Hypoimmunogenic human iPSCs expressing HLA-G, PD-L1, and PD-L2 evade innate and adaptive immunity.

BACKGROUND: The human induced pluripotent stem cells (hiPSCs) can generate all the cells composing the human body, theoretically. Therefore, hiPSCs are thought to be a candidate source of stem cells for regenerative medicine. The major challenge of allogeneic hiPSC-derived cell products is their immunogenicity. The hypoimmunogenic cell strategy is allogenic cell therapy without using immune suppressants. Advances in gene engineering technology now permit the generation of hypoimmunogenic cells to avoid allogeneic immune rejection. In this study, we generated a hypoimmunogenic hiPSC (HyPSC) clone that had diminished expression of human leukocyte antigen (HLA) class Ia and class II and expressed immune checkpoint molecules and a safety switch. METHODS: First, we generated HLA class Ia and class II double knockout (HLA class Ia/II DKO) hiPSCs. Then, a HyPSC clone was generated by introducing exogenous ß-2-microglobulin (B2M), HLA-G, PD-L1, and PD-L2 genes, and the Rapamycin-activated Caspase 9 (RapaCasp9)-based suicide gene as a safety switch into the HLA class Ia/II DKO hiPSCs. The characteristics and immunogenicity of the HyPSCs and their derivatives were analyzed. RESULTS: We found that the expression of HLA-G on the cell surface can be enhanced by introducing the exogenous HLA-G gene along with B2M gene into HLA class Ia/II DKO hiPSCs. The HyPSCs retained a normal karyotype and had the characteristics of pluripotent stem cells. Moreover, the HyPSCs could differentiate into cells of all three germ layer lineages including CD45+ hematopoietic progenitor cells (HPCs), functional endothelial cells, and hepatocytes. The HyPSCs-derived HPCs exhibited the ability to evade innate and adaptive immunity. Further, we demonstrated that RapaCasp9 could be used as a safety switch in vitro and in vivo. CONCLUSION: The HLA class Ia/II DKO hiPSCs armed with HLA-G, PD-L1, PD-L2, and RapaCasp9 molecules are a potential source of stem cells for allogeneic transplantation.

Envelopment by endothelial cells initiates translocation of avian primordial germ cell into vascular tissue.

BACKGROUND: In avian species, primordial germ cells (PGCs) migrate to the gonadal primordium through the vascular system. Because this mode of migration is reminiscent of cancer metastasis, it would be useful to elucidate the mechanisms underlying PGC migration via the bloodstream. Here, we sought to determine when, where, and how PGCs enter the vascular network by double visualization of PGCs and endothelial cells (ECs) in tie1:H2B-eYFP transgenic quails. RESULTS: In the left and right lateral germinal crescent regions corresponding to the anterior-most area vasculosa, more than 60% of PGCs were enveloped by differentiating ECs forming blood islands prior to vascular network formation. Cell morphology analysis suggested that the PGC-EC interaction was instructed by differentiating ECs. At a later developmental stage, ECs anastomosed to form a vascular network with a lumen that retained PGCs within it. As a consequence, many PGCs localized within the luminal space of the mature vascular network at later stages. CONCLUSIONS: Our findings demonstrate that the major type of avian PGC translocation into vascular tissue is not a typical intravasation, as performed by types of metastatic cancer cells, but rather a passive translocation (envelopment) mediated by differentiating ECs during early vasculogenesis.

Thalidomide and its metabolite 5-hydroxythalidomide induce teratogenicity via the cereblon neosubstrate PLZF.

