Vascularization of tissues, organoids and organ-on-chip models has been attempted using endothelial cells. However, the cultured endothelial cells lack the capacity to interact with other somatic cell types, which is distinct from developing vascular cells in vivo. Recently, it was demonstrated that blood vessel organoids (BVOs) recreate the structure and functions of developing human blood vessels. However, the tissue-specific adaptability of BVOs had not been assessed in somatic tissues. Herein, we investigated whether BVOs infiltrate human cerebral organoids and form a blood–brain barrier. As a result, vascular cells arising from BVOs penetrated the cerebral organoids and developed a vessel-like architecture composed of CD31+ endothelial tubes coated with SMA+ or PDGFR+ mural cells. Molecular markers of the blood-brain barrier were detected in the vascularized cerebral organoids. We revealed that BVOs can form neural-specific blood-vessel networks that can be maintained for over 50 days.
CRISPR effectors, which comprise a CRISPR-Cas protein and a guide (g)RNA derived from the bacterial immune system, are widely used to induce double-strand breaks in target DNA and activate the in-vivo DNA repair system for target-specific genome editing. When the gRNA recognizes genomic loci with sequences that are similar to the target, deleterious and often carcinogenic mutations can occur. Offtarget mutations with a frequency below 0.5% remain mostly undetected by current genome-wide off-target detection techniques. In this study, we developed a method to effectively detect extremely small amounts of mutated DNA based on predicted off-target-specific amplification. We used various genome editors, including CRISPR-Cpf1, Cas9, and an adenine base editor, to induce intracellular genome mutations.The CRISPR amplification method detected off-target mutations at a significantly higher rate (1.6~984 fold increase) than did an existing targeted amplicon sequencing method. In the near future, CRISPR amplification in combination with genome-wide off-target detection methods will allow to detect genome editor-induced off-target mutations with high sensitivity and in a non-biased manner.
Background and Objectives Proficient differentiation of human pluripotent stem cells (hPSCs) into specific lineages is required for applications in regenerative medicine. A growing amount of evidences had implicated hormones and hormone-like molecules as critical regulators of proliferation and lineage specification during in vivo development. Therefore, a deeper understanding of the hormones and hormone-like molecules involved in cell fate decisions is critical for efficient and controlled differentiation of hPSCs into specific lineages. Thus, we functionally and quantitatively compared the effects of diverse hormones (estradiol 17- β (E2), progesterone (P4), and dexamethasone (DM)) and a hormone-like molecule (retinoic acid (RA)) on the regulation of hematopoietic and neural lineage specification. Methods and Results We used 10 nM E2, 3 μ M P4, 10 nM DM, and 10 nM RA based on their functional in vivo developmental potential. The sex hormone E2 enhanced functional activity of hematopoietic progenitors compared to P4 and DM, whereas RA impaired hematopoietic differentiation. In addition, E2 increased CD34 + CD45 + cells with progenitor functions, even in the CD43 − population, a well-known hemogenic marker. RA exhibited lineage-biased potential, preferentially committing hPSCs toward the neural lineage while restricting the hematopoietic fate decision. Conclusions Our findings reveal unique cell fate potentials of E2 and RA treatment and provide valuable differentiation information that is essential for hPSC applications.
Universally acceptable donor cells have been developed to address the unmet need for immunotypically matched materials for regenerative medicine. Since forced expression of hypoimmunogenic genes represses the immune response, we established universal pluripotent stem cells (PSCs) by replacing endogenous β2-microglobulin (β2m) with β2m directly conjugated to human leukocyte antigen (HLA)-G, thereby simultaneously suppressing HLA-I expression and the natural killer (NK) cell-mediated immune response. These modified human PSCs retained their pluripotency and differentiation capacity; however, surface presentation of HLA-G was absent from subsequently differentiated cells, particularly cells of neural lineages, due to the downregulation of antigen processing and presentation machinery (APM) genes. Induction of APM genes by overexpression of NLR-family CARD domain-containing 5 (NLRC5) or activator subunit of nuclear factor kappa B (NF-κB) heterodimer (RelA) recovered the surface expression of HLA-G and the hypoimmunogenicity of neural cells. Our findings enhance the utility of hypoimmunogenic cells as universal donors and will contribute to the development of off-the-shelf stem-cell therapeutics.
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