Generation of OCIAD2 homozygous knockout (BJNhem20-OCIAD2-CRISPR-33) and heterozygous knockout (BJNhem20-OCIAD2-CRISPR-40) human embryonic stem cell lines using CRISPR-Cas9 mediated targeting.

Ovarian Carcinoma Immunoreactive Antigen domain containing 2 (OCIAD2) was knocked out by targeting its exon 4 through CRISPR-Cas9 paired nickase strategy to generate two OCIAD2 knockout human embryonic stem cell lines- one homozygous (BJNhem20-OCIAD2-CRISPR-33) and one heterozygous (BJNhem20-OCIAD2-CRISPR-40) for mutant ociad2. Both lines maintain pluripotency, normal karyotype, and trilineage differentiation potential.

Generation of an OCIAD2 overexpressing transgenic human embryonic stem cell line, BJNhem20-OCIAD2-OV.

Overexpression of Ovarian Carcinoma Immunoreactive Antigen Domain containing protein 2 (OCIAD2) was carried out in BJNhem20 human Embryonic Stem Cell line (hESC). A stable line was generated through nucleofection of the plasmid construct pCAG-OCIAD2.

Organelle dysfunction upon asrij depletion causes aging-like changes in mouse hematopoietic stem cells.

Aging of the blood system is characterized by increased hematopoietic stem cells (HSCs) and myeloid-biased differentiation leading to higher propensity for hematological malignancies. Unraveling cell-intrinsic mechanisms regulating HSC aging could aid reversal or slowing of aging. Asrij/OCIAD1 is an evolutionarily conserved regulator of hematopoiesis and governs mitochondrial, endosomal, and proteasomal function in mammalian stem cells.

Intact Preparation of the Lymph Gland for a Comprehensive Analysis of Larval Hematopoiesis.

Blood cells have a limited lifespan and are replenished by a small number of hematopoietic stem and progenitor cells (HSPCs). Adult vertebrate hematopoiesis occurs in the bone marrow, liver, and spleen, rendering a comprehensive analysis of the entire HSPC pool nearly impossible. The blood system is well studied and has developmental, molecular, and functional parallels with that of vertebrates.

Inclusivity and diversity: Integrating international perspectives on stem cell challenges and potential.

Regenerative medicine has great potential. The pace of scientific advance is exciting and the medical opportunities for regeneration and repair may be transformative. However, concerns continue to grow, relating to problems caused both by unscrupulous private clinics offering unregulated therapies based on little or no evidence and by premature regulatory approval on the basis of insufficient scientific rationale and clinical evidence.

A Conserved Role for Asrij/OCIAD1 in Progenitor Differentiation and Lineage Specification Through Functional Interaction With the Regulators of Mitochondrial Dynamics.

Mitochondria are highly dynamic organelles whose activity is an important determinant of blood stem and progenitor cell state. Mitochondrial morphology is maintained by continuous fission and fusion and affects stem cell proliferation, differentiation, and aging. However, the mechanism by which mitochondrial morphology and dynamics regulate cell differentiation and lineage choice remains incompletely understood.

Differential activation of JAK-STAT signaling reveals functional compartmentalization in blood progenitors.

Blood cells arise from diverse pools of stem and progenitor cells. Understanding progenitor heterogeneity is a major challenge. The larval lymph gland is a well-studied model to understand blood progenitor maintenance and recapitulates several aspects of vertebrate hematopoiesis.

Expression, Purification and Crystallization of Asrij, A Novel Scaffold Transmembrane Protein.

Asrij/OCIAD1 is a scaffold transmembrane protein belonging to the Ovarian Carcinoma Immunoreactive Antigen Domain containing protein family. In Drosophila and mouse models, Asrij localizes at the endosomal and mitochondrial membrane and is shown to regulate the stemness of hematopoietic stem cells. Interaction of Asrij with ADP Ribosylation Factor 1 (Arf1) is shown to be crucial for hematopoietic niche function and prohemocyte maintenance.

The OCIAD protein family: comparative developmental biology and stem cell application.

Over the last two decades, an exponential growth in technologies and techniques available to biologists has provided mind-boggling quantities of data and led to information overload. Yet, answers to fundamental questions such as "how are we made?" and "what keeps us ticking?" remain incomplete. Developmental biology has provided elegant approaches to address such questions leading to enlightening insights.

Rudhira/BCAS3 couples microtubules and intermediate filaments to promote cell migration for angiogenic remodeling.

Blood vessel formation requires endothelial cell (EC) migration that depends on dynamic remodeling of the cytoskeleton. Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein essential for EC migration and sprouting angiogenesis during mouse development and is implicated in metastatic disease. Here, we report that Rudhira mediates cytoskeleton organization and dynamics during EC migration.

Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence.

Inactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11% of hematological malignancies have mutant p53. Mechanisms that cause wild-type p53 dysfunction and promote leukemia are inadequately deciphered.

