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Human Wharton's jelly stem cells (HWJSC) can be efficiently isolated from the umbilical cord, and numerous reports have demonstrated that these cells can differentiate into several cell lineages. This fact, coupled with the high proliferation potential of HWJSC, makes them a promising source of stem cells for use in tissue engineering and regenerative medicine. However, their real potentiality has not been established to date. In the present study, we carried out a systematic review to determine the multilineage differentiation potential of HWJSC. After a systematic literature search, we selected 32 publications focused on the differentiation potential of these cells. Analysis of these studies showed that HWJSC display expanded differentiation potential toward some cell types corresponding to all three embryonic cell layers (ectodermal, mesodermal, and endodermal), which is consistent with their constitutive expression of key pluripotency markers such as OCT4, SOX2, and NANOG, and the embryonic marker SSEA4. We conclude that HWJSC can be considered cells in an intermediate state between multipotentiality and pluripotentiality, since their proliferation capability is not unlimited and differentiation to all cell types has not been demonstrated thus far. These findings support the clinical use of HWJSC for the treatment of diseases affecting not only mesoderm-type tissues but also other cell lineages. Impact statement Human Wharton's jelly stem cells (HWJSC) are mesenchymal stem cells that are easy to isolate and handle, and that readily proliferate. Their wide range of differentiation capabilities supports the view that these cells can be considered pluripotent. Accordingly, HWJSC are one of the most promising cell sources for clinical applications in advanced therapies.

Several models of bioartificial human urothelial mucosa (UM) have been described recently. In this study, we generated novel tubularized UM substitutes using alternative sources of cells. Nanostructured fibrin–agarose biomaterials containing fibroblasts isolated from the human ureter were used as stroma substitutes. Then, human Wharton jelly mesenchymal stromal cells (HWJSC) were used to generate an epithelial-like layer on top. Three differentiation media were used for 7 and 14 days. Results showed that the biofabrication methods used here succeeded in generating a tubular structure consisting of a stromal substitute with a stratified epithelial-like layer on top, especially using a medium containing epithelial growth and differentiation factors (EM), although differentiation was not complete. At the functional level, UM substitutes were able to synthesize collagen fibers, proteoglycans and glycosaminoglycans, although the levels of control UM were not reached ex vivo. Epithelial differentiation was partially achieved, especially with EM after 14 days of development, with expression of keratins 7, 8, and 13 and pancytokeratin, desmoplakin, tightjunction protein-1, and uroplakin 2, although at lower levels than controls. These results confirm the partial urothelial differentiative potential of HWJSC and suggest that the biofabrication methods explored here were able to generate a potential substitute of the human UM for future clinical use., CTS-115 Tissue Engineering Group and by the Spanish Plan Nacional de Investigación Científica, Desarrollo e Innovación Tecnológica, Ministry of Science and Innovation, Instituto de Salud Carlos III, grant FIS PI21/0981 (cofinanced by FEDER funds, European Union).

Current tissue engineering technology focuses on developing simple tissues, whereas multilayered structures comprising several tissue types have rarely been described. We developed a highly biomimetic multilayered palate substitute with bone and oral mucosa tissues using rabbit cells and biomaterials subjected to nanotechnological techniques based on plastic compression. This novel palate substitute was autologously grafted in vivo, and histological and histochemical analyses were used to evaluate biointegration, cell function, and cell differentiation in the multilayered palate substitute. The three-dimensional structure of the multilayered palate substitute was histologically similar to control tissues, but the ex vivo level of cell and tissue differentiation were low as determined by the absence of epithelial differentiation although cytokeratins 4 and 13 were expressed. In vivo grafting was associated with greater cell differentiation, epithelial stratification, and maturation, but the expression of cytokeratins 4, 13, 5, and 19 at did not reach control tissue levels. Histochemical analysis of the oral mucosa stroma and bone detected weak signals for proteoglycans, elastic and collagen fibers, mineralization deposits and osteocalcin in the multilayered palate substitute cultured ex vivo. However, in vivo grafting was able to induce cell and tissue differentiation, although the expression levels of these components were always significantly lower than those found in controls, except for collagen in the bone layer. These results suggest that generation of a full-thickness multilayered palate substitute is achievable and that tissues become partially differentiated upon in vivo grafting.

