Genetic lineage tracing of targeted cell populations during enthesis healing.

Rotator cuff supraspinatus tendon injuries are clinically challenging due to the high rates of failure after surgical repair. One key limitation to functional healing is the failure to regenerate the enthesis transition between tendon and bone, which heals by disorganized scar formation. Using two models of supraspinatus tendon injury in mouse (partial tear and full detachment/repair), the purpose of the study was to determine functional gait outcomes and identify the origin of the cells that mediate healing. Consistent with previous reports, enthesis injuries did not regenerate; partial tear resulted in a localized scar defect adjacent to intact enthesis, while full detachment with repair resulted in full disruption of enthesis alignment and massive scar formation between tendon and enthesis fibrocartilage. Although gait after partial tear injury was largely normal, gait was permanently impaired after full detachment/repair. Genetic lineage tracing of intrinsic tendon and cartilage/fibrocartilage cells (ScxCreERT2 and Sox9CreERT2 , respectively), myofibroblasts (αSMACreERT2 ), and Wnt-responsive stem cells (Axin2CreERT2 ) failed to identify scar-forming cells in partial tear injury. Unmineralized enthesis fibrocartilage was strongly labeled by Sox9CreERT2 while Axin2CrERT2 labeled a subset of tendon cells away from the skeletal insertion site. In contrast to the partial tear model, Axin2CreERT2 labeling showed considerable contribution of Axin2lin cells to the scar after full detachment/repair. Clinical Significance: Clinically relevant models of rotator cuff tendon injuries in mouse enable the use of genetic tools; lineage tracing suggests that distinct mechanisms of healing are activated with full detachment/repair injuries versus partial tear. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3275-3284, 2018.

Young Bone Marrow Sca-1 Cells Rejuvenate the Aged Heart by Promoting Epithelial-to-Mesenchymal Transition.

Background: To improve the regenerative capacity of aged individuals, we reconstituted bone marrow (BM) of aged mice with young Sca-1 cells, which repopulated cardiac progenitors and prevented cardiac dysfunction after a myocardial infarction (MI). However, the mechanisms involved were incompletely elucidated. This study aimed to investigate whether young, highly regenerative BM Sca-1 cells exert their cardio-protective effects on the aged heart through reactivation of the epithelial-to-mesenchymal transition (EMT) process. Methods:In vitro, BM Sca-1 cells were co-cultured with epicardial-derived cells (EPDCs) under hypoxia condition; mRNA and protein levels of EMT genes were measured along with cellular proliferation and migration. In vivo, BM Sca-1+ or Sca-1- cells from young mice (2-3 months) were transplanted into lethally-irradiated old mice (20-22 months) to generate chimeras. In addition, Sca-1 knockout (KO) mice were reconstituted with wild type (WT) BM Sca-1+ cells. The effects of BM Sca-1 cell on EMT reactivation and improvement of cardiac function after MI were evaluated. Results:In vitro, BM Sca-1+ cells increased EPDC proliferation, migration, and EMT relative to Sca-1- cells and these effects were inhibited by a TGF-ß blocker. In vivo, more young BM Sca-1+ than Sca-1- cells homed to the epicardium and induced greater host EPDC proliferation, migration, and EMT after MI. Furthermore, reconstitution of Sca-1 KO mice with WT Sca-1+ cells was associated with the reactivation of EMT and improved cardiac function after MI. Conclusions: Young BM Sca-1+ cells improved cardiac regeneration through promoting EPDC proliferation, migration and reactivation of EMT via the TGF-ß signaling pathway.

RANK Signaling Blockade Reduces Breast Cancer Recurrence by Inducing Tumor Cell Differentiation.

RANK expression is associated with poor prognosis in breast cancer even though its therapeutic potential remains unknown. RANKL and its receptor RANK are downstream effectors of the progesterone signaling pathway. However, RANK expression is enriched in hormone receptor negative adenocarcinomas, suggesting additional roles for RANK signaling beyond its hormone-dependent function. Here, to explore the role of RANK signaling once tumors have developed, we use the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT), which mimics RANK and RANKL expression patterns seen in human breast adenocarcinomas. Complementary genetic and pharmacologic approaches demonstrate that therapeutic inhibition of RANK signaling drastically reduces the cancer stem cell pool, decreases tumor and metastasis initiation, and enhances sensitivity to chemotherapy. Mechanistically, genome-wide expression analyses show that anti-RANKL therapy promotes lactogenic differentiation of tumor cells. Moreover, RANK signaling in tumor cells negatively regulates the expression of Ap2 transcription factors, and enhances the Wnt agonist Rspo1 and the Sca1-population, enriched in tumor-initiating cells. In addition, we found that expression of TFAP2B and the RANK inhibitor, OPG, in human breast cancer correlate and are associated with relapse-free tumors. These results support the use of RANKL inhibitors to reduce recurrence and metastasis in breast cancer patients based on its ability to induce tumor cell differentiation. Cancer Res; 76(19); 5857-69. ©2016 AACR.

Murine Sca1(+)Lin(-) bone marrow contains an endodermal precursor population that differentiates into hepatocytes.

The direct differentiation of hepatocytes from bone marrow cells remains controversial. Several mechanisms, including transdifferentiation and cell fusion, have been proposed for this phenomenon, although direct visualization of the process and the underlying mechanisms have not been reported. In this study, we established an efficient in vitro culture method for differentiation of functioning hepatocytes from murine lineage-negative bone marrow cells. These cells reduced liver damage and incorporated into hepatic parenchyma in two independent hepatic injury models. Our simple and efficient in vitro protocol for endodermal precursor cell survival and expansion enabled us to identify these cells as existing in Sca1(+) subpopulations of lineage-negative bone marrow cells. The endodermal precursor cells followed a sequential developmental pathway that included endodermal cells and hepatocyte precursor cells, which indicates that lineage-negative bone marrow cells contain more diverse multipotent stem cells than considered previously. The presence of equivalent endodermal precursor populations in human bone marrow would facilitate the development of these cells into an effective treatment modality for chronic liver diseases.

Murine Sca1+Lin- bone marrow contains an endodermal precursor population that differentiates into hepatocytes

The direct differentiation of hepatocytes from bone marrow cells remains controversial. Several mechanisms, including transdifferentiation and cell fusion, have been proposed for this phenomenon, although direct visualization of the process and the underlying mechanisms have not been reported. In this study, we established an efficient in vitro culture method for differentiation of functioning hepatocytes from murine lineage-negative bone marrow cells. These cells reduced liver damage and incorporated into hepatic parenchyma in two independent hepatic injury models. Our simple and efficient in vitro protocol for endodermal precursor cell survival and expansion enabled us to identify these cells as existing in Sca1+ subpopulations of lineage-negative bone marrow cells. The endodermal precursor cells followed a sequential developmental pathway that included endodermal cells and hepatocyte precursor cells, which indicates that lineage-negative bone marrow cells contain more diverse multipotent stem cells than considered previously. The presence of equivalent endodermal precursor populations in human bone marrow would facilitate the development of these cells into an effective treatment modality for chronic liver diseases.