Plasticity of photoreceptor-generating retinal progenitors revealed by prolonged retinoic acid exposure

Background

Epimorphic regeneration results in the restoration of lost tissues and structures from an aggregation of proliferating cells known as a blastema. Among amniotes the most striking example of epimorphic regeneration comes from tail regenerating lizards. Although tail regeneration is often studied in the context of ecological costs and benefits, details of the sequence of tissue-level events are lacking. Here we investigate the anatomical and histological events that characterize tail regeneration in the leopard gecko, Eublepharis macularius.

Results

Tail structure and tissue composition were examined at multiple days following tail loss, revealing a conserved pattern of regeneration. Removal of the tail results in a consistent series of morphological and histological events. Tail loss is followed by a latent period of wound healing with no visible signs of regenerative outgrowth. During this latent period basal cells of the epidermis proliferate and gradually cover the wound. An additional aggregation of proliferating cells accumulates adjacent to the distal tip of the severed spinal cord marking the first appearance of the blastema. Continued growth of the blastema is matched by the initiation of angiogenesis, followed by the re-development of peripheral axons and the ependymal tube of the spinal cord. Skeletal tissue differentiation, corresponding with the expression of Sox9, and muscle re-development are delayed until tail outgrowth is well underway.

Conclusions

We demonstrate that tail regeneration in lizards involves a highly conserved sequence of events permitting the establishment of a staging table. We show that tail loss is followed by a latent period of scar-free healing of the wound site, and that regeneration is blastema-mediated. We conclude that the major events of epimorphic regeneration are highly conserved across vertebrates and that a comparative approach is an invaluable biomedical tool for ongoing regenerative research.     

Three dimensional socket preservation: a technique for soft tissue augmentation along with socket grafting

Background

A cursory review of the current socket preservation literatures well depicts the necessity of further esthetic considerations through the corrective procedures of the alveolar ridge upon and post extraction. A new technique has been described here is a rotational pedicle combined epithelialized and connective tissue graft (RPC graft) adjunct with immediate guided tissue regeneration (GBR) procedure.

Results

We reviewed this technique through a case report and discuss it??s benefit in compare to other socket preservation procedures.

Conclusion

The main advantages of RPC graft would be summarized as follows: stable primary closure during bone remodeling, saving or crating sufficient vestibular depth, making adequate keratinized gingiva on the buccal surface, and being esthetically pleasant.     

Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis

Introduction

Mesenchymal stem cells (MSCs) can differentiate into various connective tissue cells. Several techniques have been used for the clinical application of MSCs in articular cartilage repair; however, there are many issues associated with the selection of the scaffold material, including its ability to support cell viability and differentiation and its retention and degradation in situ. The application of MSCs via a scaffold also requires a technically demanding surgical procedure. The aim of this study was to test the outcome of intra-articular transplantation of mesenchymal stem cells suspended in hyaluronic acid (HA) in the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis (OA).

Methods

Commercially available human MSCs were cultured, labeled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), suspended in either PBS or HA, and injected into the knee joints of 7-month-old animals. The control animals were injected with either PBS or HA alone. The animals were sacrificed at 1, 3, and 5 weeks post transplantation, the knee joints harvested, and fluorescent microscopic analysis was performed. Histological and immunohistochemical analysis were performed at 5 weeks post transplantation.

Results

At 5 weeks post transplantation, partial cartilage repair was noted in the HA-MSC group but not in the other groups. Examination of CFDA-SE-labeled cells demonstrated migration, differentiation, and proliferation of MSC in the HA-MSC group. There was strong immunostaining for type II collagen around both residual chondrocytes and transplanted MSCs in the OA cartilage.

Conclusion

This scaffold-free and technically undemanding technique appears to result in the regeneration of articular cartilage in the spontaneous OA animal model. Although further examination of the long-term effects of transplantation is necessary, the findings suggest that intra-articular injection of HA-MSC mixture is potentially beneficial for OA.     

Gravity, a regulation factor in the differentiation of rat bone marrow mesenchymal stem cells

Background

Stem cell therapy has emerged as a potential therapeutic option for tissue engineering and regenerative medicine, but many issues remain to be resolved, such as the amount of seed cells, committed differentiation and the efficiency. Several previous studies have focused on the study of chemical inducement microenvironments. In the present study, we investigated the effects of gravity on the differentiation of bone marrow mesenchymal stem cells (BMSCs) into force-sensitive or force-insensitive cells.

