Blastema Cells Research Articles

Immunohistochemical and western blot analysis suggest that the soluble forms of FGF1-2 and FGFR1-2 sustain tail regeneration in the lizard

Fibroblast Growth Factors 1-2 (FGF1-2) stimulate tail regeneration in lizards and therefore the distribution of their receptors, FGFR1-2, in the regenerating tail of the lizard. Podarcis muralis has been studied using immunofluorescence and western blotting. Immunoreactive protein bands at 15–16kDa for FGF1-2 in addition to those at 50–65kDa are detected in the regenerating epidermis, but weak bands at 35, 45 and 50kDa appear from the regenerating connective tissues. Strongly immunolabeled bands for FGFR1 at 32, 60, and 80kDa and less intense for FGFR2 only appear in the regenerating tail. In normal tail epidermis and dermis, higher MW forms are present at 80 and 115–140kDa, respectively, but they disappear in the regenerating epidermis and dermis where low MW forms of FGFR1-2 are found at 50–70kDa. Immunolocalization confirms that most FGFR1-2 are present in the wound epidermis, Apical Epidermal Peg, ependymal tube while immunolabeling lowers in regenerating muscles, blastema cells, cartilage and connectives tissues. The likely release of FGFs from the Apical Epidermal Peg and ependyma and the presence of their receptors in these tissues may determine the autocrine stimulation of proliferation and a paracrine stimulation of the blastema cells through their FGF Receptors.

Mechanisms of urodele limb regeneration.

This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and mononucleate cells that undergo dedifferentiation, cell cycle entry and accumulation under the apical epidermal cap. (2) The origin, phenotypic memory, and positional memory of blastema cells. (3) The role played by macrophages in the early events of regeneration. (4) The role of neural and AEC factors and interaction between blastema cells in mitosis and distalization. (5) Models of pattern formation based on the results of axial reversal experiments, experiments on the regeneration of half and double half limbs, and experiments using retinoic acid to alter positional identity of blastema cells. (6) Possible mechanisms of distalization during normal and intercalary regeneration. (7) Is pattern formation is a self‐organizing property of the blastema or dictated by chemical signals from adjacent tissues? (8) What is the future for regenerating a human limb?

Atmospheric pressure plasma accelerates tail regeneration in tadpoles Xenopus laevis

Atmospheric pressure plasma is a partially ionized gas composed of neutral and charged particles, including electrons and ions, as well as reactive oxygen species (ROS). Recently, it is utilized as possible therapy in oncology, sterilization, skin diseases, wound healing and tissue regeneration. In this study we focused on effect of plasma exposure on tail regeneration of tadpoles, Xenopus leavis with special emphasis on role of ROS, antioxidant defenses and morphological features of the regenerate. When amputated region of the tail was exposed to the helium plasma it resulted in a faster rate of growth, elevated ROS and increase in antioxidant enzymes in the regenerate compared to that of untreated control. An increase in nitric oxide (free radical) as well as activity of nitric oxide synthase(s) were observed once the cells of the regeneration blastema – a mass of proliferating cells are ready for differentiation. Microscopically the cells of the regenerate of plasma treated tadpoles show altered morphology and characteristics of cellular hypoxia and oxidative stress. We summarize that plasma exposure accelerates the dynamics of wound healing and tail regeneration through its effects on cell proliferation and differentiation as well as angiogenesis mediated through ROS signaling.

TNF signaling and macrophages govern fin regeneration in zebrafish larvae

Macrophages are essential for appendage regeneration after amputation in regenerative species. The molecular mechanisms through which macrophages orchestrate blastema formation and regeneration are still unclear. Here, we use the genetically tractable and transparent zebrafish larvae to study the functions of polarized macrophage subsets during caudal fin regeneration. After caudal fin amputation, we show an early and transient accumulation of pro-inflammatory macrophages concomitant with the accumulation of non-inflammatory macrophages which, in contrast to pro-inflammatory macrophages, remain associated to the fin until the end of the regeneration. Chemical and genetic depletion of macrophages suggested that early recruited macrophages that express TNFα are critical for blastema formation. Combining parabiosis and morpholino knockdown strategies, we show that TNFα/TNFR1 signaling pathway is required for the fin regeneration. Our study reveals that TNFR1 has a necessary and direct role in blastema cell activation suggesting that macrophage subset balance provides the accurate TNFα signal to prime regeneration in zebrafish.

