Pluripotent Stem Cells For Regeneration Research Articles

Regeneration of tracheal epithelium using mouse induced pluripotent stem cells

Conclusion The findings demonstrated the potential use of induced pluripotent stem cells for regeneration of tracheal epithelium.Objective Autologous tissue implantation techniques using skin or cartilage are often applied in cases of tracheal defects with laryngeal inflammatory lesions and malignant tumor invasion. However, these techniques are invasive with an unstable clinical outcome. The purpose of this study was to investigate regeneration in a tracheal defect site of nude rats after implantation of ciliated epithelium that was differentiated from induced pluripotent stem cells.Method Embryoid bodies were formed from mouse induced pluripotent stem cells. They were cultured with growth factors for 5 days, and then cultured at the air–liquid interface. The degree of differentiation achieved prior to implantation was determined by histological findings and the results of real-time polymerase chain reaction. Embryoid bodies including ciliated epithelium were embedded into collagen gel that served as an artificial scaffold, and then implanted into nude rats, creating an ‘air–liquid interface model’. Histological evaluation was performed 7 days after implantation.Results The ciliated epithelial structure survived on the lumen side of regenerated tissue. It was demonstrated histologically that the structure was composed of ciliated epithelial cells.

Effective embryoid body formation from induced pluripotent stem cells for regeneration of respiratory epithelium

We have previously demonstrated the potential use of induced pluripotent stem (iPS) cells for regeneration of respiratory epithelium by culturing embryoid bodies (EBs). The aim of the present study was to determine the most effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium. Experimental study. iPS cells cultured on a gelatin-coated dish were seeded on low-attachment plates for generating EBs. Under several conditions including the air-liquid interface (ALI) method, with varying cell numbers and suspension times, EBs were transferred to a gelatin-coated dish supplemented with growth factors. The shape, size, aggregation, and adhesion of EBs for iPS cell differentiation were evaluated, and the cultured tissue was histologically examined. EBs appropriate for differentiation were observed using 1,000 cells after 5 days of suspension culture. Respiratory epithelium-like tissue was histologically observed. The ciliary epithelium was confirmed immunohistologically. Based on the varying suspension times and cell numbers with the ALI method, this study presented effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium.

Cardiomyocyte differentiation of bone marrow-derived Oct-4+CXCR4+SSEA-1+ very small embryonic-like stem cells

VSELs are a population of rare Oct-4+CXCR4+ CD133+lin-CD45- cells that are deposited during early embryogenesis in developing organs/tissues as a reserve population of pluripotent stem cells for regeneration. We reported recently that they are mobilized into peripheral blood during acute myocardial infarction (AMI) in mice and humans. However, although freshly isolated VSELs in experimental AMI mouse models improve cardiac function, the number of these cells is limited and an ex vivo expansion strategy is needed to employ these cells more efficiently for cardiac regeneration. The aim of this study was to establish an efficient method to expand ex vivo BM-derived very small embryonic-like stem cells (VSELs) into cardiomyocytes. VSELs, highly purified by FACS from the bone marrow of GFP transgenic mice, were expanded over C2C12 cell line myoblasts and exposed to cardiac differentiating media. The changes in gene expression during cardiac differentiation of VSELs were evaluated by RTQ-PCR, immunostaining and gene array analysis. We developed an efficient, two-step, ex vivo expansion/differentiation model of BM-derived VSELs into cardiomyocytes. First, purified GFP+ VSELs are plated over C2C12 murine cell line myoblasts, where they expand and differentiate into characteristic spheres. Subsequently, cells from these spheres are expanded on cardiac differentiating media into cardiomyocytes. This study demonstrates that murine BM-derived VSELs can be efficiently expanded in vitro and differentiated into cardiomyocytes.

Novel Evidence That Very Small Embryonic Like Stem Cells (VSELs) Are Mobilized Into Peripheral Blood in Patients with Inflammatory Bowel Diseases – Correlation with Young Age and Severity of Disease.

