Anatomy and Cell Biology

Although many experts position statements on autologous stem cell mobilization have been published, there are some aspects that are still under discussion. A Spanish Hematologist expert group was summoned to settle on agreements and uncertainties on PBSCs mobilization, including factors not always considered; as apheresis and cytometry key factors that determine a successful PBSC collection. This document reviews critical factors that define poor mobilizer patients and the tools to better collect the desired stem cells for a successful autologous haematopoietic stem cell transplant.

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Transforming growth factor ␤ (TGF␤) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGF␤ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGF␤s shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved downregulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGF␤ signaling up-regulates Sox9 in the in vivo experimental model system in which TGF␤ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGF␤ signaling, we found that TGF␤s appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGF␤ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGF␤ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGF␤ treatments.

Repairing damaged cartilage and tendons is a major challenge of regenerative medicine. There has been great progress in the past decade toward obtaining stem cells for regenerative purposes from a variety of sources. However, the development of procedures to direct and maintain the differentiation of progenitors into cartilage or tendon is still a hurdle to overcome in regenerative medicine of the musculoskeletal system. This is because connective tissues often lack stable phenotypes and retain plasticity to return to the initial stages of differentiation or to transdifferentiate into another connective tissue cell lineage. This makes it necessary to unravel the molecular basis that is responsible for the differentiation of connective tissue cell lineages. In this review, we summarize the investigations performed in the past two decades to unravel the signals that regulate the differentiation of skeletal cell progenitors into cartilage and tendons during embryonic limb development. The data obtained in those studies demonstrate that Tgfβ, BMP, FGF, and Wnt establish a complex signaling network that directs the differentiation of skeletal cell progenitors. Remarkably, in the embryonic digit model, the divergent differentiation of progenitors depends on the temporal coordination of those signals, rather than being specified by an individual signaling pathway. Due to its potential medical relevance, we highlight the importance of the coordinate influence of the Tgfβ and BMP pathways in the differentiation of cell progenitors into tendon or cartilage.

Physiological cell death is a key mechanism that ensures appropriate development and maintenance of tissues and organs in multicellular organisms. Most structures in the vertebrate embryo exhibit defined areas of cell death at precise stages of development. In this regard the areas of interdigital cell death during limb development provide a paradigmatic model of massive cell death with an evident morphogenetic role in digit morphogenesis. Physiological cell death has been proposed to occur by apoptosis, cellular phenomena genetically controlled to orchestrate cell suicide following two main pathways, cytochrome C liberation from the mitochondria or activation of death receptors. Such pathways converge in the activation of cysteine proteases known as caspases, which execute the cell death program, leading to typical morphologic changes within the cell, termed apoptosis. According to these findings it would be expected that caspases loss of function experiments could cause inhibition of interdigital cell death promoting syndactyly phenotypes. A syndactyly phenotype is characterized by absence of digit freeing during development that, when caused by absence of interdigital cell death, is accompanied by the persistence of an interdigital membrane. However this situation has not been reported in any of the KO mice or chicken loss of function experiments ever performed. Moreover histological analysis of dying cells within the interdigit reveals the synchronic occurrence of different types of cell death. All these findings are indicative of caspase alternative and/or complementary mechanisms responsible for physiological interdigital cell death. Characterization of alternative cell death pathways is required to explain vertebrate morphogenesis. Today there is great interest in cell death via autophagy, which could substitute or act synergistically to the apoptotic pathway.

Interdigital tissue regression during embryonic development is one of the most representative model systems of morphogenetic cell death, but the degenerative cascade accounting for this process awaits clarification. Although the canonical apoptotic caspase pathway appears to be activated in the interdigital mesenchyme committed to die, neither genetic nor chemical blockage of caspases or their downstream effectors, is sufficient to prevent cell death. Hence, alternative and/or complementary dying pathways must also be responsible for this degenerative process. In this work we have chosen to study the endonucleases during the regression of the interdigital tissue of avian embryos to gain insights into the molecular mechanisms accounting for programmed cell death in this system. We show that caspase activated DNase, which is a neutral DNase associated with the caspase apoptotic pathway, appears to be the main endonuclease only at an initial phase of interdigit regression. However at peak stages of the degenerative process, the acidic DNases L-DNase II and lysosomal DNase IIB become predominant in the system and markers for cell autophagy become moderately up-regulated. Consistent with the activation of acidic endonucleases we observed that microenvironmental pH value in the interdigits decreased to levels only appropriate for acidic enzymes. Furthermore, we found that overexpression of lysosomal DNase IIB in embryonic limb mesoderm promoted cell death, which was also accompanied by up-regulation and activation of L-DNase II. Up-regulation of acidic DNases was maintained in interdigits explanted to culture dishes, where the participation of exogenous professional phagocytes of hematopoietic origin is avoided. Finally, and consistent with all our findings, up-regulation of acidic DNases was much reduced in the webbed interdigits of duck embryos, characterized by a rudimentary interdigital degenerative process. We conclude that the regression of the interdigital tissue involves a coordinated and sequential activation of the caspase and lysosomal degenerative molecular cascades.

