX9 by qRT-PCR and Western blotting. In spontaneously differentiating KIND1 cells, RING1A and SUZ12 were upregulated at day 15, while other PcG transcripts were downregulated. qRT-PCR analysis showed transcripts of RING1B, BMI1, SUZ12, EZH2 and EED were upregulated, while RING1A and CBX2 expression remained low and JARID2 was downregulated during directed differentiation of KIND1 cells. Upregulation of BMI1, EZH2 and SUZ12 during differentiation into pancreatic lineage was also confirmed by Western blotting. Histone modifications such as H3K27 trimethylation and monoubiquitinylation of H2AK119 increased during differentiation into pancreatic lineage as seen by Western blotting.ConclusionOur study shows expression of PcG proteins was distinct during spontaneous and directed differentiation. Differentiation into pancreatic lineage was achieved by directed differentiation approach and was associated with increased expression of PcG proteins RING1B, BMI1, EZH2 and SUZ12 accompanied by increase in monoubiquitinylation of H2AK119 and trimethylation of H3K27.
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