The Drosophila Duox maturation factor is a key component of a positive feedback loop that sustains regeneration signaling.

Regenerating tissue must initiate the signaling that drives regenerative growth, and sustain that signaling long enough for regeneration to complete. How these key signals are sustained is unclear. To gain a comprehensive view of the changes in gene expression that occur during regeneration, we performed whole-genome mRNAseq of actively regenerating tissue from damaged Drosophila wing imaginal discs. We used genetic tools to ablate the wing primordium to induce regeneration, and carried out transcriptional profiling of the regeneration blastema by fluorescently labeling and sorting the blastema cells, thus identifying differentially expressed genes. Importantly, by using genetic mutants of several of these differentially expressed genes we have confirmed that they have roles in regeneration. Using this approach, we show that high expression of the gene moladietz (mol), which encodes the Duox-maturation factor NIP, is required during regeneration to produce reactive oxygen species (ROS), which in turn sustain JNK signaling during regeneration. We also show that JNK signaling upregulates mol expression, thereby activating a positive feedback signal that ensures the prolonged JNK activation required for regenerative growth. Thus, by whole-genome transcriptional profiling of regenerating tissue we have identified a positive feedback loop that regulates the extent of regenerative growth.

Ultrastructure of bioscaffolds derived from bovine articular cartilage as an experimental model to support blastema cells

Decellularized cartilage extracellular matrix (ECM)-derived bioscaffolds have been designed as they provide a unique opportunity to carry out research on the mechanisms of chondrogenesis and its regulation. These scaffolds are used as a model in order to emulate different aspects relating to the formation, degeneration, and regeneration of the cartilage. This project has studied the interaction between the decellularized bovine articular cartilage and the blastema cells derived from the punched rabbit’s ear. Articular cartilage was dissected in fragments with 10 mm length and 2 mm thickness. Then, they were chemically decellularized with 2% sodium dodecyl sulfate (SDS) for 2 h. The tissue rings derived from the rabbit’s ear were assembled with the decellularized scaffolds and they were placed in a culture media for 30 days. These samples were fixed, sectioned, and microscopically studied. Micrographs of SEM electron microscopy and DAPI staining confirmed the decellularization of the scaffolds. FTIR analysis confirmed the preservation of ECM components including collagen II and proteoglycans. The optical microscopy observations confirmed migration, adherence, and penetration of the blastema cells into the scaffolds. The electron microscopy studies demonstrated that the blastema cells beside the scaffolds have triggered mutual interactions and merely progressed toward chondrogenic differentiation. The main objective is to identify and comprehend the interaction between cartilage matrix and blastema cells. The proposed model is an ideal model for fundamental studies in cartilage tissue engineering. The principles of tissue engineering must be taken in to account while studying such interactions.

Msh homeobox 1 (Msx1)- and Msx2-overexpressing bone marrow-derived mesenchymal stem cells resemble blastema cells and enhance regeneration in mice