By in situ hybridization we studied the expression patterns of Msx and Hoxd genes during the late development of the chick leg autopodium (foot) and compared them to patterns during the experimental development of interdigital extra digits. Extra digits are induced in the third interdigital space after various experimental manipulations, such as transient isolation of the interdigit, or removal of the interdigital marginal ectoderm and mesoderm. Msxl and Msx2 are normally expressed in the interdigital tissue programmed to die. Our experiment changes the fate of the interdigital tissue from cell death to chondrogenesis and provides a good model for studying Msx involvement in defining areas of programmed cell death. Among the proposed roles for Hoxd genes is their involvement in the specification of digit identity early in development. The induction of extra digits allows us to examine whether this new morphogenetic commitment of the interdigital tissue involves changes in the domains of expression of Hoxd genes. Our results show that extra digits develop without a previous modification of the normal pattern of expression of Msx or Hoxd genes. This observation does not support the correlation between the expression of Msx genes and programmed cell death and suggests a role for these genes in maintaining the interdigital tissue in an undifferentiated state. Our results show that an increased number of digits can be formed without modifications in the pattern of expression of the 5'-located Hoxd genes and suggest the existence of latent or residual digit organization mechanisms past the time when digits are normally determined, independent of Hoxd gene expression.

In view of recent evidence showing that shape and orientation of endothelial cells is determined by blood flow, the endothelium of the semilunar valves was studied in the developing chick heart using transmission and scanning electron microscopy. The results reveal significant developmental modifications of endothelial morphology and structure. These modifications can be linked to modifications of local blood flow and can also explain several aspects of valvular morphogenesis. The results substantially support the hypothesis of an involvement of hemodynamics in the development of the semilunar valves.

The composition and arrangement of the cytoskeletal elements of the endothelium of the semilunar valves have been studied in the embryonic chick heart during the stages 30 to 38. Microtubules, vimentin intermediate filaments and actin microfilaments were constant components of the valvular endothelial cells in the studied stages. Scanning electron microscopy after Triton-X-100 extraction revealed significant differences in the tridimensional arrangement of the cytoskeleton in the course of valve development. In the ventricutar face of the cusps the cytoskeletal elements displayed a progressive longitudinal alignment, while in the arterial face of the cusps the cytoskeleton maintained the appearance of a network. Transmission electron microscopy revealed that these differences were especially prominent for vimentin intermediate filaments, although a similar tendency was also observed for microtubules. Micro filaments were scarce in the endothelial cells of both faces of the cusps, and the stress fibers typical of the endothelial cells of the adult valves were not observed in the embryonic material. The significance of these results in valve morphogenesis and histogensis and a possible linkage with differences in the local characteristics of the blood flow are discussed.

The purpose of the present work was to analyze the role of hemodynamics in the morphogenesis and histogenesis of the semilunar valves. To achieve this goal we have studied the development of the chick semilunar valves in conditions of abnormal local flow. To obtain an abnormal pattern of local flow we have induced alterations of the cardiac septation process by mechanical interference of the development of the conus cordis. The malformations obtained by this procedure consisted of a spectrum of alterations in the process of incorporation of the aortic conus into the left ventricle. These malformations ranged from a simple widening of the outflow tract of the left ventricle to severe forms of double-outlet right ventricle and ventricular septal defects. Malformations of the semilunar valves consisting of extensive thickening of the leaflets and lack of maturation of the valve tissues were very often present in the malformed hearts. The malformation of the valve leaflets was more frequent and severe in the aortic valve at more advanced stages of development and in the hearts showing more severe alteration of the septation process. The absence of alterations in the semilunar valves of the control embryos and in the experimental embryos without alteration of the cardiac septation suggest a close relationship between the semilunar valves anomalies and the hemodynamic alterations present in the malformed hearts.

In the present work we have studied the mechanism of formation and the possible morphogenetic significance of the process of ectopic chondrogenesis induced by surgical removal of AER of the interdigital spaces of the chick leg bud at stage 28 30 (Hurle and Gafian 1986). Our results show that ridge removal causes condensation and rounding of the underlaying mesenchymal cells followed by chondrogensis. The long-term study of the fate of these ectopic cartilages shows that in a high percentage of the cases the cartilages undergo morphogenesis taking by day 10 of incubation the appearance of the two distal phalanges of an extra-digit. These extra-digits lack tendons and are joined by thin interdigital membranes to the neighboring digits.

