Directed reprogramming of comprehensively characterized dental pulp stem cells extracted from natal tooth.

The aim of this study was to extensively characterise natal dental pulp stem cells (nDPSC) and assess their efficiency to generate human induced pluripotent stem cells (hiPSC). A number of distinguishing features prompted us to choose nDPSC over normal adult DPSC, in that they differed in cell surface marker expression and initial doubling time. In addition, nDPSC expressed 17 out of 52 pluripotency genes we analysed, and the level of expression was comparable to human embryonic stem cells (hESC). Ours is the first group to report comprehensive characterization of nDPSC followed by directed reprogramming to a pluripotent stem cell state. nDPSC yielded hiPSC colonies upon transduction with Sendai virus expressing the pluripotency transcription factors POU5F1, SOX2, c-MYC and KLF4. nDPSC had higher reprogramming efficiency compared to human fibroblasts. nDPSC derived hiPSCs closely resembled hESC in terms of their morphology, expression of pluripotency markers and gene expression profiles. Furthermore, nDPSC derived hiPSCs differentiated into the three germ layers when cultured as embryoid bodies (EB) and by directed differentiation. Based on our findings, nDPSC present a unique marker expression profile compared with adult DPSC and possess higher reprogramming efficiency as compared with dermal fibroblasts thus proving to be more amenable for reprogramming.

Cloning of intronic sequence within DsRed2 increased the number of cells expressing red fluorescent protein.

AIM: Cloning of artificial intronic sequence within the open reading frame (ORF) of DsRed2 gene. METHOD: Splice prediction software was used to analyze DsRed2 sequence to find an ideal site for cloning artificial intronic sequence. Intron was cloned within DsRed2 using cyclic ligation assembly. Flow cytometry was used to quantify the number of cells expressing red fluorescence. RESULT: Sequencing data confirmed precise cloning of intron at the desired position using cyclic ligation assembly. Successful expression of red fluorescence after cloning of intron confirmed successful intron recognition and splicing by host cell line. Cloning of intron increased the number of cells expressing red fluorescent protein. CONCLUSION: Cloning of intronic sequence within DsRed2 has helped to increase the number of cells expressing red fluorescence by approximately four percent.

Histological Evaluation of Decellularized Skeletal Muscle Tissue Using Two Different Decellularization Agents.

The aim of the present study was to determine effect of two decellularized agents, sodium dodecyl sulphate (SDS) and Triton X-100, to the skeletal muscle tissue. Final scaffold was evaluated by several histological techniques to analyse preservation of essential structures including collagen and elastic fibres, basement membranes, glycosaminoglycans and also to confirm elimination of nuclear and cytoplasmic components which are redundant in effectively prepared decellularized scaffolds. Comparison of tissue scaffolds processed with different detergents proved that SDS is superior to Triton X-100 as it can effectively decellularize muscle tissue.

Cell Stratification, Spheroid Formation and Bioscaffolds Used to Grow Cells in Three Dimensional Cultures.

The cell culture became an invaluable tool for studying cell behaviour, development, function, gene expression, toxicity of compounds and efficacy of novel drugs. Although most results were obtained from cell cultivation in two-dimensional (2D) systems, in which cells are grown in a monolayer, three-dimensional (3D) cultures are more promising as they correspond closely to the native arrangement of cells in living tissues. In our study, we focused on three types of 3D in vitro systems used for cultivation of one cell type. Cell morphology, their spatial distribution inside of resulting multicellular structures and changes in time were analysed with histological examination of samples harvested at different time periods. In multilayered cultures of WRL 68 hepatocytes grown on semipermeable membranes and non-passaged neurospheres generated by proliferation of neural progenitor cells, the cells were tightly apposed, showed features of cell differentiation but also cell death that was observable in short-term cultures. Biogenic scaffolds composed of extracellular matrix of the murine tibial anterior muscle were colonized with C2C12 myoblasts in vitro. The recellularized scaffolds did not reach high cell densities comparable with the former systems but supported well cell anchorage and migration without any signs of cell regression.

Chemical decellularization: a promising approach for preparation of extracellular matrix.

BACKGROUND: A biological scaffold from extracellular matrix can be produced by a variety of decellularization methods whose caveat consists in efficiently eliminating cells from the treated tissue. This scaffold can be used in diverse applications for tissue engineering and organ regeneration. Preservation of the extracellular matrix ultrastructure is highly desirable because of its unique architecture, contained growth factors and decreased immunological response. All of these properties provide attachment sites and adequate environment for cells colonizing this scaffold, reconstituting the decellularized organ. This review briefly describes chemical decellularization methods, evaluation of these protocols and the role of ECM in tissue engineering. CONCLUSION: Chemical decellularization is an often used method for scaffold preparation and makes possible a well-preserved three dimensional structure of extracellular matrix.

The peripheral chimerism of bone marrow-derived stem cells after transplantation: regeneration of gastrointestinal tissues in lethally irradiated mice.

Bone marrow-derived cells represent a heterogeneous cell population containing haematopoietic stem and progenitor cells. These cells have been identified as potential candidates for use in cell therapy for the regeneration of damaged tissues caused by trauma, degenerative diseases, ischaemia and inflammation or cancer treatment. In our study, we examined a model using whole-body irradiation and the transplantation of bone marrow (BM) or haematopoietic stem cells (HSCs) to study the repair of haematopoiesis, extramedullary haematopoiesis and the migration of green fluorescent protein (GFP(+)) transplanted cells into non-haematopoietic tissues. We investigated the repair of damage to the BM, peripheral blood, spleen and thymus and assessed the ability of this treatment to induce the entry of BM cells or GFP(+) lin(-) Sca-1(+) cells into non-haematopoietic tissues. The transplantation of BM cells or GFP(+) lin(-) Sca-1(+) cells from GFP transgenic mice successfully repopulated haematopoiesis and the haematopoietic niche in haematopoietic tissues, specifically the BM, spleen and thymus. The transplanted GFP(+) cells also entered the gastrointestinal tract (GIT) following whole-body irradiation. Our results demonstrate that whole-body irradiation does not significantly alter the integrity of tissues such as those in the small intestine and liver. Whole-body irradiation also induced myeloablation and chimerism in tissues, and induced the entry of transplanted cells into the small intestine and liver. This result demonstrates that grafted BM cells or GFP(+) lin(-) Sca-1(+) cells are not transient in the GIT. Thus, these transplanted cells could be used for the long-term treatment of various pathologies or as a one-time treatment option if myeloablation-induced chimerism alone is not sufficient to induce the entry of transplanted cells into non-haematopoietic tissues.