Mice carrying a germ-line null mutation of the prolactin receptor gene have been produced by gene targeting in embryonic stem cells. Heterozygous females showed almost complete failure of lactation attributable to greatly reduced mammary gland development after their first, but not subsequent, pregnancies. Homozygous females were sterile owing to a complete failure of embryonic implantation. Moreover, they presented multiple reproductive abnormalities, including irregular cycles, reduced fertilization rates, defective preimplantation embryonic development, and lack of pseudopregnancy. Half of the homozygous males were infertile or showed reduced fertility. This work establishes the prolactin receptor as a key regulator of mammalian reproduction, and provides the first total ablation model to further study the role of the prolactin receptor and its ligands.
The mammalian SWI-SNF complex is an evolutionarily conserved, multi-subunit machine, involved in chromatin remodelling during transcriptional activation. Within this complex, the BRM (SNF2α) and BRG1 (SNF2β) proteins are mutually exclusive subunits that are believed to affect nucleosomal structures using the energy of ATP hydrolysis. In order to characterize possible differences in the function of BRM and BRG1, and to gain further insights into the role of BRM-containing SWI-SNF complexes, the mouse BRM gene was inactivated by homologous recombination. BRM -/-mice develop normally, suggesting that an observed up-regulation of the BRG1 protein can functionally replace BRM in the SWI-SNF complexes of mutant cells. Nonetheless, adult mutant mice were~15% heavier than control littermates. This may be caused by increased cell proliferation, as demonstrated by a higher mitotic index detected in mutant livers. This is supported further by the observation that mutant embryonic fibroblasts were significantly deficient in their ability to arrest in the G 0 /G 1 phase of the cell cycle in response to cell confluency or DNA damage. These studies suggest that BRM participates in the regulation of cell proliferation in adult mice.
The Ets-1 proto-oncogene is a member of a transcription factor family characterized by homology to the v-ets oncogene. In adult mice, Ets-1 is expressed predominantly in lymphoid cells where it has been implicated in regulating transcription of lymphocyte-specific genes. Following T-cell activation, the specific DNA binding activity of Ets-1 is inactivated by transient phosphorylation, suggesting a function in the transition from the resting to activated state. Ets-1 has also been suggested to cooperate with the AP-1 transcription factor complex to mediate cellular growth factor responses. Here we show, by using RAG-2-deficient blastocyst complementation, that Ets-1 deficiency has dramatic, but different, effects on development and function of T- and B-lineage cells. Ets-1-deficient T cells were present in reduced numbers and were highly susceptible to cell death in vitro. In contrast, Ets-1-deficient B cells were present in normal numbers but a large proportion were IgM plasma cells. Our data demonstrate that Ets-1 is essential for maintenance of the normal pool of resting T- and B-lineage cells.
After implantation, mouse embryos deficient for the activity of the transforming growth factor-beta member Nodal fail to form both the mesoderm and the definitive endoderm. They also fail to specify the anterior visceral endoderm, a specialized signaling center which has been shown to be required for the establishment of anterior identity in the epiblast. Our study reveals that Nodal-/- epiblast cells nevertheless express prematurely and ectopically molecular markers specific of anterior fate. Our analysis shows that neural specification occurs and regional identities characteristic of the forebrain are established precociously in the Nodal-/- mutant with a sequential progression equivalent to that of wild-type embryo. When explanted and cultured in vitro, Nodal-/- epiblast cells readily differentiate into neurons. Genes normally transcribed in organizer-derived tissues, such as Gsc and Foxa2, are also expressed in Nodal-/- epiblast. The analysis of Nodal-/-;Gsc-/- compound mutant embryos shows that Gsc activity plays no critical role in the acquisition of forebrain characters by Nodal-deficient cells. This study suggests that the initial steps of neural specification and forebrain development may take place well before gastrulation in the mouse and highlights a possible role for Nodal, at pregastrula stages, in the inhibition of anterior and neural fate determination.
Low back pain (LBP), frequently associated with intervertebral disc (IVD) degeneration, is a major public health concern. LBP is currently managed by pharmacological treatments and, if unsuccessful, by invasive surgical procedures, which do not counteract the degenerative process. Considering that IVD cell depletion is critical in the degenerative process, the supplementation of IVD with reparative cells, associated or not with biomaterials, has been contemplated. Recently, the discovery of reparative stem/progenitor cells in the IVD has led to increased interest in the potential of endogenous repair strategies. Recruitment of these cells by specific signals might constitute an alternative strategy to cell transplantation. Here, we review the status of cell-based therapies for treating IVD degeneration and emphasize the current concept of endogenous repair as well as future perspectives. This review also highlights the challenges of the mobilization/differentiation of reparative progenitor cells through the delivery of biologics factors to stimulate IVD regeneration.