Methods and results

Rat BMSCs (rBMSCs) were cultured under hypergravity or simulated microgravity (SMG) conditions with or without inducement medium. The expression levels of the characteristic proteins were measured and analyzed using immunocytochemical, RT-PCR and Western-blot analyses. After treatment with 5-azacytidine and hypergravity, rBMSCs expressed more characteristic proteins of cardiomyocytes such as cTnT, GATA4 and β-MHC; however, fewer such proteins were seen with SMG. After treating rBMSCs with osteogenic inducer and hypergravity, there were marked increases in the expression levels of ColIA1, Cbfa1 and ALP. Reverse results were obtained with SMG. rBMSCs treated with adipogenic inducer and SMG expressed greater levels of PPARgamma. Greater levels of Cbfa1- or cTnT-positive cells were observed under hypergravity without inducer, as shown by FACS analysis. These results indicate that hypergravity induces differentiation of rBMSCs into force-sensitive cells (cardiomyocytes and osteoblasts), whereas SMG induces force-insensitive cells (adipocytes).

Conclusion

Taken together, we conclude that gravity is an important factor affecting the differentiation of rBMSCs; this provides a new avenue for mechanistic studies of stem cell differentiation and a new approach to obtain more committed differentiated or undifferentiated cells.     

Uncovering the regeneration strategies of zebrafish organs: a comprehensive systems biology study on heart,cerebellum, fin,and retina regeneration

Background

Regeneration is an important biological process for the restoration of organ mass, structure, and function after damage, and involves complex bio-physiological mechanisms including cell differentiation and immune responses. We constructed four regenerative protein-protein interaction (PPI) networks using dynamic models and AIC (Akaike’s Information Criterion), based on time-course microarray data from the regeneration of four zebrafish organs: heart, cerebellum, fin, and retina. We extracted core and organ-specific proteins, and proposed a recalled-blastema-like formation model to uncover regeneration strategies in zebrafish.

Results

It was observed that the core proteins were involved in TGF-β signaling for each step in the recalled-blastema-like formation model and TGF-β signaling may be vital for regeneration. Integrins, FGF, and PDGF accelerate hemostasis during heart injury, while Bdnf shields retinal neurons from secondary damage and augments survival during the injury response. Wnt signaling mediates the growth and differentiation of cerebellum and fin neural stem cells, potentially providing a signal to trigger differentiation.

Conclusion

Through our analysis of all four zebrafish regenerative PPI networks, we provide insights that uncover the underlying strategies of zebrafish organ regeneration.

     

A Nanoconjugate Apaf-1 Inhibitor Protects Mesothelial Cells from Cytokine-Induced Injury

Background

Inflammation may lead to tissue injury. We have studied the modulation of inflammatory milieu-induced tissue injury, as exemplified by the mesothelium. Peritoneal dialysis is complicated by peritonitis episodes that cause loss of mesothelium. Proinflammatory cytokines are increased in the peritoneal cavity during peritonitis episodes. However there is scarce information on the modulation of cell death by combinations of cytokines and on the therapeutic targets to prevent desmesothelization.

Methodology

Human mesothelial cells were cultured from effluents of stable peritoneal dialysis patients and from omentum of non-dialysis patients. Mesothelial cell death was studied in mice with S. aureus peritonitis and in mice injected with tumor necrosis factor alpha and interferon gamma.Tumor necrosis factor alpha and interferon gamma alone do not induce apoptosis in cultured mesothelial cells. By contrast, the cytokine combination increased the rate of apoptosis 2 to 3-fold over control. Cell death was associated with the activation of caspases and a pancaspase inhibitor prevented apoptosis. Specific caspase-8 and caspase-3 inhibitors were similarly effective. Co-incubation with both cytokines also impaired mesothelial wound healing in an in vitro model. However, inhibition of caspases did not improve wound healing and even impaired the long-term recovery from injury. By contrast, a polymeric nanoconjugate Apaf-1 inhibitor protected from apoptosis and allowed wound healing and long-term recovery. The Apaf-1 inhibitor also protected mesothelial cells from inflammation-induced injury in vivo in mice.

Conclusion

Cooperation between tumor necrosis factor alpha and interferon gamma contributes to mesothelial injury and impairs the regenerative capacity of the monolayer. Caspase inhibition attenuates mesothelial cell apoptosis but does not facilitate regeneration. A drug targeting Apaf-1 allows protection from apoptosis as well as regeneration in the course of inflammation-induced tissue injury.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

Background

Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In Xenopus laevis tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.

