Blastema Cells Research Articles

The caudal regeneration blastema is an accumulation of rapidly proliferating stem cells in the flatworm Macrostomum lignano

BackgroundMacrostomum lignano is a small free-living flatworm capable of regenerating all body parts posterior of the pharynx and anterior to the brain. We quantified the cellular composition of the caudal-most body region, the tail plate, and investigated regeneration of the tail plate in vivo and in semithin sections labeled with bromodeoxyuridine, a marker for stem cells (neoblasts) in S-phase.ResultsThe tail plate accomodates the male genital apparatus and consists of about 3,100 cells, about half of which are epidermal cells. A distinct regeneration blastema, characterized by a local accumulation of rapidly proliferating neoblasts and consisting of about 420 cells (excluding epidermal cells), was formed 24 hours after amputation. Differentiated cells in the blastema were observed two days after amputation (with about 920 blastema cells), while the male genital apparatus required four to five days for full differentiation. At all time points, mitoses were found within the blastema. At the place of organ differentiation, neoblasts did not replicate or divide. After three days, the blastema was made of about 1420 cells and gradually transformed into organ primordia, while the proliferation rate decreased. The cell number of the tail plate, including about 960 epidermal cells, was restored to 75% at this time point.ConclusionRegeneration after artificial amputation of the tail plate of adult specimens of Macrostomum lignano involves wound healing and the formation of a regeneration blastema. Neoblasts undergo extensive proliferation within the blastema. Proliferation patterns of S-phase neoblasts indicate that neoblasts are either determined to follow a specific cell fate not before, but after going through S-phase, or that they can be redetermined after S-phase. In pulse-chase experiments, dispersed distribution of label suggests that S-phase labeled progenitor cells of the male genital apparatus undergo further proliferation before differentiation, in contrast to progenitor cells of epidermal cells. Mitotic activity and proliferation within the blastema is a feature of M. lignano shared with many other regenerating animals.

Immunolocalization of FGF1 and FGF2 in the regenerating tail of the lizard Lampropholis guichenoti: Implications for FGFs as trophic factors in lizard tail regeneration

A role for fibroblast growth factors in stimulating limb and tail regeneration in amphibians has been shown; however, it is unknown whether these growth factors are also involved in the regeneration of the tail of lizard, an amniote model for studies on tissue regeneration. The presence of fibroblast growth factor-1 (FGF1) and -2 (FGF2) in the regenerating tail of the lizard Lampropholis guichenoti has been studied using immunofluorescence labeling. The study reveals that FGF2 is mainly localized in the wound and scaling epidermis, in differentiating muscles, in spinal ganglia, regenerating nerves and spinal cord. FGF1 is also present in the wound and differentiating epidermis, but is detectable at lower levels in the regenerating muscles and spinal cord. FGF1 is present in blastema cells, while FGF2 labeling is relatively low in these cells. Fibroblasts of the forming dermis are rich in FGF1 but not in FGF2. Developing blood vessels label for both FGF1 and FGF2 while the cartilaginous, bone and fat tissues are poorly labeled or unlabeled for FGFs. The present study suggests that most FGFs in the regenerating tail are located in the nervous system, in the epidermis and muscles, and these tissues most likely require these growth factors for their differentiation and growth. The present study suggests that FGFs produced in the regenerating epidermis, spinal cord and nerves can stimulate tail regeneration in lizards.

Cells keep a memory of their tissue origin during axolotl limb regeneration

During limb regeneration adult tissue is converted into a zone of undifferentiated progenitors called the blastema that reforms the diverse tissues of the limb. Previous experiments have led to wide acceptance that limb tissues dedifferentiate to form pluripotent cells. Here we have reexamined this question using an integrated GFP transgene to track the major limb tissues during limb regeneration in the salamander Ambystoma mexicanum (the axolotl). Surprisingly, we find that each tissue produces progenitor cells with restricted potential. Therefore, the blastema is a heterogeneous collection of restricted progenitor cells. On the basis of these findings, we further demonstrate that positional identity is a cell-type-specific property of blastema cells, in which cartilage-derived blastema cells harbour positional identity but Schwann-derived cells do not. Our results show that the complex phenomenon of limb regeneration can be achieved without complete dedifferentiation to a pluripotent state, a conclusion with important implications for regenerative medicine.

