Maintenance of differentiation potential of human bone marrow mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene despite [corrected] extensive proliferation

Human bone marrow mesenchymal stem cells (hMSC) represent a population of stem cells that are capable of differentiation into multiple lineages. However, these cells exhibit senescence-associated growth arrest and phenotypic changes during long-term in vitro culture. We have recently demonstrated that overexpression of human telomerase reverse transcriptase (hTERT) in hMSC reconstitutes telomerase activity and extends life span of the cells [Nat. Biotechnol. 20 (2002) 592]. In the present study, we have performed extensive characterization of three independent cell lines derived from the parental hMSC-TERT cell line based on different plating densities during expansion in culture: 1:2 (hMSC-TERT2), 1:4 (hMSC-TERT4), and 1:20 (hMSC-TERT20). The 3 cell lines exhibited differences in morphology and growth rates but they all maintained the characteristics of self-renewing stem cells and the ability to differentiate into multiple mesoderm-type cell lineages: osteoblasts, adipocytes, chondrocytes, and endothelial-like cells over a 3-year period in culture. Also, surface marker studies using flow cytometry showed a pattern similar to that known from normal hMSC. Thus, telomerization of hMSC by hTERT overexpression maintains the stem cell phenotype of hMSC and it may be a useful tool for obtaining enough number of cells with a stable phenotype for mechanistic studies of cell differentiation and for tissue engineering protocols

Human Serum is as Efficient as Fetal Bovine Serum in Supporting Proliferation and Differentiation of Human Multipotent Stromal (Mesenchymal) Stem Cells In Vitro and In Vivo

Background: Human multipotent stromal (skeletal, mesenchymal) stem cells (hMSC) are employed in an increasing number of clinical trials for tissue regeneration of age-related degenerative diseases. However, routine use of fetal bovine sera (FBS) for their in vitro expansion is not optimal and may pose a health risk for patients. Methods: We carried out a side-by-side comparison of the effects of allogenic pooled human serum (HuS) versus FBS on hMSC proliferation and differentiation in vitro and in vivo. As a model for hMSC, we employed telomerase-immortalized hMSC; hMSC-TERT cell line. Results: hMSC-TERT exhibited similar morphology and size when cultured in HuS vs. FBS as assessed by light microscopy and FACS analysis. We did not observe any significant differences in growth rates of hMSC-TERT during short-term (10 days) and long-term (100 days) culture in media supplemented with HuS vs. FBS. hMSC-TERT or primary bone marrow derived hMSC induced to osteoblastic or adipocytic differentiation in the presence of HuS or FBS showed comparable levels of gene expression and protein production of osteoblastic markers (CBFA1/Runx2, alkaline phosphastase, collagen type I and osteocalcin) or adipocytic markers (PPAR-gamma2, lipoprotein lipase (LPL), aP2), respectively. In order to test for the functional capacity of hMSC-TERT that have been maintained in long-term cultures in the presence of HuS vs. FBS, the cells were mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) and implanted subcutaneously in immune deficient mice. hMSC maintained in HuS vs. FBS formed comparable heterotopic bone. Discussion: Human serum can support proliferation and differentiation of hMSC in vitro and can maintain their bone forming capacity in vivo. The use of human serum in cell cultures of hMSC intended for cell-based therapy is preferable

Ejby—A new H5/6 ordinary chondrite fall in Copenhagen, Denmark

On February 6, 2016 at 21:07:19 UT, a very bright fireball was seen over the eastern part of Denmark. The weather was cloudy over eastern Denmark, but many people saw the sky light up—even in the heavily illuminated Copenhagen. Two hundred and thirty three reports of the associated sound and light phenomena were received by the Danish fireball network. We have formed a consortium to describe the meteorite and the circumstances of the fall and the results are presented in this paper. The first fragment of the meteorite was found the day after the fall, and in the following weeks, a total of 11 fragments with a total weight of 8982 g were found. The meteorite is an unbrecciated, weakly shocked (S2), ordinary H chondrite of petrologic type 5/6 (Bouvier et al. 2017). The concentration of the cosmogenic radionuclides suggests that the preatmospheric radius was rather small ~20 cm. The cosmic ray exposure age of Ejby (83 ± 11 Ma) is the highest of an H chondrite and the second highest age for an ordinary chondrite. Using the preatmospheric orbit of the Ejby meteoroid (Spurny et al. 2017) locations of the recovered fragments, and wind data from the date of the fall, we have modeled the dark flight (below 18 km) of the fragments. The recovery location of the largest fragment can only be explained if aerodynamic effects during the dark flight phase are included. The recovery location of all other fragments are consistent with the dark flight modeling