Endothelial fate and angiogenic properties of human CD34+ progenitor cells in zebrafish

Objective-: The vascular competence of human-derived hematopoietic progenitors for postnatal vascularization is still poorly characterized. It is unclear whether, in the absence of ischemia, hematopoietic progenitors participate in neovascularization and whether they play a role in new blood vessel formation by incorporating into developing vessels or by a paracrine action. Methods and Results-: In the present study, human cord blood-derived CD34 + (hCD34 +) cells were transplanted into pre- and postgastrulation zebrafish embryos and in an adult vascular regeneration model induced by caudal fin amputation. When injected before gastrulation, hCD34 + cells cosegregated with the presumptive zebrafish hemangioblasts, characterized by Scl and Gata2 expression, in the anterior and posterior lateral mesoderm and were involved in early development of the embryonic vasculature. These morphogenetic events occurred without apparent lineage reprogramming, as shown by CD45 expression. When transplanted postgastrulation, hCD34 + cells were recruited into developing vessels, where they exhibited a potent paracrine proangiogenic action. Finally, hCD34 + cells rescued vascular defects induced by Vegf-c in vivo targeting and enhanced vascular repair in the zebrafish fin amputation model. Conclusion-: These results indicate an unexpected developmental ability of human-derived hematopoietic progenitors and support the hypothesis of an evolutionary conservation of molecular pathways involved in endothelial progenitor differentiation in vivo

Efficient Derivation of Excitatory and Inhibitory Neurons from Human Pluripotent Stem Cells Stably Expressing Direct Reprogramming Factors

It is essential to generate isolated populations of human neuronal subtypes in order to understand cell-type-specific roles in brain function and susceptibility to disease pathology. Here we describe a protocol for in-parallel generation of cortical glutamatergic (excitatory) and GABAergic (inhibitory) neurons from human pluripotent stem cells (hPSCs) by using the neurogenic transcription factors Ngn2 and a combination of Ascl1 and Dlx2, respectively. In contrast to the majority of neural transdifferentiation protocols that use transient lentiviral infection, the use of stable hPSC lines carrying doxycycline-inducible transcription factors allows neuronal differentiation to be initiated by addition of doxycycline and neural medium. This article presents a method to generate lentivirus from cultured mammalian cells and establish stable transcription factor–expressing cell lines (Basic Protocol 1), followed by a method for monolayer excitatory and inhibitory neuronal differentiation from the established lines (Basic Protocol 2). The resulting neurons reproducibly exhibit properties consistent with human cortical neurons, including the expected morphologies, expression of glutamatergic and GABAergic genes, and functional properties. Our approach enables the scalable and rapid production of human neurons suitable for modeling human brain diseases in a subtype-specific manner and examination of differential cellular vulnerability