Re-endothelialization of non-detergent decellularized porcine vessels.

Tissue engineering utilizes an interdisciplinary approach to generate constructs for the treatment and repair of diseased organs. Generation of small vessels as vascular grafts or as envisioned central vessel for vascularized constructs is still a challenge. Here, the decellularization of porcine vessels by a non-detergent based protocol was developed and investigated. Perfusion-decellularization with sodium hydroxide solution resulted in removal of cellular material throughout the whole length of the vessel while preserving structural and mechanical integrity. A re-endothelialization of the retrieved matrix with human umbilical vein endothelial cells and cardiac endothelial cells was achieved through rotation-based seeding employing a custom-made bioreactor. A confluent monolayer was detected on the entire luminal surface. Thus, a non-detergent-based decellularization method allowing the re-endothelialization of the luminal surface was developed in this study, thereby paving the way for future implementation of the resulting construct as vascular graft or as central vessel for tissue engineered constructs in need of a perfusion system with readily available anastomosis sites.

Characterization of Tissue Engineered Endothelial Cell Networks in Composite Collagen-Agarose Hydrogels.

Scaffolds constitute an important element in vascularized tissues and are therefore investigated for providing the desired mechanical stability and enabling vasculogenesis and angiogenesis. In this study, supplementation of hydrogels containing either MatrigelTM and rat tail collagen I (MatrigelTM/rCOL) or human collagen (hCOL) with SeaPlaqueTM agarose were analyzed with regard to construct thickness and formation and characteristics of endothelial cell (EC) networks compared to constructs without agarose. Additionally, the effect of increased rCOL content in MatrigelTM/rCOL constructs was studied. An increase of rCOL content from 1 mg/mL to 3 mg/mL resulted in an increase of construct thickness by approximately 160%. The high rCOL content, however, impaired the formation of an EC network. The supplementation of MatrigelTM/rCOL with agarose increased the thickness of the hydrogel construct by approximately 100% while supporting the formation of a stable EC network. The use of hCOL/agarose composite hydrogels led to a slight increase in the thickness of the 3D hydrogel construct and supported the formation of a multi-layered EC network compared to control constructs. Our findings suggest that agarose/collagen-based composite hydrogels are promising candidates for tissue engineering of vascularized constructs as cell viability is maintained and the formation of a stable and multi-layered EC network is supported.

Formation of three-dimensional tubular endothelial cell networks under defined serum-free cell culture conditions in human collagen hydrogels.

Implementation of tubular endothelial cell networks is a prerequisite for 3D tissue engineering of constructs with clinically relevant size as nourishment of cells is challenged by the diffusion limit. In vitro generation of 3D networks is often achieved under conditions using serum containing cell culture medium and/or animal derived matrices. Here, 3D endothelial cell networks were generated by using human umbilical vein endothelial cells (HUVECs) in combination with human adipose tissue derived stromal cells (hASCs) employing human collagen I as hydrogel and decellularized porcine small intestinal submucosa as starter matrix. Matrigel/rat tail collagen I hydrogel was used as control. Resulting constructs were cultivated either in serum-free medium or in endothelial growth medium-2 serving as control. Endothelial cell networks were quantified, tested for lumen formation, and interaction of HUVECs and hASCs. Tube diameter was slightly larger in constructs containing human collagen I compared to Matrigel/rat tail collagen I constructs under serum-free conditions. All other network parameters were mostly similar. Thereby, the feasibility of generating 3D endothelial cell networks under serum-free culture conditions in human collagen I as hydrogel was demonstrated. In summary, the presented achievements pave the way for the generation of clinical applicable constructs.

Human adipose tissue-derived stromal cells in combination with exogenous stimuli facilitate three-dimensional network formation of human endothelial cells derived from various sources.