The Role of Stromal Stem Cells in Tissue Regeneration and Wound Repair

The process of wound repair in epithelium-lined organs of mammals is complex and is influenced by numerous secreted factors including cytokines, growth factors, and chemokines. However, the cellular organizers of this process are still not understood. Recent studies of tissue regeneration in organisms with simpler development have uncovered details about the activity of stem cells in the mesenchyme (the blastema) during this process. These blastemal cells are well positioned to interpret cues from the environment and to execute decisions about the direction of wound repair. In mammalian wounds, stromal stem cells appear to be positioned to perform functions similar to those of blastemal cells, including communication with both the overlying epithelium and the inflammatory cells in the mesenchyme.

Effects of activation of hedgehog signaling on patterning, growth, and differentiation in Xenopus froglet limb regeneration

Regenerating limbs of urodele amphibians and Xenopus tadpole are reconstructed along proximal-distal, anterior-posterior (AP), and dorsal-ventral axes. In contrast, a regenerated limb of the Xenopus froglet does not have digits, and only a simple cartilaginous structure referred to as a "spike" is formed. This suggests that repatterning along the AP axis is absent in the froglet blastema. Previous studies have shown that Shh and its target genes are not expressed in the froglet blastema. In this study, we activated Hedgehog signaling in the froglet blastema and found that target genes of Shh were inducible in the mesenchyme of limb blastema. Furthermore, we found that activation of the signaling had effects on blastema cell proliferation and chondrogenesis and resulted in the formation of multiple cartilaginous structures. These findings indicate that activation of signaling that is absent in the froglet blastema is effective for improvement of limb regeneration ability in the Xenopus froglet.

Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration

An amputated cricket leg regenerates all missing parts with normal size and shape, indicating that regenerating blastemal cells are aware of both their position and the normal size of the leg. However, the molecular mechanisms regulating this process remain elusive. Here, we use a cricket model to show that the Dachsous/Fat (Ds/Ft) signalling pathway is essential for leg regeneration. We found that knockdown of ft or ds transcripts by regeneration-dependent RNA interference (rdRNAi) suppressed proliferation of the regenerating cells along the proximodistal (PD) axis concomitantly with remodelling of the pre-existing stump, making the regenerated legs shorter than normal. By contrast, knockdown of the expanded (ex) or Merlin (Mer) transcripts induced over-proliferation of the regenerating cells, making the regenerated legs longer. These results are consistent with those obtained using rdRNAi during intercalary regeneration induced by leg transplantation. We present a model to explain our results in which the steepness of the Ds/Ft gradient controls growth along the PD axis of the regenerating leg.

Expression of Hepatocyte Growth Factor and Its Receptor Met in Wilms' Tumors and Nephrogenic Rests Reflects Their Roles in Kidney Development

Hepatocyte growth factor (HGF) and its receptor Met are known to play diverse roles in both organogenesis and cancer. Wilms' tumor (WT) is a prototype for the link between abrogated development and neoplasia, with dysregulation of growth factor/receptor pathways playing key roles. Despite this, an understanding of the HGF/Met axis in the process is lacking. Observing copy number alterations at the loci for these genes in WTs and their precursor lesions nephrogenic rests, we examined protein expression by immunohistochemistry and investigated the effects of HGF on an in vitro model of kidney development. HGF was preferentially expressed in the blastemal cells of nephrogenic rests but not WTs. Met expression was infrequent and restricted to well-differentiated epithelial cells and stroma in both lesions. In an independent cohort of favorable histology WTs on a tissue microarray, HGF was expressed in 15 of 193 (8%) cases and correlated with a predominance of epithelial cells, whereas Met expression was observed in 25 of 179 (14%) cases and was associated with stromal subtypes. In a mouse mesonephric cell line model, we observed Met expression in culture conditions reflecting both mesenchymal and epithelial differentiation, whereas HGF was up-regulated in association with acquisition of a more epithelial-like phenotype. This could be mimicked by exogenous exposure of mesenchymal-like cells to recombinant HGF. These data show that the relatively infrequent expression of HGF and Met in WT tumorigenesis reflects their roles in nephrogenesis, particularly the mesenchymal-to-epithelial transition, rather than a dependence on oncogenic signaling pathways.

Presacral Extrarenal Wilms Tumor in a Child

Wilms tumor (WT) is one of the most common solid tumors in children. It also is the most frequent tumor found in the kidneys. In 5% of cases, it affects both kidneys simultaneously. Extrarenal WTs are extremely rare. The diagnosis of extrarenal WT is almost always made after surgical intervention. The tumor can be located in the retroperitoneum, uterus, cervix, testes, skin, and even the thorax. We report on a 3-year-old girl with an extrarenal WT located in the presacral region. The exact mechanism whereby a WT occurs in extrarenal tissues is unknown. The presence of ectopic metanephric blastema cells or the WT gene causing transformation of extrarenal primitive mesonephric or pronephric remnants into WT have both been considered in the embryogenesis. Although imaging examinations are helpful in the definition of retroperitoneal tumors, no characteristic findings are available to diagnose an extrarenal WT before surgery. As a rule, the diagnosis of extrarenal WT is made after surgery. Surgical excision is the treatment of choice and the same general therapeutic rules should be followed as when the kidney has been affected. The use of Stage III guidelines for chemotherapy and radiotherapy are appropriate for these patients.

