Antibodies for Venezuelan Equine Encephalitis Virus Protect Embryoid Bodies from Chikungunya Virus.

Chikungunya virus (CHIKV) is an alphavirus that causes febrile illness punctuated by severe polyarthralgia. After the emergence of CHIKV in the Western Hemisphere, multiple reports of congenital infections were published that documented neurological complications, cardiac defects, respiratory distress, and miscarriage. The Western Hemisphere is endemic to several alphaviruses, and whether antigenic cross-reactivity can impact the course of infection has not been explored. Recent advances in biomedical engineering have produced cell co-culture models that replicate the cellular interface at the maternal fetal axis. We employed a trans-well assay to determine if cross-reactive antibodies affected the movement and replication of CHIKV across placental cells and into an embryoid body. The data showed that antibodies to Venezuelan equine encephalitis virus significantly reduced CHIKV viral load in embryoid bodies. The data highlighted the fact that viral pathogenesis can be cell-specific and that exploiting antigenic cross-reactivity could be an avenue for reducing the impact of congenital CHIKV infections.

Remyelination Is Correlated with Regulatory T Cell Induction Following Human Embryoid Body-Derived Neural Precursor Cell Transplantation in a Viral Model of Multiple Sclerosis.

We have recently described sustained clinical recovery associated with dampened neuroinflammation and remyelination following transplantation of neural precursor cells (NPCs) derived from human embryonic stem cells (hESCs) in a viral model of the human demyelinating disease multiple sclerosis. The hNPCs used in that study were derived by a novel direct differentiation method (direct differentiation, DD-NPCs) that resulted in a unique gene expression pattern when compared to hNPCs derived by conventional methods. Since the therapeutic potential of human NPCs may differ greatly depending on the method of derivation and culture, we wanted to determine whether NPCs differentiated using conventional methods would be similarly effective in improving clinical outcome under neuroinflammatory demyelinating conditions. For the current study, we utilized hNPCs differentiated from a human induced pluripotent cell line via an embryoid body intermediate stage (EB-NPCs). Intraspinal transplantation of EB-NPCs into mice infected with the neurotropic JHM strain of mouse hepatitis virus (JHMV) resulted in decreased accumulation of CD4+ T cells in the central nervous system that was concomitant with reduced demyelination at the site of injection. Dampened neuroinflammation and remyelination was correlated with a transient increase in CD4+FOXP3+ regulatory T cells (Tregs) concentrated within the peripheral lymphatics. However, compared to our earlier study, pathological improvements were modest and did not result in significant clinical recovery. We conclude that the genetic signature of NPCs is critical to their effectiveness in this model of viral-induced neurologic disease. These comparisons will be useful for understanding what factors are critical for the sustained clinical improvement.

Enrichment of Scleroderma Vascular Disease-Associated Autoantigens in Endothelial Lineage Cells.

OBJECTIVE: Scleroderma patients with autoantibodies to CENPs and/or interferon-inducible protein 16 (IFI-16) are at increased risk of severe vascular complications. This study was undertaken to determine whether these autoantigens are enriched in cells of the vasculature. METHODS: Successive stages of embryoid bodies (EBs) as well as vascular progenitors were used to evaluate the expression of scleroderma autoantigens IFI-16 and CENP by immunoblotting. CD31 was included to mark early blood vessels. IFI-16 and CD31 expression were defined in paraffin-embedded skin sections from scleroderma patients and from healthy controls. IFI-16 expression was determined by flow cytometric analysis in circulating endothelial cells (CECs) and circulating hematopoietic progenitor cells. RESULTS: Expression of CENP-A, IFI-16, and CD31 was enriched in EBs on days 10 and 12 of differentiation, and particularly in cultures enriched in vascular progenitors (IFI-16, CD31, and CENPs A and B). This pattern was distinct from that of comparator autoantigens. Immunohistochemical staining of paraffin-embedded skin sections showed enrichment of IFI-16 in CD31-positive vascular endothelial cells in biopsy specimens from scleroderma patients and normal controls. Flow cytometric analysis revealed IFI-16 expression in circulating hematopoietic progenitor cells but minimal expression in CECs. CONCLUSION: Our findings indicate that expression of the scleroderma autoantigens IFI-16 and CENPs, which are associated with severe vascular disease, is increased in vascular progenitors and mature endothelial cells. High level, lineage-enriched expression of autoantigens may explain the striking association between clinical phenotypes and the immune targeting of specific autoantigens.

Control of Immune Response to Allogeneic Embryonic Stem Cells by CD3 Antibody-Mediated Operational Tolerance Induction.

Implantation of embryonic stem cells (ESCs) and their differentiated derivatives into allogeneic hosts triggers an immune response that represents a hurdle to clinical application. We established in autoimmunity and in transplantation that CD3 antibody therapy induces a state of immune tolerance. Promising results have been obtained with CD3 antibodies in the clinic. In this study, we tested whether this strategy can prolong the survival of undifferentiated ESCs and their differentiated derivatives in histoincompatible hosts. Recipients of either mouse ESC-derived embryoid bodies (EBs) or cardiac progenitors received a single short tolerogenic regimen of CD3 antibody. In immunocompetent mice, allogeneic EBs and cardiac progenitors were rejected within 20-25 days. Recipients treated with CD3 antibody showed long-term survival of implanted cardiac progenitors or EBs. In due course, EBs became teratomas, the growth of which was self-limited. Regulatory CD4(+)FoxP3(+) T cells and signaling through the PD1/PDL1 pathway played key roles in the CD3 antibody therapeutic effect. Gene profiling emphasized the importance of TGF-ß and the inhibitory T cell coreceptor Tim3 to the observed effect. These results demonstrate that CD3 antibody administered alone promotes prolonged survival of allogeneic ESC derivatives and thus could prove useful for enhancing cell engraftment in the absence of chronic immunosuppression.