In natural tissues, the nutrition of cells and removal of waste products is facilitated by a dense capillary network which is generated during development. This perfusion system is also indispensable for tissue formation in vitro. Nutrition depending solely on diffusion is not sufficient to generate tissues of clinically relevant dimensions, which is a core aim in tissue engineering research. In this study, the establishment of a vascular network was investigated in a self-assembling approach employing endothelial and mural cells. The process of vascularization was analyzed in constructs based on a carrier matrix of decellularized porcine small intestinal submucosa (SIS). A three-dimensional hydrogel containing Matrigel™, collagen, and respective cells was casted on top of the SIS. Various types of human endothelial cells (hECs), e.g. HUVECs, cardiac tissue ECs (hCECs), pulmonary artery ECs (hPAECs), and iPSC-derived ECs, were co-cultured with human adipose tissue-derived stromal cells (hASCs) within the hydrogel. Analyzed hECs were able to self-assemble and form three-dimensional networks harboring small caliber lumens within the hydrogel constructs in the presence of hASCs as supporting cells. Additionally, microvessel assembling required exogenous growth factor supplementation. This study demonstrates the development of stable vascularized hydrogels applying hASCs as mural cells in combination with various types of hECs, paving the way for the generation of clinically applicable tissue engineered constructs.

Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles.

In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

Effects of combined cryopreservation and decellularization on the biomechanical, structural and biochemical properties of porcine pulmonary heart valves.

UNLABELLED: Non-fixed, decellularized allogeneic heart valve scaffolds seem to be the best choice for heart valve replacement, their availability, however, is quite limited. Cryopreservation could prolong their shelf-life, allowing for their ideal match to a recipient. In this study, porcine pulmonary valves were decellularized using detergents, either prior or after cryopreservation, and analyzed. Mechanical integrity was analyzed by uniaxial tensile testing, histoarchitecture by histological staining, and composition by DNA, collagen (hydroxyproline) and GAG (chondroitin sulfate) quantification. Residual sodium dodecyl sulfate (SDS) in the scaffold was quantified by applying a methylene blue activation assay (MBAS). Cryopreserved decellularized scaffolds (DC) and scaffolds that were decellularized after cryopreservation (CD) were compared to fresh valves (F), cryopreserved native valves (C), and decellularized only scaffolds (D). The E-modulus and tensile strength of decellularized (D) tissue showed no significant difference compared to DC and CD. The decellularization resulted in an overall reduction of DNA and GAG, with DC containing the lowest amount of GAGs. The DNA content in the valvular wall of the CD group was higher than in the D and DC groups. CD valves showed slightly more residual SDS than DC valves, which might be harmful to recipient cells. In conclusion, cryopreservation after decellularization was shown to be preferable over cryopreservation before decellularization. However, in vivo testing would be necessary to determine whether these differences are significant in biocompatibility or immunogenicity of the scaffolds. STATEMENT OF SIGNIFICANCE: Absence of adverse effects on biomechanical stability of acellular heart valve grafts by cryopreservation, neither before nor after decellularization, allows the identification of best matching patients in a less time pressure dictated process, and therefore to an optimized use of a very limited, but best-suited heart valve prosthesis.

Directing Cardiomyogenic Differentiation and Transdifferentiation By Ectopic Gene Expression - Direct Transition Or Reprogramming Detour?

Cardiovascular disorders and associated morbidities remain the leading cause of premature death worldwide. Since the regeneration of diseased hearts is very limited and the insufficient supply of donor organs persists, hopes rely on new therapies for heart repair. Reviving the proliferation of endogenous cardiomyocytes (CMs) or the administration of adult stem cells to the heart was of limited curative success to date. Thus, the administration of in vitro generated CMs is under investigation to replenish loss of functional heart muscle tissue. This requires a sustainable source of CMs. Induced pluripotent stem cells (iPSC) have raised hopes for developing autologous cell therapies. To serve for heart repair, efficient and safe iPSC differentiation protocols for CMs production are required. iPSC differentiation into CMs and even functional subtypes was indeed achieved in recent years, either by the ectopic expression of cardiac transcription factors or the supplementation of chemical pathway modulators. An alternative approach aims at the direct transdifferentiation of fibroblasts, which are present in the interstitial tissue of many organs, into functional lineage-specific cell types. As a result the formation of induced cardiomyocyte-like cells (iCMs) by the ectopic expression of specific transcription factors combinations has been demonstrated in vitro and in vivo. This is an important proof-of-concept that the intermediate state of iPSC induction is dispensable. However, most of the early experiments were conducted in mice and translation to more relevant large animal models and subsequently to the clinic are challenging. Progress, drawbacks, and perspectives in this field will be discussed.

In vitro maturation of large-scale cardiac patches based on a perfusable starter matrix by cyclic mechanical stimulation.