Proliferative Potential of a Spinal Nephroblastoma in a Young Dog

The proliferative potential of a spinal nephroblastoma was studied in a young dog. A 4-month-old, female golden retriever showed developing deterioration in her gait and subsequent paralysis of her hind legs. At necropsy, a well-demarcated grayish brown tumor mass was found in the lumbar spinal cord segments between L2 and L3. Histologically, a blastemal cell tumor with a tubule- or glomeruli-like structure was found to be infiltrating intradurally. Proliferating cells at the S-phase, assessed using the bromodeoxyuridine (BrdU) labeling method, were seen occasionally in the tubular cells and glomeruli-like structures and were frequently seen in the blastemal cells. Immunohistochemically, the tubular epithelial cells were positive for cytokeratin, and the blastemal cells were positive for vimentin. The present tumor showed a high potential for growth and invasion, which suggests that it the potential to expand into the adjacent spinal cord.

Short communication. Evaluation of presumptive caudal fin blastema cells as candidate donors in intraspecies zebrafish (Danio rerio) chimaeras

The blastema is a regenerative tissue with remarkable pluripotency. The aim of this work done on zebrafish (Danio rerio) was to define technical procedures required for obtaining and integrating blastema cells into embryos at the mid blastula transition stage (MBT) and the effect on survival, as well as the capacity to produce pigmented chimaeras. Wild type blastema cells were injected into gold type MBT embryos (E). Wild MBT blastomere cells were also injected into gold type MBT embryos as a control (C1). A second control group, C2, was not subjected to any manipulation. Survival was evaluated at 24, 48 and 72 h after performing the chimaerism, and the rate of adult chimaeras evaluated. The results showed significant differences in embryo survival between the E and C1 groups in embryo survival at 24 and 48 h postchimaerism (24 h: E-83.49% vs C1-54.8%, p

Regulation of dermal fibroblast dedifferentiation and redifferentiation during wound healing and limb regeneration in the Axolotl

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The Accessory Limb Model (ALM) in the axolotl allows for the identification of signals from the wound epidermis, nerves and dermal fibroblasts that interact to regenerate a limb. In the present study, we have used the ALM to identity the axolotl (Ambystoma mexicanum) orthologue of Twist (AmTwist), a basic helix-loop-helix transcription factor that is involved in the regeneration of the dermis during limb regeneration. AmTwist is expressed during the blastema stages in regeneration, but is inhibited by signals from the nerve during the early stages when dermal fibroblasts dedifferentiate to form blastema cells. As the dermis regenerates, AmTwist is expressed in association with the synthesis of type I collagen in the proximal region of the blastema. Exogenous bone morphogenetic protein-2 leads to an increase in AmTwist expression, and therefore may function as an endogenous regulator of AmTwist expression and dermis regeneration. The nerve appears to have a dual function in regeneration by coordinately regulating dedifferentiation and redifferentiation of dermal fibroblasts.

Gene expression and functional analysis of zebrafish larval fin fold regeneration

Teleost fish have a remarkable ability to regenerate their body parts compared to many higher vertebrates including humans. To facilitate molecular and genetic approaches for regeneration, we previously established an assay using the fin fold of zebrafish larvae. Here, we performed transcriptional profiling and identified genes differentially controlled during regeneration. From up-regulated transcripts, we identified a number of genes with localized expressions. Strikingly, all identified genes were also induced in the regenerating adult fin, which has a different tissue origin from the larval fin fold. This result supports the commonality of regeneration irrespective of tissue type and stage. Importantly, our analysis suggested that the regenerating tissue had many more compartments than generally assumed ones, the blastema and wound epidermis. By pharmacological and genetic approaches, we further evaluated functional involvement of induced molecules. Inhibition of Mmp9 function impaired proper morphological restoration without disturbing cell proliferation. Genetic mutations of blastema genes, hspa9 and smarca4, disrupted the fin fold regeneration by impairing the blastema cell proliferation. Thus, our results demonstrate that the regeneration model of juvenile zebrafish offers a powerful assay to dissect the regeneration processes.

