Pleiotropic Impacts of Macrophage and Microglial Deficiency on Development in Rats with Targeted Mutation of the Csf1r Locus.

We have produced Csf1r-deficient rats by homologous recombination in embryonic stem cells. Consistent with the role of Csf1r in macrophage differentiation, there was a loss of peripheral blood monocytes, microglia in the brain, epidermal Langerhans cells, splenic marginal zone macrophages, bone-associated macrophages and osteoclasts, and peritoneal macrophages. Macrophages of splenic red pulp, liver, lung, and gut were less affected. The pleiotropic impacts of the loss of macrophages on development of multiple organ systems in rats were distinct from those reported in mice. Csf1r-/- rats survived well into adulthood with postnatal growth retardation, distinct skeletal and bone marrow abnormalities, infertility, and loss of visceral adipose tissue. Gene expression analysis in spleen revealed selective loss of transcripts associated with the marginal zone and, in brain regions, the loss of known and candidate novel microglia-associated transcripts. Despite the complete absence of microglia, there was little overt phenotype in brain, aside from reduced myelination and increased expression of dopamine receptor-associated transcripts in striatum. The results highlight the redundant and nonredundant functions of CSF1R signaling and of macrophages in development, organogenesis, and homeostasis.

Reprogramming pig fetal fibroblasts reveals a functional LIF signaling pathway.

Distinct signaling pathways are reported to maintain pluripotency in embryo-derived stem cells. Mouse embryonic stem cells (ESCs) respond to leukemia inhibitory factor (LIF) and bone morphogenetic protein (BMP)-mediated activity, whereas human ESCs depend upon Fibroblast growth factor (FGF) and activin signaling. In the majority of mammals investigated, however, the signals that support stem cell pluripotency are not well defined, as is evident by the persistent difficulties in maintaining authentic stable ESC lines. Induction of pluripotency by transcription factor-mediated reprogramming could provide an alternative way to produce ESC-like cells from nonpermissive species, and facilitate identification of core ESC signaling requirements. To evaluate the effectiveness of this approach in pigs, we transduced porcine foetal fibroblasts with retroviruses expressing Oct4, Sox2, Klf4, and c-Myc, and maintained the resulting cultures in medium containing either LIF or FGF2. Alkaline phosphatase positive colonies with compact, mouse ESC-like morphology were preferentially recovered using serum-free medium supplemented with LIF. These cell lines expressed the endogenous stem cell transcription factors, OCT4, NANOG, and SOX2, and the cell surface marker SSEA-4, consistent with acquisition of an undifferentiated state. However, restricted differentiation potential, and persistent expression of retroviral transgenes indicated that reprogramming was incomplete. Interestingly, LIF activated both the transcription factor STAT3 and its target gene SOCS3, and stimulated cell growth, indicating functional coupling of the signaling pathway in these cells. This demonstration of LIF-dependence in reprogrammed pig cells supports the notion that the connection between LIF/STAT3 signaling and the core regulatory network of pluripotent stem cells is a conserved pathway in mammals.

Ovine-induced pluripotent stem cells can contribute to chimeric lambs.

Pluripotential stem cells from livestock offer an exciting prospect for the biotechnology industry. Applying strategies established for the derivation of murine induced pluripotential stem cells (iPSCs), we have isolated ovine iPSCs that can give rise to cells characteristic of all three germ cell layers both in vitro from embryoid bodies and in teratomas in vivo. Furthermore, although at a low level, these ovine iPS cells can contribute to live-born chimeric lambs. Colonies derived from ovine embryonic fibroblasts transfected with murine cMyc, Klf4, Oct4, and Sox2 displayed smooth domes with sharp edges when grown in human embryonic stem cell (ESC) medium but not in mouse ESC medium. These ovine iPSCs were alkaline phosphatase positive, expressed Nanog, and had a normal karyotype. These cells represent an important step in the understanding of mechanistic nature of pluripotency in ungulates.

Ablation of undifferentiated human embryonic stem cells: exploiting innate immunity against the Gal alpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) epitope.

Although undifferentiated human embryonic stem cells (hESCs) are tumorigenic, this capacity is lost after differentiation, and hESCs are being widely investigated for applications in regenerative medicine. To engineer protection against the unintentional transplantation of undifferentiated cells, we generated hESCs carrying a construct in which the alpha1,3-galactosyltransferase (GalT) open reading frame was transcribed from the hTERT promoter (pmGT). Because the endogenous GalT gene is inactive, GalT expression was limited to undifferentiated cells. A second chimeric construct (pmfGT) differed by replacement of the GalT leader sequence for that of the fucosyltransferase gene. Two subclones containing stable integrations of pmGT and pmfGT (M2 and F11, respectively) were assessed for their response to human serum containing antibodies to the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitope. The low-variegation line, M2, and to a lesser extent the more variegated line F11, were sensitive to human serum when exposed in the undifferentiated state. However, M2 cells were largely insensitive after differentiation and retained both a normal karyotype and the ability to differentiate into derivatives of the three germ layers in severe combined immunodeficient mice. These data exemplify a method of protection against residual, undifferentiated hESCs prior to engraftment and may provide ongoing immune surveillance after engraftment against dedifferentiation or against de novo tumorigenesis involving hTERT reactivation. Untransfected H9 cells were not sensitive to the human serum used in this study. Hence, in our system, interactions of hESCs with other circulating antibodies, such as anti-Neu5Gc, were not observed.

Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors.

Human and mouse embryonic stem (ES) cells have the capacity to differentiate into derivatives of all three germ layers, suggesting novel therapies for degenerative, metabolic, and traumatic disorders. ES-based regenerative medicine will be further advanced by the development of reliable methods for transgene introduction and expression. Here, we show infection of human and mouse embryonic stem (ES) cells with two of the most popular vectors in gene transfer, adenovirus type 5 (Ad5) and adeno-associated virus (AAV; serotypes 2, 4, and 5). All vectors express the nuclear-localized marker gene beta-galactosidase expressed from the Rous Sarcoma Virus long terminal repeat (RSV-LTR). Both Ad5 and AAV2 infected human and mouse ES cells and gave rise to beta-galactosidase expression. AAV4 and 5 did not yield detectable levels of beta-galactosidase expression. Quantitative PCR analysis of virally infected human and mouse ES cells revealed that only Ad5 and AAV2 are capable of transducing both cell-types. No viral DNA was detected in cells infected with either AAV4 or AAV5. Infection and subsequent differentiation of mouse and human ES cells with Ad5 showed that beta-galactosidase-expressing cells were restricted to cells in the interior of the embryoid body mass. No beta-galactosidase expression was observed in AAV-infected cells following differentiation. There was no difference in morphology or differentiation patterns between infected and noninfected differentiating mouse and human ES cells. Differentiation of hES cells prior to infection led to transduction of neuronally differentiated cells with good efficiency using all vectors. These data show that Ad5- and AAV2-based vectors are capable of infecting both human and mouse ES cells, in both their undifferentiated and differentiated states, whereas AAV4 and AAV5 can infect human and mouse ES cells only following differentiation.