Differences in neural stem cell identity and differentiation capacity drive divergent regenerative outcomes in lizards and salamanders.

While lizards and salamanders both exhibit the ability to regenerate amputated tails, the outcomes achieved by each are markedly different. Salamanders, such as Ambystoma mexicanum, regenerate nearly identical copies of original tails. Regenerated lizard tails, however, exhibit important morphological differences compared with originals. Some of these differences concern dorsoventral patterning of regenerated skeletal and spinal cord tissues; regenerated salamander tail tissues exhibit dorsoventral patterning, while regrown lizard tissues do not. Additionally, regenerated lizard tails lack characteristically roof plate-associated structures, such as dorsal root ganglia. We hypothesized that differences in neural stem cells (NSCs) found in the ependyma of regenerated spinal cords account for these divergent regenerative outcomes. Through a combination of immunofluorescent staining, RT-PCR, hedgehog regulation, and transcriptome analysis, we analyzed NSC-dependent tail regeneration. Both salamander and lizard Sox2+ NSCs form neurospheres in culture. While salamander neurospheres exhibit default roof plate identity, lizard neurospheres exhibit default floor plate. Hedgehog signaling regulates dorsalization/ventralization of salamander, but not lizard, NSCs. Examination of NSC differentiation potential in vitro showed that salamander NSCs are capable of neural differentiation into multiple lineages, whereas lizard NSCs are not, which was confirmed by in vivo spinal cord transplantations. Finally, salamander NSCs xenogeneically transplanted into regenerating lizard tail spinal cords were influenced by native lizard NSC hedgehog signals, which favored salamander NSC floor plate differentiation. These findings suggest that NSCs in regenerated lizard and salamander spinal cords are distinct cell populations, and these differences contribute to the vastly different outcomes observed in tail regeneration.

Lizard tail skeletal regeneration combines aspects of fracture healing and blastema-based regeneration.

Lizards are amniotes with the remarkable ability to regenerate amputated tails. The early regenerated lizard tail forms a blastema, and the regenerated skeleton consists of a cartilage tube (CT) surrounding the regenerated spinal cord. The proximal, but not distal, CT undergoes hypertrophy and ossifies. We hypothesized that differences in cell sources and signaling account for divergent cartilage development between proximal and distal CT regions. Exogenous spinal cord implants induced ectopic CT formation in lizard (Anolis carolinensis) blastemas. Regenerated spinal cords expressed Shh, and cyclopamine inhibited CT induction. Blastemas containing vertebrae with intact spinal cords formed CTs with proximal hypertrophic regions and distal non-hypertrophic regions, whereas removal of spinal cords resulted in formation of proximal CT areas only. In fate-mapping studies, FITC-labeled vertebra periosteal cells were detected in proximal, but not distal, CT areas. Conversely, FITC-labeled blastema cells were restricted to distal CT regions. Proximal cartilage formation was inhibited by removal of periosteum and could be recapitulated in vitro by periosteal cells treated with Ihh and BMP-2. These findings suggest that proximal CTs are directly derived from vertebra periosteal cells in response to BMP and Ihh signaling, whereas distal CTs form from blastema cells in response to Shh signals from regenerated spinal cords.

Lizard tail regeneration as an instructive model of enhanced healing capabilities in an adult amniote.

The ability to regenerate damaged or lost tissues has remained the lofty goal of regenerative medicine. Unfortunately, humans, like most mammals, suffer from very minimal natural regenerative capabilities. Certain non-mammalian animal species, however, are not so limited in their healing capabilities, and several have attracted the attention of researchers hoping to recreate enhanced healing responses in humans. This review focuses on one such animal group with remarkable regenerative abilities, the lizards. As the closest relatives of mammals that exhibit enhanced regenerative abilities as adults, lizards potentially represent the most relevant model for direct comparison and subsequent improvement of mammalian healing. Lizards are able to regenerate amputated tails and exhibit adaptations that both limit tissue damage in response to injury and initiate coordinated regenerative responses. This review summarizes the salient aspects of lizard tail regeneration as they relate to the overall regenerative process and also presents the relevant information pertaining to regrowth of specific tissues, including skeletal, muscular, nervous, and vascular tissues. The goal of this review is to introduce the topic of lizard tail regeneration to new audiences with the hope of expanding the knowledge base of this underutilized but potentially powerful model organism.

Lizard tail regeneration: regulation of two distinct cartilage regions by Indian hedgehog.