Proteomics of Asrij Perturbation in Lymph Glands for Identification of New Regulators of Hematopoiesis.

Hematopoiesis is the process of differentiation of precursor blood cells into mature blood cells that is controlled by a complex set of molecular interactions. Understanding hematopoiesis is important for the study of hematological disorders. However, a comprehensive understanding of how physiological and genetic mechanisms regulate blood cell precursor maintenance and differentiation is lacking.

Synthetic Host-Guest Assembly in Cells and Tissues: Fast, Stable, and Selective Bioorthogonal Imaging via Molecular Recognition.

Bioorthogonal strategies are continuing to pave the way for new analytical tools in biology. Although a significant amount of progress has been made in developing covalent reaction based bioorthogonal strategies, balanced reactivity, and stability are often difficult to achieve from these systems. Alternatively, despite being kinetically beneficial, the development of noncovalent approaches that utilize fully synthetic and stable components remains challenging due to the lack of selectivity in conventional noncovalent interactions in the living cellular environment.

OCIAD1 Controls Electron Transport Chain Complex I Activity to Regulate Energy Metabolism in Human Pluripotent Stem Cells.

Pluripotent stem cells (PSCs) derive energy predominantly from glycolysis and not the energy-efficient oxidative phosphorylation (OXPHOS). Differentiation is initiated with energy metabolic shift from glycolysis to OXPHOS. We investigated the role of mitochondrial energy metabolism in human PSCs using molecular, biochemical, genetic, and pharmacological approaches.

A double helical motif in OCIAD2 is essential for its localization, interactions and STAT3 activation.

The Ovarian Carcinoma Immunoreactive Antigen domain (OCIAD) - containing proteins OCIAD1/Asrij and OCIAD2, are implicated in several cancers and neurodegenerative diseases. While Asrij has a conserved role in facilitating STAT3 activation for JAK/STAT signaling, the expression and function of OCIAD2 in non-cancerous contexts remains unknown. Here, we report that ociad2 neighbors ociad1/asrij in most vertebrate genomes, and the two genes likely arose by tandem gene duplication, probably somewhere between the Ordovician and Silurian eras.

Rudhira/BCAS3 is essential for mouse development and cardiovascular patterning.

Rudhira/Breast Carcinoma Amplified Sequence 3 (BCAS3) is a cytoskeletal protein that promotes directional cell migration and angiogenesis in vitro and is implicated in human carcinomas and coronary artery disease. To study the role of Rudhira during development in vivo, we generated the first knockout mouse for rudhira and show that Rudhira is essential for mouse development. Rudhira null embryos die at embryonic day (E) 9.

Networks that link cytoskeletal regulators and diaphragm proteins underpin filtration function in Drosophila nephrocytes.

Insect nephrocytes provide a valuable model for kidney disease, as they are structurally and functionally homologous to mammalian kidney podocytes. They possess an exceptional macromolecular assembly, the nephrocyte diaphragm (ND), which serves as a filtration barrier and helps maintain tissue homeostasis by filtering out wastes and toxic products. However, the elements that maintain nephrocyte architecture and the ND are not understood.

Report of the International Stem Cell Banking Initiative Workshop Activity: Current Hurdles and Progress in Seed-Stock Banking of Human Pluripotent Stem Cells.

This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells.

Differential modulation of the cellular and humoral immune responses in Drosophila is mediated by the endosomal ARF1-Asrij axis.

How multicellular organisms maintain immune homeostasis across various organs and cell types is an outstanding question in immune biology and cell signaling. In Drosophila, blood cells (hemocytes) respond to local and systemic cues to mount an immune response. While endosomal regulation of Drosophila hematopoiesis is reported, the role of endosomal proteins in cellular and humoral immunity is not well-studied.

Generation of a transgenic human embryonic stem cell line ectopically expressing the endosomal protein Asrij that regulates pluripotency in mouse embryonic stem cells: BJNhem20-Asrij.

Asrij is an endocytic protein expressed in mouse embryonic stem cells (mESCs) and is essential for maintenance of stemness of mESCs (Mukhopadhyay et al., 2003; Sinha et al., 2013).

Generation of human embryonic stem cell line expressing green fluorescent protein.

Human embryonic stem cell line BJNhem20 pCAG-EGFP was generated using non-viral method (Fig. 1). The construct pCAG-EGFP was transfected using microporation procedure.

Generation of a heterozygous knockout human embryonic stem cell line for the OCIAD1 locus using CRISPR/CAS9 mediated targeting: BJNhem20-OCIAD1-CRISPR-39.

Ovarian carcinoma immuno-reactive antigen domain containing 1 (OCIAD1) single copy was knocked out generating an OCIAD1 heterozygous knockout human embryonic stem line named BJNhem20-OCIAD1-CRISPR-39. The line was generated using CRISPR-Cas9D10A double nickase knockout strategy (Mali et al., 2013).