The ultimate goal of tissue engineering is the generation of implants similar to native tissue. Thus, it is essential to utilize physiological stimuli to improve the quality of engineered constructs. Numerous publications reported that mechanical stimulation of small-sized, non-perfusable, tissue engineered cardiac constructs leads to a maturation of immature cardiomyocytes like neonatal rat cardiomyocytes or induced pluripotent stem cells/embryonic stem cells derived self-contracting cells. The aim of this study was to investigate the impact of mechanical stimulation and perfusion on the maturation process of large-scale (2.5×4.5cm), implantable cardiac patches based on decellularized porcine small intestinal submucosa (SIS) or Biological Vascularized Matrix (BioVaM) and a 3-dimensional construct containing neonatal rat heart cells. Application of cyclic mechanical stretch improved contractile function, cardiomyocyte alignment along the stretch axis and gene expression of cardiomyocyte markers. The development of a complex network formed by endothelial cells within the cardiac construct was enhanced by cyclic stretch. Finally, the utilization of BioVaM enabled the perfusion of the matrix during stimulation, augmenting the beneficial influence of cyclic stretch. Thus, this study demonstrates the maturation of cardiac constructs with clinically relevant dimensions by the application of cyclic mechanical stretch and perfusion of the starter matrix. STATEMENT OF SIGNIFICANCE: Considering the poor endogenous regeneration of the heart, engineering of bioartificial cardiac tissue for the replacement of infarcted myocardium is an exciting strategy. Most techniques for the generation of cardiac tissue result in relative small-sized constructs insufficient for clinical applications. Another issue is to achieve cardiomyocytes and tissue maturation in culture. Here we report, for the first time, the effect of mechanical stimulation and simultaneous perfusion on the maturation of cardiac constructs of clinical relevant dimensions, which are based on a perfusable starter matrix derived from porcine small intestine. In response to these stimuli superior organization of cardiomyocytes and vascular networks was observed in contrast to untreated controls. The study provides substantial progress towards the generation of implantable cardiac patches.

Large-scale production of human pluripotent stem cell derived cardiomyocytes.

Regenerative medicine, including preclinical studies in large animal models and tissue engineering approaches as well as innovative assays for drug discovery, will require the constant supply of hPSC-derived cardiomyocytes and other functional progenies. Respective cell production processes must be robust, economically viable and ultimately GMP-compliant. Recent research has enabled transition of lab scale protocols for hPSC expansion and cardiomyogenic differentiation towards more controlled processing in industry-compatible culture platforms. Here, advanced strategies for the cultivation and differentiation of hPSCs will be reviewed by focusing on stirred bioreactor-based techniques for process upscaling. We will discuss how cardiomyocyte mass production might benefit from recent findings such as cell expansion at the cardiovascular progenitor state. Finally, remaining challenges will be highlighted, specifically regarding three dimensional (3D) hPSC suspension culture and critical safety issues ahead of clinical translation.

BioVaM in the rat model: a new approach of vascularized 3D tissue for esophageal replacement.

INTRODUCTION: A major obstacle in tissue engineering is to create a surgically implantable tissue with long-term viability. Several promising techniques have focused on biological vascularized matrices (BioVaM) with preserved vascular pedicles in the porcine model. However, the handling of this model is time-consuming and expensive. Therefore, our aim was to establish a BioVaM in the rat. MATERIALS AND METHODS: Small bowel segments of Sprague-Dawley rats were isolated and perfused via cannulation of the superior mesenteric artery and the portal vein. All cellular matrix components were removed by sequential treatment with sodium dodecyl sulfate, sodium deoxycholate, and DNase. Quality of decellularization was investigated by histology and potential residual DNA by spectrophotometry. Primary endothelial cells (ECs) isolated from the major vessels of Sprague-Dawley rats. Cells were labeled with fluorescent cell tracker and injected into the vascular pedicles of the matrix. Attachment of ECs was assessed using fluorescence microscopy of the whole mount. RESULTS: Decellularized matrix demonstrated the absence of cellular components but conserved matrix architecture as determined by immune fluorescent, pentachrome, and hematoxylin and eosin stains. DNA content was reduced by more than 99%. ECs were characterized by specific staining against endothelial nitric oxide synthase and von Willebrand factor; when injected, ECs attached along the vessel walls including the capillaries of the intestinal wall. CONCLUSIONS: Rat small bowel segments harvested with intact vascular pedicles and associated vascular network can be successfully decellularized and re-endothelialized ex vivo. This model is an inexpensive and easy to handle alternative and appears to be a promising approach for establishing vascularized tissue constructs.