Nodal, a secreted factor known for its conserved functions in cell-fate specification and the establishment of embryonic axes, is also required in mammals to maintain the pluripotency of the epiblast, the tissue that gives rise to all fetal lineages. Although Nodal is expressed as early as E3.5 in the mouse embryo, its regulation and functions at pre- and peri-implantation stages are currently unknown. Sensitive reporter transgenes for two Nodal cis-regulatory regions, the PEE and the ASE, exhibit specific expression profiles before implantation. Mutant and inhibitor studies find them respectively regulated by Wnt/β-catenin signaling and Activin/Nodal signaling, and provide evidence for localized and heterogeneous activities of these pathways in the inner cell mass, the epiblast and the primitive endoderm. These studies also show that Nodal and its prime effector, FoxH1, are not essential to preimplantation Activin/Nodal signaling. Finally, a strong upregulation of the ASE reporter in implanting blastocysts correlates with a downregulation of the pluripotency factor Nanog in the maturing epiblast. This study uncovers conservation in the mouse blastocyst of Wnt/β-catenin and Activin/Nodal-dependent activities known to govern Nodal expression and the establishment of polarity in the blastula of other deuterostomes. Our results indicate that these pathways act early on to initiate distinct cell-specification processes in the ICM derivatives. Our data also suggest that the activity of the Activin/Nodal pathway is dampened by interactions with the molecular machinery of pluripotency until just before implantation, possibly delaying cell-fate decisions in the mouse embryo.
Degenerative disc disease (DDD) primarily affects the central part of the intervertebral disc namely the nucleus pulposus (NP). DDD explains about 40% of low back pain and is characterized by massive cellular alterations that ultimately result in the disappearance of resident NP cells. Thus, repopulating the NP with regenerative cells is a promising therapeutic approach and remains a great challenge. The objectives of this study were to evaluate the potential of growth factor-driven protocols to commit human adipose stromal cells (hASCs) toward NP-like cell phenotype and the involvement of Smad proteins in this differentiation process. Here, we demonstrate that the transforming growth factor-b1 and the growth differentiation factor 5 synergistically drive the nucleopulpogenic differentiation process. The commitment of the hASCs was robust and highly specific as attested by the expression of NP-related genes characteristic of young healthy human NP cells. In addition, the engineered NP-like cells secreted an abundant aggrecan and type II collagen rich extracellular matrix comparable with that of native NP. Furthermore, we demonstrate that these in vitro engineered cells survived, maintained their specialized phenotype and secretory activity after in vivo transplantation in nude mice subcutis. Finally, we provide evidence suggesting that the Smad 2/3 pathway mainly governed the acquisition of the NP cell molecular identity while the Smad1/5/8 pathway controlled the NP cell morphology. This study offers valuable insights for the development of biologically-inspired treatments for DDD by generating adapted and exhaustively characterized autologous regenerative cells. STEM CELLS 2016;34:653-667 SIGNIFICANCE STATEMENTIn the present manuscript, we investigated whether human adipose stromal cells (hASCs) can be a clinically relevant source of stem cells for the generation of phenotypically stable and biologically active NPCyte-like cells. We successfully generated NPCyte-like cells from hASCs using a reproducible, robust and accurate growth factor-based induction protocol. We also demonstrated by experimental organogenesis in nude mice subcutis, that NPCyte-like cells seeded in an instructive hydrogel survived, maintained their specialized phenotype and presented secretory activities in vivo. In addition to highlighting the robustness and reproducibility of our protocol, we also document the temporal role of Smad pathways towards NP commitment by using specific chemical inhibitors. These data strengthen the specificity of the NP cell commitment by demonstrating new insights into the role of Smad proteins.
In mammals, cloning by nuclear transfer (NT) into an enucleated oocyte is a very inefficient process, even if it can generate healthy adults. We show that blastocysts derived from embryonic stem (ES) donor cells develop at a high rate, correctly express the pluripotential marker gene Oct4 in ICM cells and display normal growth in vitro. Moreover, the majority of them implant in the uterus of recipient females. We combine embryological studies, gene expression analysis during gastrulation and generation of chimaeric embryos to identify the developmental origin (stage and tissue affected) of NT embryo mortality. The majority died before mid-gestation from defects arising early, either at peri-implantation stages or during the gastrulation period. The first type of defect is a non-cell autonomous defect of the epiblast cells and is rescued by complementation of NT blastocysts with normal ES or ICM cells. The second type of defect affects growth regulation and the shape of the embryo but does not directly impair the initial establishment of the patterning of the embryo. Only chimaeras formed by the aggregation of NT and tetraploid embryos reveal no growth abnormalities at gastrulation. These studies indicate that the trophoblast cell lineage is the primary source of these defects. These embryological studies provide a solid basis for understanding reprogramming errors in NT embryos. In addition, they unveil new aspects of growth regulation while increasing our knowledge on the role of crosstalk between the extra-embryonic and the embryonic regions of the conceptus in the control of growth and morphogenesis.
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