Abstract 4595 IntroductionWe have hypothesized that circulating pluripotent stem cells could play a protective role in the pathogenesis of inflammatory bowel diseases (IBD) and participate in regeneration of damaged intestinal eipthelium. Recently, our group has identified in murine and human BM as well as other organs a population of very small embryonic like stem cells (VSELs) (Leukemia 2006, 20, 857-69). These are cells deposited in BM during development, differentiate into cells from all three germ layers and are capable of long term repopulation of hematopoiesis. They also play an important role in turnover of tissue-specific/committed stem cells in various organs. We have also shown that the number of VSELs circulating in peripheral blood (PB) increases during stress and tissue/organ injury e.g., in patients after stroke (Stroke 2009, 40, 1237-1244) and myocardial infarction (JACC 2009, 53, 1-9). It is currently unknown whether VSELs are mobilized into PB in patients with IBD. Materials and MethodsWe evaluated the mobilization of VSELs into PB in patients with active IBD. There were twenty patients (n=20) enrolled with de novo diagnosed or untreated flare-ups with mild to moderate ulcerative colitis (Truelove and Witt's criteria) and Crohn's disease (Crohns Disease Activity Index) as well as age-matched healthy subjects (n=10). Blood was sampled on admission, erythrocytes were lysed and CXCR4+ CD133+ lin- CD45- VSELs were evaluated in PB by fluorescence-activated cell sorting analysis (FACS), direct immunofluorescence staining and real-time quantitative polymerase chain reaction to detect expression of developmentally early genes. ResultsIn IBD patients, we found an increase in the number of circulating cells expressing stem cells-associated antigens such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4+ CD133+ in- CD45- cells - smaller than erythrocytes) (median 3.7 [range 0.9 to 8.9] cells/ml; p < 0.001) in comparison to healthy individuals (median 1.33 [range 0.2 – 1.45] cells/ml; p < 0.001 [Mann–Whitney test Friedman's ANOVA followed by Wilcoxon signed-rank test]). The mobilization of VSELs was higher in patients with active Crohn's disease and younger patients. Of note antimicrobial treatment also influenced the mobilization process. Circulating VSELs express pluripotent stem cell markers (Oct-4, SSEA-4, Nanog), CXCR4 receptor and respond robust to stromal derived factor-1 (SDF-1) gradient. We also noticed that the number of mobilized/circulating VSELs correlated in IBD patients with elevated serum level of CXCR4 receptor ligand - stromal derived factor -1 (SDF-1). ConclusionsUsing multiparameter analysis, we have demonstrated for the first time that VSELs could be detected in circulating PB of patients with active IBD. The number of this cell positively correlated with intensity of disease and young age. This strongly supports a potential involvement of circulating pluripotent stem cells in regeneration of damaged intestinal epithelium. However, the biological significance as well as potential application of these cells in regeneration of damaged gut tissue needs further investigations and is currently tested in animal models in our laboratories. Disclosures:No relevant conflicts of interest to declare.

An Evidence That Murine Marrow-Derived CXCR4+ SSEA-1+ Oct-4+ Very Small Embryonic-Like (VSEL) Stem Cells Are Pluripotent and Express Several Primordial Germ Cell (PGC) Markers - Hypotesis for Developmental Deposition of PGC in Various Organs.

Recently we identified in murine BM a homogenous population of rare (~0.01% of BMMNC) Sca-1+ lin− CD45− cells that express by RQ-PCR and immunhistochemistry markers of pluripotent stem cells (PSC) such as SSEA-1, Oct-4, Nanog and Rex-1 and highly express Rif-1 telomerase protein (Leukemia 2006; 20,857–869). Direct electronmicroscopical analysis revealed that these cells display several features typical for embryonic stem cells such asi) small size (2–4 um in diameter),ii) large nuclei surrounded by a narrow rim of cytoplasm, andiii) open-type chromatin (euchromatin).We also found that VSELs may be released from BM and circulate in peripheral blood during tissue/organ injuries (e.g., heart infarct, stroke). Recently we noticed that ~5–10% of purified VSELs if plated over a C2C12 murine sarcoma cell feeder layer are able to form spheres that resemble embryoid bodies. Cells from these VSEL-derived spheres (VSEL-DS) are composed of immature cells with large nuclei containing euchromatin, and similarly as purified VSELs are CXCR4+SSEA-1+Oct-4+. Furthermore, VSEL-DS after replating over C2C12 cells may again (up to 5–7 passages) grow new spheres or if plated into cultures promoting tissue differentiation expand into cells from all three germ-cell layers. The formation of VSEL-DS was observed in a presence of C2C12 cells obtained from different sources. Furthermore, VSELs isolated from GFP+ mice grew GFP+ VSEL-DS which show a diploid content of DNA. This suggests that VSEL-DS are in fact derived from VSELs and not from the supportive C2C12 cell line as well as excludes the possibility of cell fusion to the observed phenomenon. Similar spheres were also formed by VSELs isolated from murine fetal liver, spleen and thymus. Interestingly formation of VSEL-DS was associated with a young age, and no VSEL-DS were observed by cells isolated from old mice (> 2 years). We also found that cells isolated from VSEL-DS similarly as embryonic stem cells grow tumors after injection into immunodeficient NOD/SCID mice (51/52 inoculated mice). Since VSELs isolated by us express several markers of primordial germ cells (fetal-type alkaline phosphatase, Oct-4, SSEA-1, CXCR4, Mvh, Stella, Fragilis, Nobox, Hdac6) we hypothesize that VSELs are closely related to a population of primordial germ cells. These cells are specified during early gastrulation in the proximal epiblast and subsequently migrate in a CXCR4-SDF-1 dependent manner through the embryo proper to their final destination in genital ridges. It is possible that some of these cells or a population of cells closely related to them migrate astray being chemoattracted by SDF-1 to fetal liver and subsequently, during the third trimester of gestation seed together with hematopoietic stem cells in bone marrow and perhaps other organs as well. In conclusion, we postulate that VSELs identified by us and purified at the single cell level could become an important source of pluripotent stem cells for regeneration.