Successful re-endothelialization of a perfusable biological vascularized matrix (BioVaM) for the generation of 3D artificial cardiac tissue.

Generating cellularized 3D constructs with clinical relevant dimensions is challenged by nutrition supply. This is of utmost importance for cardiac tissue engineering, since cardiomyocytes are extremely sensitive to malnutrition and hypoxia in vitro and after implantation. To develop a perfusable myocardial patch, we have focused on seeding a decellularized biological vascularized matrix (BioVaM) with endothelial cells. BioVaM is produced by decellularization of porcine small intestinal segments with preserved arterial and venous pedicles, which can be connected to a perfusion system in vitro or the host vasculature in vivo. The BioVaM vessel bed was re-seeded with porcine primary endothelial cells (pCEC). Seeding efficiency was influenced by detergent composition used for decellularization (sodium dodecyl sulfate (SDS) and/or Triton X-100) and the medium composition used for re-seeding. After decellularization, residual SDS was detected in the matrix affecting the survival of pCEC which showed a low tolerance to SDS and Triton X-100. Sensitivity to detergents was attenuated by supplementation of the medium with bovine serum albumin (BSA) or fetal calf serum (FCS). Pre-conditioning of the BioVaM with 20% FCS was not sufficient to attain pCEC survival in the vascular bed. However, re-cellularization was achieved by prolonged FCS supplementation during cultivation, resulting in a perfusable, re-endothelialized matrix of 11 cm2 in size. This achievement represents a promising step towards engineering of perfusable, 3D cardiac constructs with clinically relevant dimensions.

Generation of bioartificial heart tissue by combining a three-dimensional gel-based cardiac construct with decellularized small intestinal submucosa.

The in vitro generation of a bioartificial cardiac construct (CC) represents a promising tool for the repair of ischemic heart tissue. Several approaches to engineer cardiac tissue in vitro have been conducted. The main drawback of these studies is the insufficient size of the resulting construct for clinical applications. The focus of this study was the generation of an artificial three-dimensional (3D), contractile, and suturable myocardial patch by combining a gel-based CC with decellularized porcine small intestinal submucosa (SIS), thereby engineering an artificial tissue of 11 cm² in size. The alignment and morphology of rat neonatal cardiomyocytes (rCMs) in SIS-CC complexes were investigated as well as the re-organization of primary endothelial cells which were co-isolated in the rCM preparation. The ability of a rat heart endothelial cell line (RHE-A) to re-cellularize pre-existing vessel structures within the SIS or a biological vascularized matrix (BioVaM) was determined. SIS-CC contracted spontaneously, uniformly, and rhythmically with an average rate of 200 beats/min in contrast to undirected contractions observed in CC without SIS support. rCM exhibited an elongated morphology with well-defined sarcomeric structures oriented along the longitudinal axis in the SIS-CC, whereas round-shaped and random-arranged rCM were observed in CC. Electric coupling of rCM was demonstrated by microelectrode array measurements. A dense network of CD31⁺/eNOS⁺ cells was detected as permeating the whole construct. Superficial supplementation of RHE-A cells to SIS-CC led to the migration of these cells through the CC, resulting in the re-population of pre-existing vessel structures within the decelluarized SIS. By infusion of RHE-A cells into the BioVaM venous and arterial pedicles, a re-population of the BioVaM vessel bed as well as distribution of RHE-A cells throughout the CC was achieved. Rat endothelial cells within the CC were in contact with RHE-A cells. Ingrowth and formation of a network by endothelial cells infused through the BioVaM represent a promising step toward engineering a functional perfusion system, enabling the engineering of vascularized and well-nourished 3D CC of dimensions relevant for therapeutic heart repair.

Small intestinal submucosa segments as matrix for tissue engineering: review.