High Cyclin E Staining Index in Blastemal, Stromal or Epithelial Cells Is Correlated with Tumor Aggressiveness in Patients with Nephroblastoma

PurposeIdentifying among nephroblastoma those with a high propensity for distant metastases using cell cycle markers: cyclin E as a regulator of progression through the cell cycle and Ki-67 as a tumor proliferation marker, since both are often deregulated in many human malignancies.Methodology/Principal FindingsA staining index (SI) was obtained by immunohistochemistry using anti-cyclin E and anti-Ki-67 antibodies in paraffin sections of 54 postchemotherapy nephroblastoma including 42 nephroblastoma without metastasis and 12 with metastases. Median cyclin E and Ki-67 SI were 46% and 33% in blastemal cells, 30% and 10% in stromal cells, 37% and 29.5% in epithelial cells. The highest values were found for anaplastic nephroblastoma. A correlation between cyclin E and Ki-67 SI was found for the blastemal component and for the epithelial component. Univariate analysis showed prognostic significance for metastases with cyclin E SI in stromal cells, epithelial cells and blastemal cells (p = 0.03, p = 0.01 and p = 0.002, respectively) as well as with Ki-67 SI in blastema (p<10−4). The most striking data were that both cyclin E SI and blastemal Ki-67 SI discriminated between patients with metastases and patients without metastasis among intermediate-risk nephroblastoma.ConclusionsOur findings show that a high cyclin E SI in all components of nephroblastoma is correlated with tumor aggressiveness and metastases, and that assessment of its expression may have prognostic value in the categorization of nephroblastoma.

Three genes control the timing, the site and the size of blastema formation in Drosophila

Regeneration is a vital process to maintain and repair tissues. Despite the importance of regeneration, the genes responsible for regenerative growth remain largely unknown. In Drosophila, imaginal disc regeneration can be induced either by fragmentation and in vivo culture or in situ by ubiquitous expression of wingless ( wg/ wnt1). Imaginal discs, like appendages in lower vertebrates, initiate regeneration by wound healing and proliferation at the wound site, forming a regeneration blastema. Most blastema cells maintain their disc-specific identity during regeneration; a few cells however, exhibit stem-cell like properties and switch to a different fate, in a phenomenon known as transdetermination. We identified three genes, regeneration ( rgn), augmenter of liver regeneration ( alr) and Matrix metalloproteinase-1 ( Mmp1) expressed specifically in blastema cells during disc regeneration. Mutations in these genes affect both fragmentation- and wg-induced regeneration by either delaying, reducing or positioning the regeneration blastema. In addition to the modifications of blastema homeostasis, mutations in the three genes alter the rate of regeneration-induced transdetermination. We propose that these genes function in regenerative proliferation, growth and regulate cellular plasticity.

Analysis of the expression and function of Wnt‐5a and Wnt‐5b in developing and regenerating axolotl (Ambystoma mexicanum) limbs

Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.

Immunohistochemical Profiling of Wilms Tumor

Wilms tumor (WT) usually shows a bi-phasic or tri-phasic morphology comprised of blastemal, stromal, and epithelial cells. Other biphasic renal tumors that can mimic WT may pose diagnostic dilemmas especially in preoperative needle biopsy samples. This tissue microarray study was designed to investigate the immunohistochemical features that may prove useful in the accurate diagnosis of WT in small biopsy samples. Eight punches from each paraffin block of 45 cases of WTs were used to construct 5 tissue microarray blocks. Immunohistochemical reactions of blastemal, stromal, and epithelial cells of each core to a panel of 37 antibodies were evaluated. Blastemal elements expressed CD56 (22, 57%), CD57 (19, 55%), cytokeratin 22 (CK22) (12, 27%), and CK8 (9, 21%). Epithelial cells were stained mostly with CK22 (17, 94%), CK18 (12, 66%), CK8 (14, 70%), CD57 (10, 76%), CD56 (6, 43%), EMA (7, 44%), and CK19 (5, 25%). Stromal cells expressed SMA (21, 50%), actin (18, 48%), desmin (9, 20%), CD34 (7, 24%), CD57 (5, 18%), and CD56 (5, 15%). Only one case was positive for CK5/6, CK13, CK14, and CK20. Calretinin expression was seen in the stromal cells of 3 and placental alkaline phosphatase expression was observed in 1 case. All 3 components were negative for CK1, CK7, myoglobin, Myf-4, MyoD1, HMB45, chromogranin A, synaptophysin, Melan A, beta-HCG, alpha-HCG, alpha-Inhibin, renal cell carcinoma antigen, glycophorin A, PSA, and estrogen and progesterone receptors. CD56, CD57, CK22, CK18, CK8, EMA, SMA, and actin are useful markers for an accurate diagnosis of WT in small biopsy samples.