Thalidomide causes teratogenic effects by inducing protein degradation via cereblon (CRBN)-containing ubiquitin ligase and modification of its substrate specificity. Human P450 cytochromes convert thalidomide into two monohydroxylated metabolites that are considered to contribute to thalidomide effects, through mechanisms that remain unclear. Here, we report that promyelocytic leukaemia zinc finger (PLZF)/ZBTB16 is a CRBN target protein whose degradation is involved in thalidomide- and 5-hydroxythalidomide-induced teratogenicity. Using a human transcription factor protein array produced in a wheat cell-free protein synthesis system, PLZF was identified as a thalidomide-dependent CRBN substrate. PLZF is degraded by the ubiquitin ligase CRL4CRBN in complex with thalidomide, its derivatives or 5-hydroxythalidomide in a manner dependent on the conserved first and third zinc finger domains of PLZF. Surprisingly, thalidomide and 5-hydroxythalidomide confer distinctly different substrate specificities to mouse and chicken CRBN, and both compounds cause teratogenic phenotypes in chicken embryos. Consistently, knockdown of Plzf induces short bone formation in chicken limbs. Most importantly, degradation of PLZF protein, but not of the known thalidomide-dependent CRBN substrate SALL4, was induced by thalidomide or 5-hydroxythalidomide treatment in chicken embryos. Furthermore, PLZF overexpression partially rescued the thalidomide-induced phenotypes. Our findings implicate PLZF as an important thalidomide-induced CRBN neosubstrate involved in thalidomide teratogenicity.

Melanosome transfer to keratinocyte in the chicken embryonic skin is mediated by vesicle release associated with Rho-regulated membrane blebbing.

During skin pigmentation in amniotes, melanin synthesized in the melanocyte is transferred to keratinocytes by a particle called the melanosome. Previous studies, mostly using dissociated cultured cells, have proposed several different models that explain how the melanosome transfer is achieved. Here, using a technique that labels the plasma membrane of melanocytes within a three-dimensional system that mimics natural tissues, we have visualized the plasma membrane of melanocytes with EGFP in chicken embryonic skin. Confocal time-lapse microscopy reveals that the melanosome transfer is mediated, at least in part, by vesicles produced by plasma membrane. Unexpectedly, the vesicle release is accompanied by the membrane blebbing of melanocytes. Blebs that have encapsulated a melanosome are pinched off to become vesicles, and these melanosome-containing vesicles are finally engulfed by neighboring keratinocytes. For both the membrane blebbing and vesicle release, Rho small GTPase is essential. We further show that the membrane vesicle-mediated melanosome transfer plays a significant role in the skin pigmentation. Given that the skin pigmentation in inter-feather spaces in chickens is similar to that in inter-hair spaces of humans, our findings should have important consequences in cosmetic medicine.

Hedgehog-BMP signalling establishes dorsoventral patterning in lateral plate mesoderm to trigger gonadogenesis in chicken embryos.

The gonad appears in the early embryo after several events: cells at the lateral plate mesoderm (LPM) undergo ingression, begin gonadal differentiation and then retain primordial germ cells (PGCs). Here we show that in the chicken embryo, these events are triggered on the basis of dorsoventral patterning at the medial LPM. Gonadal progenitor cells (GPCs) at the ventromedial LPM initiate gonadogenesis by undergoing ingression, whereas mesonephric capsule progenitor cells (MCPCs) at the dorsomedial LPM do not. These contrasting behaviours are caused by Hedgehog signalling, which is activated in GPCs but not in MCPCs. Inhibiting Hedgehog signalling prevents GPCs from forming gonadal structures and collecting PGCs. When activated by Hedgehog signalling, MCPCs form an ectopic gonad. This Hedgehog signalling is mediated by BMP4. These findings provide insight into embryonic patterning and gonadal initiation in the chicken embryo.

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos.

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin-containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross-talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus-mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication-Competent Avian sarcoma-leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back-transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)(+) Neo(r+) -electroporated melanocytes from Hypeco nera were selectively grown in G418-supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS-mOrange, mOrange(+) keratinocytes and transplanted EGFP(+) melanocytes were frequently juxtaposed each other. High-resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.

The dorsal aorta initiates a molecular cascade that instructs sympatho-adrenal specification.

The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel-specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell-derived factor-1 (SDF -1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.