Tissue engineering (TE) is an emerging interdisciplinary field aiming at the restoration or improvement of impaired tissue function. A combination of cells, scaffold materials, engineering methods, and biochemical and physiological factors is employed to generate the desired tissue substitute. Scaffolds often play a pivotal role in the engineering process supporting a three-dimensional tissue formation. The ideal scaffold should mimic the native extracellular environment providing mechanical and biological properties to allow cell attachment, migration, and differentiation, as well as remodeling by the host organism. The scaffold should be nonimmunogenic and should ideally be resorbed by the host over time, leaving behind only the regenerated tissue. More than 40 years ago, a preparation of the small intestine was introduced for the replacement of vascular structures. Since then the small intestinal submucosa (SIS) has gained a lot of interest in TE and subsequent clinical applications, as this material exhibits key features of a highly supportive scaffold. This review will focus on the general properties of the SIS and its applications in therapeutical approaches as well as in generating tissue substitutes in vitro. Furthermore, the main problem of TE, which is the insufficient nourishment of cells within three-dimensional, artificial tissues exceeding certain dimensions is addressed. To solve this issue the implementation of another small intestine-derived preparation, the biological vascularized matrix (BioVaM), could be a feasible option. The BioVaM comprises in addition to SIS the arterial and venous mesenteric pedicles and exhibits thereby a perfusable vessel bed that is preserved after decellularization.

Gene expression analysis of human red blood cells.

Understanding of molecular mechanisms governing the enucleating phenomena of human erythrocytes is of major importance in both fundamental and applied studies. Total RNA (n=7) from human RBCs (purity of erythrocyte preparation >99,99%) was tested using 2100 Bioanalyzer (Agilent, USA), and transcribed to cDNA. Microarray analysis was performed with the Human Genome Focus GeneChip (Affymetrix, USA), containing 8500 transcripts corresponding to 8400 human genes. Here we report that human RBCs contain typical eukaryotic RNA with 28S- and18S-rRNA standard bands. Microarray studies revealed the presence of transcripts of 1019 different genes in erythrocytic RNA. Gene Ontology analysis recognized 859 genes involved in general biological processes: 529 genes for cellular metabolism, 228 genes for signal transduction, 104 genes for development, 107 genes for immune response, 62 genes for protein localization, 53 genes for programmed cell death, and 5 genes for autophagy. A number of genes responsible for transcription, translation, RNA-stabilisation as well as for apoptosis and anti-apoptosis have been identified for the first time in circulating human RBCs. The presented data shed new light on the genetic determination of erythropoiesis, apoptosis and may have implications on the pathophysiology and diagnosis of various diseases involving red blood cells.

The pro-domain of the zebrafish Nodal-related protein Cyclops regulates its signaling activities.

Nodal proteins are secreted signaling factors of the transforming growth factor beta (TGFbeta) family with essential roles in embryonic development in vertebrates. Mutations affecting the Nodal factors have severe consequences in mammals and fish. Furthermore, increased Nodal levels have been associated with melanoma tumor progression. Like other TGFbeta-related proteins, Nodal factors consist of a pro-domain and a mature domain. The pro-domain of mouse Nodal protein stabilizes its precursor. However, the mechanisms by which the pro-domains exert their activities are unknown. Here, we characterize the zebrafish Nodal-related factor Cyclops (Cyc) and find unexpected functions for the pro-domain in regulating Cyc activity. We identified a lysosome-targeting region in the Cyc pro-domain that destabilizes the precursor and restricts Cyc activity, revealing the molecular basis for the short-range signaling activities of Cyc. We show that both the pro- and mature-domains of Cyc regulate its stability. We also characterize a mutation in the pro-domain of human NODAL (hNODAL) that underlies congenital heterotaxia. Heterologous expression of mutant hNODAL increases expression of Nodal-response genes. Our studies reveal unexpected roles for the pro-domain of the Nodal factors and provide a possible mechanism for familial heterotaxia.

Expression of the human Cathepsin L inhibitor hurpin in mice: skin alterations and increased carcinogenesis.

The serine protease inhibitor (serpin) hurpin (serpin B13) is a cross class-specific inhibitor of the cysteine protease Cathepsin (Cat) L. Cat L is involved in lysosomal protein degradation, hair follicle morphogenesis, epidermal differentiation and epitope generation of antigens. Hurpin is a 44 kDa protein which is expressed predominantly in epidermal cells. In psoriatic skin samples, hurpin was strongly overexpressed when compared with normal skin. Keratinocytes overexpressing hurpin showed increased resistance towards UVB-induced apoptosis. To further analyse the functional importance of this inhibitor, we have generated transgenic mice with deregulated Cat L activity by expressing human hurpin in addition to the endogenous mouse inhibitor. The three independent transgenic lines generated were characterized by identical effects excluding insertional phenotypes. Macroscopically, mice expressing human hurpin are characterized by abnormal abdominal fur. The number of apoptotic cells and caspase-3 positive cells was reduced after UV-irradiation in transgenic animals compared with wild-type mice. Interestingly, after chemical carcinogenesis, transgenic mice showed an increased susceptibility to develop skin cancer. Array analysis of gene expression revealed distinct differences between wild-type and hurpin-transgenic mice. Among others, differentially expressed genes are related to antigen presentation and angiogenesis. These results suggest an important role of Cat L regulation by hurpin which might be of clinical relevance in human skin diseases.