Results

Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and in vivo cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.

Conclusions

Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with in vivo cell tracing experiments to better understand the regenerative mechanisms.     

Origin of tendon stem cells <Emphasis Type="Italic">in situ</Emphasis>

Background

Adult stem cells are surveillance repositories capable of supplying a renewable source of progenitors for tissue repair and regeneration to maintain tissue homeostasis throughout life. Many tissue-resident stem cells have been identified in situ, which lays the foundation for studying them in their native microenvironment, i.e. the niche. Within the musculoskeletal system, muscle stem cells have been unequivocally identified in the mouse, which have led to considerable advances in understanding their role in muscle homeostasis and regeneration. On the other hand, for bone and tendon progenitor cells, mesenchymal stem cells have been used as the main in vitro cell model as they can differentiate into osteogenic, chondrogenic and tenogenic fates. Despite considerable efforts and employment of modern tools, the in vivo origins of bone and tendon stem cells remain debated. Tendon regeneration via stem cells is understudied and deserves attention as tendon damage is noted for a bleak, time-consuming recovery and the repaired tendon seldom regains the structural integrity and strength of the native, uninjured state.

Objective

Here we review the past efforts and recent studies toward defining adult tendon stem cells and understanding tendon regeneration instead of tendon development. The focus is on adult tendon resident cells in situ and the uncertainty of their roles in regeneration.

Methods

A systematic literature search using the Pubmed search engine was conducted encompassing the seminal papers in the tendon field.

Conclusion

Investigation of tendon stem cells in situ is in its infancy mainly due to lack of necessary tools and standardized injury model. We propose a concerted effort toward establishing a comprehensive cell atlas of the tendon, making genetic tools and choosing a reliable injury model for coordinated studies among different laboratories. Increasing our basic understanding should aid future therapeutic innovations to shorten and enhance the tendon repair/regeneration process.

     

Effect of connective tissue growth factor (CCN2/CTGF) on proliferation and differentiation of mouse periodontal ligament-derived cells

Background

CCN2/CTGF is known to be involved in tooth germ development and periodontal tissue remodeling, as well as in mesenchymal tissue development and regeneration. In this present study, we investigated the roles of CCN2/CTGF in the proliferation and differentiation of periodontal ligament cells (murine periodontal ligament-derived cell line: MPL) in vitro.

Results

In cell cultures of MPL, the mRNA expression of the CCN2/CTGF gene was stronger in sparse cultures than in confluent ones and was significantly enhanced by TGF-β. The addition of recombinant CCN2/CTGF (rCCN2) to MPL cultures stimulated DNA synthesis and cell growth in a dose-dependent manner. Moreover, rCCN2 addition also enhanced the mRNA expression of alkaline phosphatase (ALPase), type I collagen, and periostin, the latter of which is considered to be a specific marker of the periosteum and periodontium; whereas it showed little effect on the mRNA expression of typical osteoblastic markers, e.g., osteopontin and osteocalcin. Finally, rCCN2/CTGF also stimulated ALPase activity and collagen synthesis.

Conclusion

These results taken together suggest important roles of CCN2/CTGF in the development and regeneration of periodontal tissue including the periodontal ligament.     

Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

A functional genomics screen identifies an Importin-α homolog as a regulator of stem cell function and tissue patterning during planarian regeneration

Background

Planarians are renowned for their regenerative capacity and are an attractive model for the study of adult stem cells and tissue regeneration. In an effort to better understand the molecular mechanisms underlying planarian regeneration, we performed a functional genomics screen aimed at identifying genes involved in this process in Schmidtea mediterranea.

Methods

We used microarrays to detect changes in gene expression in regenerating and non-regenerating tissues in planarians regenerating one side of the head and followed this with high-throughput screening by in situ hybridization and RNAi to characterize the expression patterns and function of the differentially expressed genes.