Amputation of the proximal region in mammals is not followed by regeneration because blastema cells (BCs) and expression of regenerative genes, such as Msh homeobox (Msx) genes, are absent in this animal group. The lack of BCs and positional information in other cells is therefore the main obstacle to therapeutic approaches for limb regeneration. Hence, this study aimed to create blastema-like cells (BlCs) by overexpressing Msx1 and Msx2 genes in mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to regenerate a proximally amputated digit tip. We transduced mBMSCs with Msx1 and Msx2 genes and compared osteogenic activity and expression levels of several Msx-regulated genes (Bmp4, Fgf8, and keratin 14 (K14)) in BlC groups, including MSX1, MSX2, and MSX1/2 (in a 1:1 ratio) with those in mBMSCs and BCs in vitro and in vivo following injection into the amputation site. We found that Msx gene overexpression increased expression of specific blastemal markers and enhanced the proliferation rate and osteogenesis of BlCs compared with mBMSCs and BCs via activation of Fgf8 and Bmp4 Histological analyses indicated full regrowth of digit tips in the Msx-overexpressing groups, particularly in MSX1/2, through endochondral ossification 6 weeks post-injection. In contrast, mBMSCs and BCs formed abnormal bone and nail. Full digit tip was regenerated only in the MSX1/2 group and was related to boosted Bmp4, Fgf8, and K14 gene expression and to limb-patterning properties resulting from Msx1 and Msx2 overexpression. We propose that Msx-transduced cells that can regenerate epithelial and mesenchymal tissues may potentially be utilized in limb regeneration.

Regeneration of the nervous and muscular system after caudal amputation in the polychaete Alitta virens (Annelida: Nereididae)

Regenerating segments in polychaetes offer a vivid example of epimorphic recovery of the lost organs and tissues. It is also a promising object for studying positional information and the mechanisms maintaining the body integrity. With the aim to develop a convenient standardized model, we described the dynamics of recovery of the major anatomical structures and created a staging system for the caudal regeneration in Alitta virens. In average the normal organization of the posterior body end is restored within 10 days after amputation (dpa). The whole regenerative process was divided into 5 stages: (1) wound healing (0–1 dpa), (2) blastema formation (1–2 dpa), (3) patterning and growth of the blastema (2–3 dpa), (4) differentiation of the first regenerated segment (3–5 dpa), (5) formation and differentiation of the subsequent 5–6 segments (5–10 dpa). The regeneration is carried out mainly by epimorphosis, although the elements of intercalary growth as well as the morphallactic transformation of the stump have been noted. Terminal structures of the pygidium (muscles of the anal sphincter, pygidial cavity, pygidial ring nerve, pygidial cirri) appear at stages 1–3, and then (from stage 3) the formation of new metameres begins in front of the pygidium. Differentiation of the first newborn segment is associated with the tissue remodeling in the last old segment. Formation of the next segments resembles accelerated postlarval growth. The neural elements of the regenerative bud are developing faster than the surrounding muscles. The neurites extending from the CNS and PNS come to the surface of the wound epithelium at stage 1. Later, nerve fibers from the CNS lengthen and thicken along with the growth of the regenerative bud. Ganglion, parapodial nerves, oblique muscles and coeloms of the first segment are detected at stage 4. Longitudinal muscles regenerate in anterior to posterior progression, being constantly in contact with the corresponding fibers of the old tissues. All other muscles differentiate from blastemal cells in isolation from the old musculature of the stump. Our data promote the further using of the posterior body end regeneration in A. virens as an experimental model for resolving crucial problems of developmental biology.

Multilocular Cystic Nephroma in a Child: A Rare Case

Multilocular cystic nephroma (MLCN) is classically described as a benign slow-growing renal tumor. The aim of this study was to evaluate MLCN in a child in the West of Iran. The ultrasound examination showed a multicystic mass measuring 6.6x6.5 cm with multiple internal septae in the middle and lower left kidney in a 19-month-old boy. Another sonography revealed multicystic mass 7.4x6.6cm in left renal pole with a diagnosis of multicystic nephroma. The lab data were within normal limits, except for hemoglobin level, that was 11.6 g/dl. The gross examination revealed kidney tissue measuring 10x8x7 cm including multiloculated cyst measuring up to 8 cm and the microscopic examination revealed multiple cysts lined by flat epithelium with spindle cell stroma containing abortive tubular elements and rare foci of small round cells. In conclusion, MLCN occures more in male children and female adults. The results showed that low hemoglobin level had association with some cases of MLCN. Inflammation may be seen in MLCN, but presence of any amount of blastemal cells is diagnosed as cystic partially differentiated nephroblastoma by most experts.