Although several studies have been published regarding the morphology and anatomical variations of the human shoulder joint, most have dealt with adult individuals. Those looking into the development of the joint have been focused on specific structures or have observed specimens in advanced gestational stages. The goal of this paper is to perform a complete analysis of the embryonic and early fetal development of the elements in the shoulder joint, and to clarify some contradictory data in the literature. In our study, serial sections of 32 human embryos (Carnegie stages 16-23) and 26 fetuses (9-13 weeks) were analyzed. The chondrogenic anlagen of the humerus and the medial border of the scapula can be observed from as early as Carnegie stage 17, whereas that of the rest of the scapula appears at stage 18. The osteogenic process begins in week 10 for the humeral head and week 11 for the scapula. At stage 19 the interzone becomes apparent, which will form the glenohumeral joint. In the next stage the glenohumeral joint will begin delaminating and exhibiting a looser central band. Denser lateral bands will join the humeral head (caput humeri) and the margins of the articular surface of the scapula, thus forming the glenoid labrum, which can be fully appreciated by stage 22. In 24-mm embryos (stage 21) we can observe, for the first time, the long head of the biceps tendon (which is already inserted in the glenoid labrum by week 9), and the intertubercular sulcus, whose depth is apparent since week 12. Regarding ligamentous structures, the coracohumeral ligament is observed at the end of Carnegie stage 23, whereas the primitive glenohumeral ligament already appeared in week 10. The results of this study provide a detailed description of the morphogenesis, origin and chronological order of appearance of the main intrinsic structures of the human shoulder joint during late embryonic and early fetal development. We expect these results to help explain several functional aspects of the shoulder joint, and to clarify some contradictory data in the literature regarding this complex anatomical and biomechanical structure, helping future researchers in their efforts.

The development of the human wrist joint has been studied widely, with the main focus on carpal chondrogenesis, ligaments and triangular fibrocartilage. However, there are some discrepancies concerning the origin and morphogenetic time-table of these structures, including nerves, muscles and vascular elements. For this study we used serial sections of 57 human embryonic (n = 30) and fetal (n = 27) specimens from O’Rahilly stages 17–23 and 9–14 weeks, respectively. The following phases in carpal morphogenesis have been established: undifferentiated mesenchyme (stage 17), condensated mesenchyme (stages 18 and 19), pre-chondrogenic (stages 19 and 20) and chondrogenic (stages 21 and over). Carpal chondrification and osteogenic processes are similar, starting with capitate and hamate (stage 19) and ending with pisiform (stage 22). In week 14, a vascular bud penetrates into the lunate cartilaginous mold, early sign of the osteogenic process that will be completed after birth. In stage 18, median, ulnar and radial nerves and thenar eminence appear in the hand plate. In stage 21, there are indications of the interosseous muscles, and in stage 22 flexor digitorum superficialis, flexor digitorum profundus and lumbrical muscles, transverse carpal ligament and collateral ligaments emerge. In stage 23, the articular disc, radiocarpal and ulnocarpal ligaments and deep palmar arterial arch become visible. Radiate carpal and interosseous ligaments appear in week 9, and in week 10, dorsal radiocarpal ligament and articular capsule are evident. Finally, synovial membrane is observed in week 13. We have performed a complete analysis of the morphogenesis of the structures of the human wrist joint. Our results present new data on nervous and arterial elements and provide the basis for further investigations on anatomical pathology, comparative morphology and evolutionary anthropology.

The origin of the styloglossus muscle was histologically studied bilaterally in nine human fetuses (18 sides). In all cases, the muscle originated in Reichert's cartilage, which gives rise to the temporal styloid process. We identified three types of variation: type A, an accessory muscle fascicle originating from the mandibular angle, found in 7 cases (12 sides); type B, where the styloglossus muscle was attached to the mandibular angle by fibrous tracts, found in three cases (4 sides); and type C, where an accessory muscle fascicle arose from the fibrous tract connecting Reichert's cartilage to the mandibular angle; found in one case. In all cases (2 sides), the styloglossus muscle was innervated by the hypoglossal nerve. Relationships between the styloglossus muscle and vasculonervous elements of the prestyloid and retrostyloid spaces were analysed.

The juxtaoral organ was studied using light microscopy in 55 human embryos and 90 fetuses at different stages of development. The juxtaoral organ arises from the epithelium at the bottom of the transverse opening of the primitive mouth during O'Rahilly stage 16 and becomes detached from the epithelium after O'Rahilly stage 18. The juxtaoral organ is innervated by the buccal nerve from O'Rahilly stage 20 onward, and its connective tissue capsule is clearly visible after week 11 of development. This study enabled us to describe three main periods of juxtaoral organ development: (1) the period of epithelial condensation and invagination, at O'Rahilly stages 16–17; (2) the period during which the juxtaoral organ becomes detached from the oral epithelium and is innervated, at O'Rahilly stages 18–23; and (3) the period during which the connective tissue capsule is formed, after week 11 of development. We also analysed the juxtaoral organ of five additional fetuses by immunohistochemistry with anti-NF-200 to verify their innervation. The results show that the juxtaoral organ may have a function in the mechanical activity of the region.

This study was performed on 50 human embryos and fetuses between 7 and 17 weeks of development. Reichert's cartilage is formed in the second pharyngeal arch in two segments. The longer cranial or styloid segment is continuous with the otic capsule; its inferior end is angulated and is situated very close to the oropharynx. The smaller caudal segment is in contact with the body and greater horn of the hyoid cartilaginous structure. No cartilage forms between these segments. The persistent angulation of the inferior end of the cranial or styloid segment of Reichert's cartilage and its important neurovascular relationships may help explain the symptomatology of Eagle's syndrome.