Results

Along with five previously characterized genes (Smed-cycD, Smed-morf41/mrg-1, Smed-pdss2/dlp1, Smed-slbp, and Smed-tph), we identified 20 additional genes necessary for stem cell maintenance (Smed-sart3, Smed-smarcc-1, Smed-espl1, Smed-rrm2b-1, Smed-rrm2b-2, Smed-dkc1, Smed-emg1, Smed-lig1, Smed-prim2, Smed-mcm7, and a novel sequence) or general regenerative capability (Smed-rbap46/48-2, Smed-mcm2, Smed-ptbp1, and Smed-fen-1) or that caused tissue-specific defects upon knockdown (Smed-ddc, Smed-gas8, Smed-pgbd4, and Smed-b9d2). We also found that a homolog of the nuclear transport factor Importin-α plays a role in stem cell function and tissue patterning, suggesting that controlled nuclear import of proteins is important for regeneration.

Conclusions

Through this work, we described the roles of several previously uncharacterized genes in planarian regeneration and implicated nuclear import in this process. We have additionally created an online database to house our in situ and RNAi data to make it accessible to the planarian research community.

     

Mesenchymal‐like stem cells in canine ovary show high differentiation potential

Objectives

Recent studies have reported the existence of stem cells in ovarian tissue that show enhanced proliferative and differentiation potential compared to other adult tissues. Based on this evidence, we hypothesized that ovarian tissue contained mesenchymal‐like stem cells (MSC) that could be isolated using a novel rapid plastic adhesion technique.

Materials and methods

We established MSC lines derived from ovarian and adipose tissue based on their ability to rapidly adhere to plastic culture dishes in the first 3 hours after plating and studied their potentiality in terms of molecular markers and differentiation capacity.

Results

Morphological and kinetic properties of in vitro cultured ovarian MSC were similar to adipose‐derived MSC, and both reached senescence after similar passage numbers. Ovarian‐derived MSC expressed mesenchymal (CD90 and CD44) but not haematopoietic markers (CD34 and CD45), indicating similarity to adipose‐derived MSC. Moreover, ovarian‐derived MSC expressed NANOG, TERT, SOX2, OCT4 and showed extensive capacity to differentiate not only into adipogenic, osteogenic and chondrogenic tissue but also towards neurogenic and endodermal lineages and even precursors of primordial germ cells.

Conclusion

These results show for the first time the derivation of ovarian cells with the molecular properties of MSC as well as wide differentiation potential. Canine ovarian tissue is accessible, expandable, multipotent and has high plasticity, holding promise for applications in regenerative medicine.

     

A molecular map of mesenchymal tumors

Background

Bone and soft tissue tumors represent a diverse group of neoplasms thought to derive from cells of the mesenchyme or neural crest. Histological diagnosis is challenging due to the poor or heterogenous differentiation of many tumors, resulting in uncertainty over prognosis and appropriate therapy.

Results

We have undertaken a broad and comprehensive study of the gene expression profile of 96 tumors with representatives of all mesenchymal tissues, including several problem diagnostic groups. Using machine learning methods adapted to this problem we identify molecular fingerprints for most tumors, which are pathognomonic (decisive) and biologically revealing.

Conclusion

We demonstrate the utility of gene expression profiles and machine learning for a complex clinical problem, and identify putative origins for certain mesenchymal tumors.     

Mangrove community structure and regeneration potential on a rapidly expanding,river delta in Java

Key message

Mangroves in rapidly expanding Southeast Asian river deltas form floristically simple zones dominated by a few highly regenerative species adaptable or tolerant to rapid sedimentation and extensive river flooding.

Abstract

The size class distribution, community composition and spatial structure of five representative mangrove forests in the rapidly expanding Cimanuk river delta on Java were determined. These deltaic forests are species-poor (eight true mangrove species) and spatially segregated into three distinct floristic zones: (1) a fringing, low intertidal zone co-dominated by Avicennia marina and A. officinalis, with less abundant Bruguiera parviflora, Rhizophora apiculata, and R. mucronata; (2) a zone transitional between the low and mid intertidal in which Avicennia and Rhizophora spp. co-dominate; and (3) a mid intertidal zone dominated by R. mucronata and R. apiculata. Numerically dominated by seedlings (52,500–73,500 seedlings ha^?1) and saplings (5,268–5,660 saplings ha^?1), all five forests are relatively young and actively regenerating. Positive correlations of tree stem diameter and tree height with soil organic matter and P concentrations, salinity, the soil C/N ratio, pH, and silt/clay composition highlight the importance of soil factors in sustaining forest growth. The low diversity and relative structural simplicity of these rapidly growing and regenerating forests may be attributed to adaptation or tolerance to flooding and the rapid sedimentation and seaward expansion of the delta.     