Switch from actin alpha1 to alpha2 expression and upregulation of biomarkers for pressure overload and cardiac hypertrophy in taurine-deficient mouse heart.

Taurine is the most abundant free amino acid in heart muscle and protects against heart failure. In the present study, the consequences of hereditary taurine deficiency on cardiac gene expression were examined in 2- and 15-16-month-old taurine transporter knockout (taut(-/-)) mice using a mouse-specific DNA microarray. This oligonucleotide-based microarray contains probes for 251 genes with relevance for heart function. Of these, 163 probes exhibited a reproducible hybridization signal and were analyzed. alpha-Actin type 1 mRNA levels were 70% lower in the heart of young and older taut(-/-) mice compared to wild-type controls. Interestingly, the hearts of taut(-/-) mice showed a switch from alpha-actin 1 to alpha-actin 2 expression, as confirmed by real-time PCR and Western blot analysis. In addition, mRNA levels of biomarkers for pressure overload and hypertension were upregulated in taut(-/-) hearts, i.e., atrial natriuretic factor (+848%), brain natriuretic peptide (+90%), cardiac ankyrin repeat protein (+118%), and procollagen 1a1, 1a2 and 3a1 (+40% at least). These results point to a stress situation in the heart of taut(-/-) mice under laboratory conditions, and it can be speculated that taut(-/-) hearts may be even more susceptible to failure in the wild when under exogenous stress.

Mouse Pop1 is required for muscle regeneration in adult skeletal muscle.

Popeye (Pop) genes are a novel gene family encoding putative transmembrane proteins predominantly present in striated and smooth muscle cells. In this study, a null mutation of Pop1 was generated by replacing the first coding exon of the Pop1 gene with the lacZ reporter gene. Homozygous mice lacking Pop1 were fertile and had a normal life span without any apparent phenotype. LacZ staining of tissues of heterozygous and homozygous Pop1-LacZ mice revealed strong expression in embryonic and fetal hearts. Pop1-LacZ was also expressed in the myotome and in myogenic progenitor cells within the limb and in smooth muscle cells of various organs. In the heart, Pop1-LacZ activity was downregulated postnatally in heterozygous mice but not in homozygous mice. Administration of the beta-adrenergic agonist isoproterenol led to a rapid increase in Pop1-LacZ activity in heterozygotes without induction at the transcriptional level, suggesting stabilization of the protein. No difference, however, was observed between homozygous and heterozygous mice in the ability to develop cardiac hypertrophy in response to isoproterenol. The capacity to regenerate skeletal muscle was tested after cardiotoxin injection into the hind limbs of hetero- and homozygous mice. In activated satellite cells of both genotypes, rapid activation of Pop1-LacZ expression was observed. In heterozygous animals, LacZ activity was only transiently elevated in muscle precursor cells undergoing fusion and in newly formed myotubes. In homozygotes, persistence of LacZ expression and a retarded ability to regenerate skeletal muscle were apparent, suggesting that Pop1 plays a role in muscle regeneration.

Molecular and functional analysis of Popeye genes: A novel family of transmembrane proteins preferentially expressed in heart and skeletal muscle.

Popeye (Pop) genes encode novel transmembrane proteins, of which three family members are present in vertebrates, while in Drosophila a single gene is found. By northern blot analysis a restricted expression pattern is observed; Pop genes are predominantly expressed in the heart, skeletal and smooth muscle. Using homologous recombination, a null mutation was generated in the case of Pop1. The homozygous mutants are viable and do not display any obvious phenotype. They display an impaired ability to regenerate skeletal muscle while the hypertropic response of the heart after isoproterenol infusion revealed no difference between genotypes. Recently a function for Pop1 as a prototype of a novel class of cell adhesion molecules was proposed. Further work is required to substantiate these findings and to extend it to other members of the family.