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Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta-gonad-mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)-cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA-CREB and BMP pathways in isolated AGM VE-cadherin(+) cells from mid-gestation embryos, we demonstrate that PKA-CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1-null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA-CREB-BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

Soft tissue coverage of the exposed Achilles tendon is a unique reconstructive challenge. In this report, we describe the management of a large posterior leg wound with exposed Achilles tendon using a free anterolateral thigh (ALT) flap. A careful review of alternative reconstructive options is included, along with their respective advantages and disadvantages. A 32-year-old white man suffered a fulminant right lower extremity soft tissue infection requiring extensive debridement of the entire posterior surface of the right leg. The resulting large soft tissue defect included exposure of the Achilles tendon. Reconstruction of the defect was achieved with an ALT flap and split-thickness skin graft for coverage of the Achilles tendon and gastrocnemius muscle, respectively. The patient was able to ambulate independently within 2 months of the procedure.

Author(s): Alejandro De Los Angeles [1, 2, 3]; Francesco Ferrari [4]; Yuko Fujiwara [1, 2, 3]; Ronald Mathieu [5]; Soohyun Lee [4]; Semin Lee [4]; Ho-Chou Tu [1, 2, 3]; [...]

Summary: The first hematopoietic stem cells (HSCs) that engraft irradiated adult mice arise in the aorta-gonad-mesonephros (AGM) on embryonic day 11.5 (E11.5). However, at this stage, there is a discrepancy between the apparent frequency of HSCs depicted with imaging and their rarity when measured with limiting dilution transplant. We have attempted to reconcile this difference using neonatal recipients, which are more permissive for embryonic HSC engraftment. We found that embryonic HSCs from E9.5 and E10.5 preferentially engrafted neonates, whereas developmentally mature, definitive HSCs from E14.5 fetal liver or adult bone marrow (BM) more robustly engrafted adults. Neonatal engraftment was enhanced after treating adult BM-derived HSCs with interferon. Adult BM-derived HSCs preferentially homed to the liver in neonatal mice yet showed balanced homing to the liver and spleen in adults. These findings emphasize the functional differences between nascent and mature definitive HSCs. [Copyright &y& Elsevier]

Hematopoietic stem and progenitor cells (HSPCs), first specified from hemogenic endothelium (HE) in the ventral dorsal aorta (VDA), support lifelong hematopoiesis. Their de novo production promises significant therapeutic value; however, current in vitro approaches cannot efficiently generate multipotent long-lived HSPCs. Presuming this reflects a lack of extrinsic cues normally impacting the VDA, we devised a human dorsal aorta-on-a-chip platform that identified Yes-activated protein (YAP) as a cyclic stretch-induced regulator of HSPC formation. In the zebrafish VDA, inducible Yap overexpression significantly increased runx1 expression in vivo and the number of CD41+ HSPCs downstream of HE specification. Endogenous Yap activation by lats1/2 knockdown or Rho-GTPase stimulation mimicked Yap overexpression and induced HSPCs in embryos lacking blood flow. Notably, in static human induced pluripotent stem cell (iPSC)-derived HE culture, compound-mediated YAP activation enhanced RUNX1 levels and hematopoietic colony-forming potential. Together, our findings reveal a potent impact of hemodynamic Rho-YAP mechanotransduction on HE fate, relevant to de novo human HSPC production. • Biomechanical strain impacts human HSPC formation in vitro • YAP drives endothelial-to-hematopoietic transition downstream of blood flow in vivo • Rho-GTPase stimulation promotes HSPC production in vitro and in vivo via YAP activity Hemodynamic forces are required for embryonic hematopoietic stem and progenitor cell (HSPC) formation; however, transcriptional mediators of this process are unknown. Using in vitro microfluidics-mediated stimulation of human induced pluripotent stem cells and in vivo functional analyses in zebrafish embryos, Lundin et al. show cyclic stretch-mediated biophysical activation of YAP facilitates HSPC production. [ABSTRACT FROM AUTHOR]

Human cerebral organoids are unique in their development of progenitor-rich zones akin to ventricular zones from which neuronal progenitors differentiate and migrate radially. Analyses of cerebral organoids thus far have been performed in sectioned tissue or in superficial layers due to their high scattering properties. Here, we demonstrate label-free three-photon imaging of whole, uncleared intact organoids (~2 mm depth) to assess early events of early human brain development. Optimizing a custom-made three-photon microscope to image intact cerebral organoids generated from Rett Syndrome patients, we show defects in the ventricular zone volumetric structure of mutant organoids compared to isogenic control organoids. Long-term imaging live organoids reveals that shorter migration distances and slower migration speeds of mutant radially migrating neurons are associated with more tortuous trajectories. Our label-free imaging system constitutes a particularly useful platform for tracking normal and abnormal development in individual organoids, as well as for screening therapeutic molecules via intact organoid imaging., Competing Interests: MY, CD, DF, VP, SC, JI, AN, LT, GM, PS, MS No competing interests declared, (© 2022, Yildirim et al.)

Human pluripotent stem cells (hPSCs) represent a promising source of patient-specific cells for disease modeling, drug screens, and cellular therapies. However, the inability to derive engraftable human hematopoietic stem and progenitor cells (HSPCs) has limited their characterization to in vitro assays. We report a strategy to respecify lineage-restricted CD34(+)CD45(+) myeloid precursors derived from hPSCs into multilineage progenitors that can be expanded in vitro and engrafted in vivo. HOXA9, ERG, and RORA conferred self-renewal and multilineage potential in vitro and maintained primitive CD34(+)CD38(-) cells. Screening cells via transplantation revealed that two additional factors, SOX4 and MYB, conferred engraftment. Progenitors specified with all five factors gave rise to reproducible short-term engraftment with myeloid and erythroid lineages. Erythroid precursors underwent hemoglobin switching in vivo, silencing embryonic and activating adult globin expression. Our combinatorial screening approach establishes a strategy for obtaining transcription-factor-mediated engraftment of blood progenitors from human pluripotent cells., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Soft tissue coverage of the exposed Achilles tendon is a unique reconstructive challenge. In this report, we describe the management of a large posterior leg wound with exposed Achilles tendon using a free anterolateral thigh (ALT) flap. A careful review of alternative reconstructive options is included, along with their respective advantages and disadvantages. A 32-year-old white man suffered a fulminant right lower extremity soft tissue infection requiring extensive debridement of the entire posterior surface of the right leg. The resulting large soft tissue defect included exposure of the Achilles tendon. Reconstruction of the defect was achieved with an ALT flap and split-thickness skin graft for coverage of the Achilles tendon and gastrocnemius muscle, respectively. The patient was able to ambulate independently within 2 months of the procedure.