Efficient differentiation of steroidogenic and germ-like cells from epigenetically-related iPSCs derived from ovarian granulosa cells.

To explore restoration of ovarian function using epigenetically-related, induced pluripotent stem cells (iPSCs), we functionally evaluated the epigenetic memory of novel iPSC lines, derived from mouse and human ovarian granulosa cells (GCs) using c-Myc, Klf4, Sox2 and Oct4 retroviral vectors. The stem cell identity of the mouse and human GC-derived iPSCs (mGriPSCs, hGriPSCs) was verified by demonstrating embryonic stem cell (ESC) antigen expression using immunocytochemistry and RT-PCR analysis, as well as formation of embryoid bodies (EBs) and teratomas that are capable of differentiating into cells from all three germ layers. GriPSCs' gene expression profiles associate more closely with those of ESCs than of the originating GCs as demonstrated by genome-wide analysis of mRNA and microRNA. A comparative analysis of EBs generated from three different mouse cell lines (mGriPSCs; fibroblast-derived iPSC, mFiPSCs; G4 embryonic stem cells, G4 mESCs) revealed that differentiated mGriPSC-EBs synthesize 10-fold more estradiol (E2) than either differentiated FiPSC- or mESC-EBs under identical culture conditions. By contrast, mESC-EBs primarily synthesize progesterone (P4) and FiPSC-EBs produce neither E2 nor P4. Differentiated mGriPSC-EBs also express ovarian markers (AMHR, FSHR, Cyp19a1, ER and Inha) as well as markers of early gametogenesis (Mvh, Dazl, Gdf9, Boule and Zp1) more frequently than EBs of the other cell lines. These results provide evidence of preferential homotypic differentiation of mGriPSCs into ovarian cell types. Collectively, our data support the hypothesis that generating iPSCs from the desired tissue type may prove advantageous due to the iPSCs' epigenetic memory.

Reduced immunogenicity of induced pluripotent stem cells derived from Sertoli cells.

Sertoli cells constitute the structural framework in testis and provide an immune-privileged environment for germ cells. Induced pluripotent stem cells (iPS cells) resemble embryonic stem cells (ES cells) and are generated from somatic cells by expression of specific reprogramming transcription factors. Here, we used C57BL/6 (B6) Sertoli cells to generate iPS cells (Ser-iPS cells) and compared the immunogenicity of Ser-iPS cells with iPS cells derived from mouse embryonic fibroblast (MEF-iPS cells). Ser-iPS cells were injected into syngeneic mice to test for their in vivo immunogenicity in teratoma assay. Teratoma assay allows assessing in vivo immunogenicity of iPS cells and of their differentiated progeny simultaneously. We observed that early-passage Ser-iPS cells formed more teratomas with less immune cell infiltration and tissue damage and necrosis than MEF-iPS cells. Differentiating Ser-iPS cells in embryoid bodies (EBs) showed reduced T cell activation potential compared to MEF-iPS cells, which was similar to syngeneic ES cells. However, Ser-iPS cells lost their reduced immunogenicity in vivo after extended passaging in vitro and late-passage Ser-iPS cells exhibited an immunogenicity similar to MEF-iPS cells. These findings indicate that early-passage Ser-iPS cells retain some somatic memory of Sertoli cells that impacts on immunogenicity of iPS cells and iPS cell-derived cells in vivo and in vitro. Our data suggest that immune-privileged Sertoli cells might represent a preferred source for iPS cell generation, if it comes to the use of iPS cell-derived cells for transplantation.

Antibacterial capacity of differentiated murine embryonic stem cells during defined in vitro inflammatory conditions.

Embryonic stem (ES) cells are a powerful model for the development of cells responsible for the cellular immune response. Therefore, we analyzed the defense and phagocytic capacity of embryoid bodies (EBs) derived from ES cells using in the vitro inflammatory conditions caused by Escherichia coli. Further, we used this phagocytic activity to purify activated immune cells. Our data show that spontaneously differentiated 18-day-old EBs of the cell line CGR8 contained immune cells, which were positive for CD45, CD68, CD11b, F4/80, and CD19. Exposure of these EBs to E. coli with defined infection doses of bacterial colony-forming units (CFUs) led to a significant time-dependent reduction of CFUs, indicating the immune responses exerted by EBs. This was paralleled by an upregulation of inflammatory cytokines, that is, IL-1ß and TNF-α. Western blot analysis of infected EBs indicated an upregulation of CD14 and cytochrome b-245 heavy chain (NOX2). Silencing of NOX2 significantly reduced the antibacterial capacity of EBs, which was partially explained by reduction of F4/80-positive cells. To identify, isolate, and further cultivate phagocytic active cells from differentiated EBs, a cocultivation assay of differentiated ES cells with green fluorescent protein (GFP)-labeled E. coli was established. Colocalization of GFP-labeled E. coli with cells positive for CD45, CD68, and F4/80 revealed time-dependent phagocytotic uptake, which was underlined by colocalization with the LysoTracker-Red(®) dye as well as preincubation with cytochalasin D. In conclusion, a primitive immune response with efficient phagocytosis was responsible for the antibacterial capacity of differentiated EBs.