Extracellular control of muscle regeneration

In urodele amphibian limb regeneration, the muscle provides a reservoir for uncommitted stem‐like blastema cells that will participate in the replacement of the lost structure. However, it is currently not clear what signals activate muscle‐associated satellite cells and/or dedifferentiation of multinucleate myofibers. Cultures of newt muscle fibers allow us to follow these physiological changes at the cellular level using time‐lapse video microscopy.Our studies on extracellular matrix (ECM) distribution during blastema formation demonstrate upregulation of hyaluronic acid (HA), fibronectin (FN), and Tenascin C, suggesting dynamic changes in the ECM composition. Contemporaneous with ECM reorganization, histological analyses of regenerating blastemas reveal increased satellite cell numbers and migration along with myofiber fragmentation. In order to gain a better understanding of the signaling properties of the ECM in regeneration, we are investigating various ECMs and their effects on cell physiology and morphology. Our data indicate that HA induces fragmentation of myotubes and limits adhesion whereas FN or laminin‐containing matrigel stimulates formation of myotubes.The establishment of a physiologically relevant environment and its manipulation in vitro will provide new insights into the control of the differentiated state of the cell by its ECM.Supported by DARPA, NIH, Searle Funds

Malignant Nephroblastoma in a Common Marmoset (Callithrix jacchus)

Necropsy of a 17-month-old male common marmoset (Callithrix jacchus) with a history of increased abdominal girth resulted in the finding of a unilateral polycystic renal neoplasm. Detailed histopathologic and immunohistochemical investigations revealed different tissue types within the tumor including stromal connective tissue and fusiform mesenchymal cell formations surrounding blastemal cells as well as different developmental stages of organ-specific epithelial cells accompanied by extensive cyst formation. Metastases were not observed. In consideration of the macroscopic, histologic, and immunohistochemical findings, the tumor was classified as a nephroblastoma closely resembling the so-called Wilms' tumor, a malignant embryonic renal tumor frequently observed in humans, especially in young children. In contrast, this tumor entity has rarely been observed in nonhuman primates. This report represents the first documented case of a cystic variant of nephroblastoma in a nonhuman primate.

Origin and proliferation of blastema cells during regeneration of Drosophila wing imaginal discs

Following a period of neglect, there has been a resurgence of interest in Drosophila imaginal discs as a model with which to analyze the relationships between growth and pattern formation during regeneration. To broaden our understanding of this process, we used cell lineage techniques to trace the origin of blastema cells and the early and late boundaries of the blastema in regenerating 3/4 wing disc fragments, examined the distribution of S-phase, mitotic and dead cells, and undertook clonal analysis to determine the topology of cell proliferation and its relationship to pattern formation. Using lineage tagging with the JNK phosphatase puckered (puc), we demonstrate that a substantial number of blastema cells arise from cells in which JNK is activated. Furthermore, we show that DNA synthesis and mitosis are activated well before wound healing is completed, in a region where the JNK pathway is activated; later, DNA synthesis and mitosis are observed in scattered cells throughout the blastema. Finally, clonal analysis shows a close relationship between the size and shape of clones and disparities in the positional values of the apposed surfaces.

Development and regeneration of the neonatal digit tip in mice

The digit tips of children and rodents are known to regenerate following amputation. The skeletal structure that regenerates is the distal region of the terminal phalangeal bone that is associated with the nail organ. The terminal phalanx forms late in gestation by endochondral ossification and continues to elongate until sexual maturity (8 weeks of age). Postnatal elongation at its distal end occurs by appositional ossification, i.e. direct ossification on the surface of the terminal phalanx, whereas proximal elongation results from an endochondral growth plate. Amputation through the middle of the terminal phalanx regenerates whereas regenerative failure is observed following amputation to remove the distal 2/3 of the bone. Regeneration is characterized by the formation of a blastema of proliferating cells that appear undifferentiated and express Bmp4. Using chondrogenic and osteogenic markers we show that redifferentiation does not occur by endochondral ossification but by the direct ossification of blastema cells that form the rudiment of the digit tip. Once formed the rudiment elongates by appositional ossification in parallel with unamputated control digits. Regenerated digits are consistently shorter than unamputated control digits. Finally, we present a case study of a child who suffered an amputation injury at a proximal level of the terminal phalanx, but failed to regenerate despite conservative treatment and the presence of the nail organ. These clinical and experimental findings expand on previously published observations and initiate a molecular assessment of a mammalian regeneration model.