Emergent mechanical control of vascular morphogenesis.

Vascularization is driven by morphogen signals and mechanical cues that coordinately regulate cellular force generation, migration, and shape change to sculpt the developing vascular network. However, it remains unclear whether developing vasculature actively regulates its own mechanical properties to achieve effective vascularization. We engineered tissue constructs containing endothelial cells and fibroblasts to investigate the mechanics of vascularization. Tissue stiffness increases during vascular morphogenesis resulting from emergent interactions between endothelial cells, fibroblasts, and ECM and correlates with enhanced vascular function. Contractile cellular forces are key to emergent tissue stiffening and synergize with ECM mechanical properties to modulate the mechanics of vascularization. Emergent tissue stiffening and vascular function rely on mechanotransduction signaling within fibroblasts, mediated by YAP1. Mouse embryos lacking YAP1 in fibroblasts exhibit both reduced tissue stiffness and develop lethal vascular defects. Translating our findings through biology-inspired vascular tissue engineering approaches will have substantial implications in regenerative medicine.

A reappraisal and revision of the numbering of the pharyngeal arches.

The pharyngeal arches are a prominent and significant feature of vertebrate embryos. These are visible as a series of bulges on the lateral surface of the embryonic head. In humans, and other amniotes, there are five pharyngeal arches numbered 1, 2, 3, 4 and 6; note the missing '5'. This is the standard scheme for the numbering of these structures, and it is a feature of modern anatomy textbooks. In this article, we discuss the rationale behind this odd numbering, and consider its origins. One reason given is that there is a transient 5th arch that is never fully realized, while another is that this numbering reflects considerations from comparative anatomy. We show here, however, that neither of these reasons has substance. There is no evidence from embryology for a '5th' arch, and the comparative argument does not hold as it does not apply across the vertebrates. We conclude that there is no justification for this strange numbering. We suggest that the pharyngeal arches should simply be numbered 1, 2, 3, 4 and 5 as this would be in keeping with the embryology and with the general numbering of the pharyngeal arches across the vertebrates.

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kitpos) cells. The adult heart indeed contains a heterogeneous mixture of c-kitpos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kitpos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kitpos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kitpos sorting. The blood/endothelial lineage-committed (Lineagepos) CD45posc-kitpos cardiac cells were compared to CD45neg(Lineageneg/Linneg) c-kitpos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kitpos cardiac cells are blood/endothelial lineage-committed CD45posCD31posc-kitpos cells. In contrast, the LinnegCD45negc-kitpos cardiac cell cohort, which represents ⩽10% of the total c-kitpos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kitneg and the blood/endothelial lineage-committed c-kitpos cardiac cells. Single Linnegc-kitpos cell-derived clones, which represent only 1-2% of total c-kitpos myocardial cells, when stimulated with TGF-ß/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Linnegc-kitpos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kitpos cardiac cells were injected. Thus, among the cardiac c-kitpos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

Skeletal muscle-derived interstitial progenitor cells (PICs) display stem cell properties, being clonogenic, self-renewing, and multi-potent in vitro and in vivo.

BACKGROUND: The development of cellular therapies to treat muscle wastage with disease or age is paramount. Resident muscle satellite cells are not currently regarded as a viable cell source due to their limited migration and growth capability ex vivo. This study investigated the potential of muscle-derived PW1+/Pax7- interstitial progenitor cells (PICs) as a source of tissue-specific stem/progenitor cells with stem cell properties and multipotency. METHODS: Sca-1+/PW1+ PICs were identified on tissue sections from hind limb muscle of 21-day-old mice, isolated by magnetic-activated cell sorting (MACS) technology and their phenotype and characteristics assessed over time in culture. Green fluorescent protein (GFP)-labelled PICs were used to determine multipotency in vivo in a tumour formation assay. RESULTS: Isolated PICs expressed markers of pluripotency (Oct3/4, Sox2, and Nanog), were clonogenic, and self-renewing with >60 population doublings, and a population doubling time of 15.8 ± 2.9 h. PICs demonstrated an ability to generate both striated and smooth muscle, whilst also displaying the potential to differentiate into cell types of the three germ layers both in vitro and in vivo. Moreover, PICs did not form tumours in vivo. CONCLUSION: These findings open new avenues for a variety of solid tissue engineering and regeneration approaches, utilising a single multipotent stem cell type isolated from an easily accessible source such as skeletal muscle.