Nephroblastoma with lymphatic metastasis in an Angus calf: scientific communication

Nephroblastoma is a congenital renal neoplasm composed of metanephric blastema and stromal cells. A one-year-old male Angus calf presented with diarrhea, nasal discharge, cough and weight loss. Serum levels of creatinine and urea were increased, with reduction in the level of albumin. After eight days, clinical condition got worse and the calf died. On necropsy, the cranial pole of the left kidney presented a firm whitish mass (30 x 21 x 14 cm). Multifocal to coalescing firm whitish nodules were observed on the serosa of the large intestine, in the mesentery and in the adventitia of the urinary bladder. All lung lobes contained multifocal to coalescing firm whitish elevated nodules (0.5 to 1 cm of diameter). Histologically, renal mass consisted of neoplastic proliferation of embryonal epithelial and mesenchymal cells, intermixed with large amounts of blastema cells. The nodules on large intestine, mesentery, urinary bladder and lungs were composed mainly of blastema cells and mild amount of mesenchymal cells. Blastema cells were also observed within lymphatic vessels in lung parenchyma. Embryonal epithelial, mesenchymal and blastema cells showed, respectively, positive immunostain for cytokeratin, vimentin and S100. Based on pathological and immunohistochemical findings, the tumor was diagnosed as nephroblastoma in a calf with metastasis through lymphatic vessels to lungs, large intestine, mesentery and urinary bladder.

WITHDRAWN: Sacroanterior extrarenal Wilms tumor: Case report and literature review

Abstract Extrarenal Wilms tumor (ERWT) is a rare neoplasm, which often occurs in retroperitoneum and inguinal region. We report a case of a 3-year old Chinese boy who had a mass located at the sacroanterior region with a few concurrent anomalies. The patient underwent operation and pathology study gave the diagnosis of ERWT with classical triphasic components of blastemal, stromal and epithelial cells. He received postoperative adjuvant chemotherapy and radiotherapy and was well after 6 months follow-up. We discuss the diagnosis, treatment, pathogenesis and the malformations in ERWT and give a short literature review.

Serum Proteases Potentiate BMP-Induced Cell Cycle Re-entry of Dedifferentiating Muscle Cells during Newt Limb Regeneration

Limb amputation in the newt induces myofibers to dedifferentiate and re-enter the cell cycle to generate proliferative myogenic precursors in the regeneration blastema. Here we show that bone morphogenetic proteins (BMPs) and mature BMPs that have been further cleaved by serum proteases induce cell cycle entry by dedifferentiating newt muscle cells. Protease-activated BMP4/7 heterodimers that are present in serum strongly induced myotube cell cycle re-entrywith protease cleavage yielding a 30-fold potency increase of BMP4/7 compared with canonical BMP4/7. Inhibition of BMP signaling via muscle-specificdominant-negative receptor expression reduced cell cycle entry invitro and invivo. Invivo inhibition of serine protease activity depressed cell cycle re-entry, which in turn was rescued by cleaved-mimic BMP. This work identifies a mechanism of BMP activation that generates blastema cells from differentiated muscle.

Transcriptome analysis of the regenerating tail vs. the scarring limb in lizard reveals pathways leading to successful vs. unsuccessful organ regeneration in amniotes.

Lizards are amniotes regenerating the tail but not the limb, and no information on their different gene expression is available. Transcriptomes of regenerating tail and limb blastemas show differences in gene expression between the two organs. In tail blastemal, snoRNAs and Wnt signals appear up-regulated probably in association with the apical epidermal peg (AEP), an epithelial region that sustains tail regeneration but is absent in the limb. A balance between pro-oncogenes and tumor suppressors is likely present in tail blastema allowing a regulated proliferation. Small collagens, protease inhibitors, embryonic keratins are up-regulated in the regenerating tail blastema but not in the limb where Wnt inhibitors, inflammation-immune and extracellular matrix proteins depress cell growth. The AEP and the spinal cord in the tail maintains Wnt and fibroblast growth signaling that stimulate blastema cell proliferation and growth while these signals are absent in the limb as a consequence of the intense inflammation. Regeneration of amniote appendages requires a control of cell proliferation and inflammatory-immune reactions to form an apical epidermal cap. Genes that control cell proliferation and inflammation, addressing regeneration and not tumor formation in the tail and scarring in the limb are discussed for future studies. Developmental Dynamics 246:116-134, 2017. © 2016 Wiley Periodicals, Inc.