The anatomy of the human aortic valve was studied by SEM in 36 subjects without cardiac pathology who had died of various accidental causes. Villous and lamellar tissue excrescences were observed at the node of Arantius and at the limit between the lunules and the load-bearing portion of the leaflets. The morphology of these structures suggests that they represent areas in which valve tissue becomes detached into the bloodstream. Fenestrations were present in the lunules of 14 specimens, with a higher incidence in specimens from subjects who were middle-aged or older. Our observations suggest that fenestrations appear initially as small perforations which then coalesce to form larger apertures.

The development of the semilunar valves takes place in association with septation of the outflow tract in the embryonic heart. Although numerous studies have focused on this process, the causal mechanisms of valvular development remain obscure. This paper reports an immunohistochemical analysis of tenascin and type-I collagen distribution in developing chick heart valves. Tenascin is a glycoprotein that is present on some embryonic extracellular matrices. It plays several significant roles in tissue differentiation, cell growth, and tissue interactions; it is also important for the formation of specific zones of connective tissue that fulfill mechanical functions. Our results show that tenascin is present during valvular morphogenesis and histogenesis, and that its distribution is associated with zones specialized in bearing mechanical loads.

In the final stages of limb morphogenesis, autopodial cells leaving the progress zone differentiate into cartilage or undergo apoptotic cell death, depending on whether they are incorporated into the digital rays or interdigital spaces. Most evidence indicates that these two opposite fates of the autopodial mesoderm are controlled by BMP signaling. However, the molecular basis for these two distinct actions of BMPs, including the receptors involved in the process, is controversial. In this study we have addressed this question by exploring the presence in the developing autopod of diffusible signals able to modulate BMP function and by analyzing the effects of their exogenous administration on the pattern of expression of BMP receptor genes. Our findings show that tgf␤2 and noggin genes are expressed in the condensing region of the developing digital rays in addition to the well-known distribution in the autopodial tissues of FGFs (apical ectodermal ridge, AER) and BMPs (AER, progress zone mesoderm, and interdigital regions). Exogenous administration of all the factors causes changes in the expression of the bmpR-1b gene which are followed by parallel alterations of the skeletal phenotype: FGFs inhibit the expression of bmpR-1b compatible with their function in the maintenance of the progress zone mesoderm in an undifferentiated state; and TGF␤s induce the expression of bmpR-1b and promote ectopic chondrogenesis, compatible with a function in the establishment of the position of the digital rays. In addition we provide evidence for the occurrence of an interactive loop between BMPs and noggin accounting for the spatial distribution of bmpR-1b which may control the size and shape of the skeletal pieces. In contrast to the bmpR-1b gene, the bmpR-1a gene is expressed at low levels in the autopodial mesoderm and its expression is not modified by any of the tested factors regardless of their effects on chondrogenesis or cell death. Finally, the role of BMPs in programmed cell death is confirmed here by the intense inhibitory effect of noggin on apoptosis, but the lack of correlation between changes in the pattern of cell death induced by treatment with the studied factors and the expression of either bmpR-1a or bmpR-1b genes suggest that a still-unidentified BMP receptor may account for this BMP function.

In order to investigate whether endocardial cells (EC) in chick embryo hearts have polarity of shape and structure, the position of the Golgi apparatus, silver impregnated, and the shape of the cells were studied using light and TEM and SEM microscopy. The EC have a prominent Golgi apparatus located lateral to the nucleus and show a statistically significant polarization.

Previous studies have shown that three members of the Wnt signaling pathway, the ligand WNT5A, the receptor FZ4, and the Wnt antagonist FRZB1, are implicated in the formation and differentiation of the digits. In this study, we have attempted to establish a functional correlation between them by comparing their expression patterns and their regulation by the signals controlling proliferation and differentiation of the limb mesoderm during formation of the avian digits in vivo and in micromass cultures. In vivo Wnt5a and Fz4 are expressed in the undifferentiated mesoderm of the autopod and in the differentiating digit cartilages. In the undifferentiated mesoderm, the expression of both genes is regulated positively by FGFs and negatively by bone morphogenetic proteins (BMPs). As chondrogenic differentiation starts, Fz4 becomes intensely up-regulated in the aggregate and in the developing perichondrium, whereas transcripts of Wnt5a are excluded from the core of the aggregate but maintained in the surrounding mesenchyme and perichondrium. In addition, at this stage, the expression of both genes become positively regulated by BMPs. These changes in expression and regulation are coincident with the induction of Frzb1 in the chondrogenic aggregate, which is expressed under the positive control of BMPs. Our findings fit with a role of Wnt5a/Fz4 negatively regulating in vivo the initiation and progression of cartilage differentiation. In vitro, only Frzb1 is expressed and regulated in a manner resembling that observed in vivo. Wnt5a and Fz4 are both expressed in the differentiating mesenchyme of micromass cultures, but their expression is not significantly regulated by the addition of FGF-2 or BMP-7 to the culture medium.