Wnt3a promotes differentiation of human bone marrow-derived mesenchymal stem cells into cementoblast-like cells

Cementum is a calcified, avascular connective tissue that laminates the root of a tooth and plays a pivotal role in the development, homeostasis, and regeneration of a periodontal tissue. As a potential treatment for periodontal tissue defects in the patient with chronic periodontitis, much attention has been paid to tissue engineering combined with mesenchymal stem cells for regenerating periodontal tissues including cementum. However, limited information is available for the molecular factors that have impacts on the differentiation of mesenchymal stem cells into cementoblasts. Here, we focus on the effect of Wnt3a as a potential inducer and tested the effect of this protein in vitro using human bone marrow-derived mesenchymal stem cells. It was found that, when cells were cultured in an osteogenic medium containing Wnt3a, cementoblast-specific genes, such as cementum protein 1 and cementum attachment protein, as well as bone-related genes were significantly upregulated. These results suggest that Wnt3a promotes differentiation of the cells into cementoblast-like cells. Further experiments were carried out using inhibitors to gain deeper insights into molecular mechanisms underlying the observed differentiation. As a result, we conclude that Wnt3a-triggered differentiation into cementoblast-like cells is the consequence of the activation of the canonical Wnt signaling pathway with possible involvement of the non-canonical pathway.     

Proteomic analysis of regenerating mouse liver following 50% partial hepatectomy

Background

Although 70% (or 2/3) partial hepatectomy (PH) is the most studied model for liver regeneration, the hepatic protein expression profile associated with lower volume liver resection (such as 50% PH) has not yet been reported. Therefore, the aim of this study was to determine the global protein expression profile of the regenerating mouse liver following 50% PH by differential proteomics, and thereby gaining some insights into the hepatic regeneration mechanism(s) under this milder but clinically more relevant condition.

Results

Proteins from sham-operated mouse livers and livers regenerating for 24 h after 50% PH were separated by SDS-PAGE and analyzed by nanoUPLC-Q-Tof mass spectrometry. Compared to sham-operated group, there were totally 87 differentially expressed proteins (with 50 up-regulated and 37 down-regulated ones) identified in the regenerating mouse livers, most of which have not been previously related to liver regeneration. Remarkably, over 25 differentially expressed proteins were located at mitochondria. Several of the mitochondria-resident proteins which play important roles in citric acid cycle, oxidative phosphorylation and ATP production were found to be down-regulated, consistent with the recently-proposed model in which the reduction of ATP content in the remnant liver gives rise to early stress signals that contribute to the onset of liver regeneration. Pathway analysis revealed a central role of c-Myc in the regulation of liver regeneration.

Conclusions

Our study provides novel evidence for mitochondria as a pivotal organelle that is connected to liver regeneration, and lays the foundation for further studies on key factors and pathways involved in liver regeneration following 50% PH, a condition frequently used for partial liver transplantation and conservative liver resection.     

Low melting point agarose beads as a standard method for plantlet regeneration from protoplasts within the Cichorium genus

Key message

A standard method has been developed with which we are able to fully regenerate protoplasts of different Cichorium species. For the first time, endive protoplasts have been regenerated into plantlets.

Abstract

Protoplast regeneration is essential for somatic hybridizations. In this study, a standard method for plantlet regeneration from Cichorium protoplasts was developed. We evaluated the effect of the low melting point agarose (LMPA) bead technique on the regeneration capacity of protoplasts of seven C. intybus and four C. endivia genotypes. The LMPA bead technique was more efficient than culture in liquid or solid medium and allowed us to obtain plating efficiencies up to 4.9?% in C. intybus genotypes and efficiencies of up to 0.7?% in C. endivia genotypes. Moreover, the LMPA bead technique offers great advantages over liquid and solid culture systems: the media can be readily refreshed, protoplasts can be monitored separately, and microcalli can easily be removed from the beads. This increased efficiency was observed for all of the 11 Cichorium genotypes tested. Shoot formation was induced more efficiently when using 0.5?mg?l^?1 indole-3-acetic acid-enriched medium (up to 87.5?% of the protoplast-derived calli started shoot development) compared to 1-naphthaleneacetic acid-enriched medium. The LMPA bead technique optimized in this study enabled for the first time the full plantlet regeneration from protoplasts of C. endivia genotypes and increased the protoplast regenerating ability in other Cichorium species. This fine-tuned LMPA bead technique can therefore be applied for protoplast regeneration after protoplast fusions of the genus Cichorium.