Transplantation of Allogeneic PW1pos/Pax7neg Interstitial Cells Enhance Endogenous Repair of Injured Porcine Skeletal Muscle.

Skeletal muscle-derived PW1pos/Pax7neg interstitial cells (PICs) express and secrete a multitude of proregenerative growth factors and cytokines. Utilizing a porcine preclinical skeletal muscle injury model, delivery of allogeneic porcine PICs (pPICs) significantly improved and accelerated myofiber regeneration and neocapillarization, compared with saline vehicle control-treated muscles. Allogeneic pPICs did not contribute to new myofibers or capillaries and were eliminated by the host immune system. In conclusion, allogeneic pPIC transplantation stimulated the endogenous stem cell pool to bring about enhanced autologous skeletal muscle repair and regeneration. This allogeneic cell approach is considered a cost-effective, easy to apply, and readily available regenerative therapeutic strategy.

Mode of reduction in the number of pharyngeal segments within the sarcopterygians.

BACKGROUND: Pharyngeal segmentation is a defining feature of vertebrate embryos and is apparent as a series of bulges found on the lateral surface of the embryonic head, the pharyngeal arches. The ancestral condition for gnathostomes is to have seven pharyngeal segments: jaw, hyoid, and five posterior branchial arches. However, within the sarcopterygians, the pharyngeal region has undergone extensive remodelling that resulted in a reduction in the number of pharyngeal segments, such that amniotes have only five pharyngeal arches. The aim of this study is to probe the developmental basis of this loss of pharyngeal segments. RESULTS: We have therefore compared the development of the pharyngeal arches in an amniote, the chick, which has five segments, with those of a chondrichthyan, the catshark, which has seven segments. We have analysed the early phase of pharyngeal segmentation and we find that in both the most anterior segments form first with the posterior segments being added sequentially. We also documented the patterns of innervation of the pharynx in several vertebrates and note that the three most anterior segments receive distinct innervation: the first arch being innervated by the Vth nerve, the second by the VIIth and the third by the IXth. Finally, we have analysed Hox gene expression, and show that the anterior limit of Hoxa2 aligns with the second pouch and arch in both chick and catshark, while Hoxa3 is transiently associated with the third arch and pouch. Surprisingly, we have found that Hoxb1 expression is spatially and temporally dynamic and that it is always associated with the last most recently formed pouch and that this domains moves caudally as additional pouches are generated. CONCLUSION: We propose that the first three pharyngeal segments are homologous, as is the posterior limit of the pharynx, and that the loss of segments occurred between these two points. We suggest that this loss results from a curtailment of the posterior expansion of the pharyngeal endoderm in amniotes at relatively earlier time point, and thus the generation of fewer segments.

Endodermal/ectodermal interfaces during pharyngeal segmentation in vertebrates.

A key event in the formation of the pharyngeal arches is the outpocketing of the endodermal pharyngeal pouches and the establishment of contact with the overlying ectoderm. However, relatively little is known about how the endoderm and ectoderm relate to each other at these points of contact and the extent to which this differs between the pouches. We have therefore detailed the interactions between the pharyngeal pouches and ectoderm in the chick embryo. Unlike the other pouches, the first pouch does not sustain direct contact with the ectoderm but separates after initial contact. Contrastingly, a perforation is formed between the second pouch and cleft that creates an external opening into the pharynx. Finally, the third and fourth pouch endoderm can be seen to bulge outwards through the ectoderm, although external openings to the pharyngeal lumen are not established. To understand whether these behaviours represent derived or ancestral features, we characterised the pharyngeal ectodermal-endodermal interfaces in the shark embryo. We found that the pouches of the posterior gill-bearing arches in this species also displayed the outward bulging of the endoderm into the ectoderm, although openings were established. We further used genetic tools to detail unambiguously the relationship between the endoderm and ectoderm in zebrafish and mouse embryos and again found that the posterior pouches break through the ectoderm. Thus different pharyngeal pouches establish different topological relationships with the overlying ectoderm and the posterior pouches initiate the developmental programme for the formation of gills, be they amniotes or anamniotes.