Mesenchymal stem cells (MSCs) are often known to have a therapeutic potential in the cell-mediated repair for fatal or incurable diseases. In this study, canine umbilical cord MSCs (cUC-MSCs) were isolated from umbilical cord matrix (n = 3) and subjected to proliferative culture for 5 consecutive passages. The cells at each passage were characterized for multipotent MSC properties such as proliferation kinetics, expression patterns of MSC surface markers and self-renewal associated markers, and chondrogenic differentiation. In results, the proliferation of the cells as determined by the cumulative population doubling level was observed at its peak on passage 3 and stopped after passage 5, whereas cell doubling time dramatically increased after passage 4. Expression of MSC surface markers (CD44, CD54, CD61, CD80, CD90 and Flk-1), molecule (HMGA2) and pluripotent markers (sox2, nanog) associated with self-renewal was negatively correlated with the number of passages. However, MSC surface marker (CD105) and pluripotent marker (Oct3/4) decreased with increasing the number of subpassage. cUC-MSCs at passage 1 to 5 underwent chondrogenesis under specific culture conditions, but percentage of chondrogenic differentiation decreased with increasing the number of subpassage. Collectively, the present study suggested that sequential subpassage could affect multipotent properties of cUC-MSCs and needs to be addressed before clinical applications.

Monocytes and macrophages that originate from common myeloid progenitors perform various crucial roles in the innate immune system. Stimulation with LPS combined with TLR4 drives the production of pro-inflammatory cytokines through MAPKs and NF-κB pathway in different cells. However, the difference in LPS susceptibility between monocytes and macrophages is poorly understood. In this study, we found that pro-inflammatory cytokines—IL-1β, IL-6 and TNFα showed greater induction in phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 cells than in THP-1 cells. To determine the difference in cytokine expression, the surface proteins such as TLR4-related proteins and intracellular adaptor proteins were more preserved in PMA-differentiated THP-1 cells than in THP-1 cells. MyD88 is a key molecule responsible for the difference in LPS susceptibility. Moreover, MAPKs and NF-κB pathway-related molecules showed higher levels of phosphorylation in PMA-differentiated THP-1 cells than in THP-1 cells. Upon MyD88 depletion, there was no difference in the phosphorylation of MAPK pathway-related molecules. Therefore, these results demonstrate that the difference in LPS susceptibility between THP-1 cells and PMA-differentiated THP-1 cells occur as a result of gap between the activated MAPKs and NF-κB pathways via changes in the expression of LPS-related receptors and MyD88.

The testis-derived male germ-line stem (GS) cell, the in vitro counterpart of spermatogonial stem cell (SSC), can initiate donor-derived spermatogenesis in recipient testes and therefore, has been viewed as a future therapeutic modality for treatment of male infertility in azoospermic patients and in cancer patients who are expecting chemotherapy. Upon extended in vitro culture, GS cells also generate a second cell type called multipotent adult germ-line stem (maGS) cell which, upon testicular transplantation, produces teratoma instead of initiating spermatogenesis. Here, we show that expressions of both Let-7a and Let-7d were consistently higher while that of miR-294 (embryonic stem cell-cycle-regulating miRNA; ESCC) was lower in GS cells than in maGS cells. Furthermore, among several putative targets of Let-7 identified by in silico bioinformatics, expressions of Igf2 and H19 mRNA targets significantly differed between GS and maGS cells. However, although the CTCF binding factor (a component of DNA methylation machinery at Igf2-H19 cluster) was also a putative target for Let-7, the difference in expressions of Igf2 and H19 between GS and maGS cells was not mediated through a change in DNA methylation. Both GS and maGS cells maintained androgenetic imprinting at the Igf2-H19 imprinting control region and Peg1 differentially methylated region. In conclusion, our study suggests that high Let-7 expression may be a unique property of GS cells and expressions of Let-7 and ESCC miRNAs may serve as miRNA signatures to distinguish them from maGS cells during clinical transplantation, to avoid the likelihood of teratoma formation due to maGS cells generated during extended in vitro culture of GS cells.