Immunolocalization of β-catenin, E-cadherin and N-cadherin in neonate and adult rat kidney.

β-catenin, E-cadherin and N-cadherin are adhesion molecules that play important roles in organogenesis, tissue homeostasis, renal epithelial integrity and polarity. The present study demonstrated their immunolocalization in adult and neonate rat kidney. Membranous or cytoplasmic expression of β-catenin, E-cadherin and N-cadherin were seen in adult and developing renal tubular epithelial cells. Particularly, in adult kidney, E-cadherin and β-catenin were intensively expressed in distal renal tubules, whereas N-cadherin was expressed in proximal renal tubules. In neonate rat kidney on 1 and 4 days old, developing renal tubular epithelial cells were mainly reacted with E-cadherin and very weakly expressed N-cadherin; β-catenin was expressed in developing renal tubules and mesenchymal blastemal cells. Interestingly, β-catenin-positive renal tubular epithelial cells simultaneously expressed E-cadherin in the kidney of adult and developing rats. Collectively, the adhesion molecules were differentially distributed in the renal tubules of adult rats and β-catenin and E-cadherin are predominant adhesion molecules in developing kidney. The present findings would provide the basic information of evaluating renal tubular toxicity using rats, in addition to renal genesis, in terms of adhesion molecules.

Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin.

Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies ( Shibata et al., 2016 ; Yoshinari et al., 2012 ) is a useful approach for tracking and manipulating the blastema cells during fish fin regeneration.

Microscopic observations show invasion of inflammatory cells in the limb blastema and epidermis in pre-metamorphic frog tadpoles which destroy the Apical Epidermal CAP and impede regeneration

Some limb regeneration in tadpoles of Rana dalmatina occurs at stages 44–48 when small hind-limbs are present while scarring occurs at stages 51–52 when forelimbs have developed and metamorphosis is approaching. Ultrastructural analysis of cells forming the regenerating blastema detects mesenchymal cells and an Apical Epidermal Cap (AEC) in regenerating limb blastema 5–6 days post-amputation at stages 46–48. In contrast, granulocytes and numerous macrophages and lymphocytes prevail over mesenchymal cells in limb blastema at stages 51–52, which are destined to form scars. An increase in inflammatory cells in limb blastema prior to metamorphosis suggests a negative influence of immune cells on limb regeneration. Inflammatory cells invade the apical wound epidermis where stem keratinocytes are likely destroyed, impeding the formation of an AEC, the microregion which leads to limb regeneration. The invasion of immune cells, however, may also represent a physiological consequence of the death of cell populations in the tadpoles occurring with approaching metamorphosis. The passage from an aquatic to a terrestrial life in this frog elicits the typical amniote scarring reaction after wounding, and the limb cannot regenerate. The present observations sustain the hypothesis that the evolution of the adaptive immunity in tetrapods while efficiently preserving adult self-condition, determined the loss of tissue regeneration since the embryonic antigens evocated in blastema cells are removed by immune cells of the adult.

Live Monitoring of Blastemal Cell Contributions during Appendage Regeneration

The blastema is a mass of progenitor cells that enables regeneration of amputated salamander limbs or fish fins. Methodology to label and track blastemal cell progeny has been deficient, restricting our understanding of appendage regeneration. Here, we created a system for clonal analysis and quantitative imaging of hundreds of blastemal cells and their respective progeny in living adult zebrafish undergoing fin regeneration. Amputation stimulates resident cells within a limited recruitment zone to reset proximodistal (PD) positional information and assemble the blastema. Within the newly formed blastema, the spatial coordinates of connective tissue progenitors are predictive of their ultimate contributions to regenerated skeletal structures, indicating early development of an approximate PD pre-pattern. Calcineurin regulates size recovery by controlling the average number of progeny divisions without disrupting this pre-pattern. Our longitudinal clonal analyses of regenerating zebrafish fins provide evidence that connective tissue progenitors are rapidly organized into a scalable blueprint of lost structures.

Live Imaging of Axolotl Digit Regeneration Reveals Spatiotemporal Choreography of Diverse Connective Tissue Progenitor Pools

SummaryConnective tissues—skeleton, dermis, pericytes, fascia—are a key cell source for regenerating the patterned skeleton during axolotl appendage regeneration. This complexity has made it difficult to identify the cells that regenerate skeletal tissue. Inability to identify these cells has impeded a mechanistic understanding of blastema formation. By tracing cells during digit tip regeneration using brainbow transgenic axolotls, we show that cells from each connective tissue compartment have distinct spatial and temporal profiles of proliferation, migration, and differentiation. Chondrocytes proliferate but do not migrate into the regenerate. In contrast, pericytes proliferate, then migrate into the blastema and give rise solely to pericytes. Periskeletal cells and fibroblasts contribute the bulk of digit blastema cells and acquire diverse fates according to successive waves of migration that choreograph their proximal-distal and tissue contributions. We further show that platelet-derived growth factor signaling is a potent inducer of fibroblast migration, which is required to form the blastema.

Diethylnitrosamine-induced expression of germline-specific genes and pluripotency factors, including vasa and oct4, in medaka somatic cells

Various methods have been developed to reprogram mammalian somatic cells into pluripotent cells as well as to directly reprogram somatic cells into other cell lineages. We are interested in applying these methods to fish, and here, we examined whether mRNA expression of germline-specific genes (vasa, nanos2, -3) and pluripotency factors (oct4, sox2, c-myc, nanog) is inducible in somatic cells of Japanese medaka (Oryzias latipes). We found that the expression of vasa is induced in the gut and regenerating fin by exposure to a carcinogen, diethylnitrosamine (DEN). Induction of vasa in the gut started on the 5th day of treatment with >50 ppm DEN. In addition, nanos2, -3, oct4, sox2, klf4, c-myc, and nanog were also expressed simultaneously in some vasa-positive gut and regenerating fin samples. Vasa-positive cells were detected by immunohistochemistry (IHC) in the muscle surrounding the gut and in the wound epidermis, blastema, and fibroblast-like cells in regenerating fin. In vasa:GFP transgenic medaka, green fluorescent protein (GFP) fluorescence appeared in the wound epidermis and fibroblast-like cells in the regenerating fin following DEN exposure, in agreement with the IHC data. Our data show that mRNA expression of genes relevant to germ cell specification and pluripotency can be induced in fish somatic cells by exposure to DEN, suggesting the possibility of efficient and rapid cell reprogramming of fish somatic cells.

Fgf signalling controls diverse aspects of fin regeneration.

Studies have shown that fibroblast growth factor (Fgf) signalling is necessary for appendage regeneration, but its exact function and the ligands involved during regeneration have not yet been elucidated. Here, we performed comprehensive expression analyses and identified fgf20a and fgf3/10a as major Fgf ligands in the wound epidermis and blastema, respectively. To reveal the target cells and processes of Fgf signalling, we performed a transplantation experiment of mesenchymal cells that express the dominant-negative Fgf receptor 1 (dnfgfr1) under control of the heat-shock promoter. This mosaic knockdown analysis suggested that Fgf signalling is directly required for fin ray mesenchyme to form the blastema at the early pre-blastema stage and to activate the regenerative cell proliferation at a later post-blastema stage. These results raised the possibility that the early epidermal Fgf20a and the later blastemal Fgf3/10a could be responsible for these respective processes. We demonstrated by gain-of-function analyses that Fgf20a induces the expression of distal blastema marker junbl, and that Fgf3 promotes blastema cell proliferation. Our study highlights that Fgfs in the wound epidermis and blastema have distinct functions to regulate fin regeneration cooperatively.