Lizards capable of caudal autotomy exhibit the remarkable ability to "drop" and then regenerate their tails. However, the regenerated lizard tail (RLT) is known as an "imperfect replicate" due to several key anatomical differences compared to the original tail. Most striking of these "imperfections" concerns the skeleton; instead of the vertebrae of the original tail, the skeleton of the RLT takes the form of an unsegmented cartilage tube (CT). Here we have performed the first detailed staging of skeletal development of the RLT CT, identifying two distinct mineralization events. CTs isolated from RLTs of various ages were analyzed by micro-computed tomography to characterize mineralization, and to correlate skeletal development with expression of endochondral ossification markers evaluated by histology and immunohistochemistry. During early tail regeneration, shortly after CT formation, the extreme proximal CT in direct contact with the most terminal vertebra of the original tail develops a growth plate-like region that undergoes endochondral ossification. Proximal CT chondrocytes enlarge, express hypertrophic markers, including Indian hedgehog (Ihh), apoptose, and are replaced by bone. During later stages of tail regeneration, the distal CT mineralizes without endochondral ossification. The sub-perichondrium of the distal CT expresses Ihh, and the perichondrium directly calcifies without cartilage growth plate formation. The calcified CT perichondrium also contains a population of stem/progenitor cells that forms new cartilage in response to TGF-ß stimulation. Treatment with the Ihh inhibitor cyclopamine inhibited both proximal CT ossification and distal CT calcification. Thus, while the two mineralization events are spatially, temporally, and mechanistically very different, they both involve Ihh. Taken together, these results suggest that Ihh regulates CT mineralization during two distinct stages of lizard tail regeneration.

Wdpcp, a PCP protein required for ciliogenesis, regulates directional cell migration and cell polarity by direct modulation of the actin cytoskeleton.

Planar cell polarity (PCP) regulates cell alignment required for collective cell movement during embryonic development. This requires PCP/PCP effector proteins, some of which also play essential roles in ciliogenesis, highlighting the long-standing question of the role of the cilium in PCP. Wdpcp, a PCP effector, was recently shown to regulate both ciliogenesis and collective cell movement, but the underlying mechanism is unknown. Here we show Wdpcp can regulate PCP by direct modulation of the actin cytoskeleton. These studies were made possible by recovery of a Wdpcp mutant mouse model. Wdpcp-deficient mice exhibit phenotypes reminiscent of Bardet-Biedl/Meckel-Gruber ciliopathy syndromes, including cardiac outflow tract and cochlea defects associated with PCP perturbation. We observed Wdpcp is localized to the transition zone, and in Wdpcp-deficient cells, Sept2, Nphp1, and Mks1 were lost from the transition zone, indicating Wdpcp is required for recruitment of proteins essential for ciliogenesis. Wdpcp is also found in the cytoplasm, where it is localized in the actin cytoskeleton and in focal adhesions. Wdpcp interacts with Sept2 and is colocalized with Sept2 in actin filaments, but in Wdpcp-deficient cells, Sept2 was lost from the actin cytoskeleton, suggesting Wdpcp is required for Sept2 recruitment to actin filaments. Significantly, organization of the actin filaments and focal contacts were markedly changed in Wdpcp-deficient cells. This was associated with decreased membrane ruffling, failure to establish cell polarity, and loss of directional cell migration. These results suggest the PCP defects in Wdpcp mutants are not caused by loss of cilia, but by direct disruption of the actin cytoskeleton. Consistent with this, Wdpcp mutant cochlea has normal kinocilia and yet exhibits PCP defects. Together, these findings provide the first evidence, to our knowledge, that a PCP component required for ciliogenesis can directly modulate the actin cytoskeleton to regulate cell polarity and directional cell migration.

Endothelial and cancer cells interact with mesenchymal stem cells via both microparticles and secreted factors.

Tightly associated with blood vessels in their perivascular niche, human mesenchymal stem cells (MSCs) closely interact with endothelial cells (ECs). MSCs also home to tumours and interact with cancer cells (CCs). Microparticles (MPs) are cell-derived vesicles released into the extracellular environment along with secreted factors. MPs are capable of intercellular signalling and, as biomolecular shuttles, transfer proteins and RNA from one cell to another. Here, we characterize interactions among ECs, CCs and MSCs via MPs and secreted factors in vitro. MPs and non-MP secreted factors (Sup) were isolated from serum-free medium conditioned by human microvascular ECs (HMEC-1) or by the CC line HT1080. Fluorescently labelled MPs were prepared from cells treated with membrane dyes, and cytosolic GFP-containing MPs were isolated from cells transduced with CMV-GFP lentivirus. MSCs were treated with MPs, Sup, or vehicle controls, and analysed for MP uptake, proliferation, migration, activation of intracellular signalling pathways and cytokine release. Fluorescently labelled MPs fused with MSCs, transferring the fluorescent dyes to the MSC surface. GFP was transferred to and retained in MSCs incubated with GFP-MPs, but not free GFP. Thus, only MP-associated cellular proteins were taken up and retained by MSCs, suggesting that MP biomolecules, but not secreted factors, are shuttled to MSCs. MP and Sup treatment significantly increased MSC proliferation, migration, and MMP-1, MMP-3, CCL-2/MCP-1 and IL-6 secretion compared with vehicle controls. MSCs treated with Sup and MPs also exhibited activated NF-κB signalling. Taken together, these results suggest that MPs act to regulate MSC functions through several mechanisms.

Stem cell-based microphysiological osteochondral system to model tissue response to interleukin-1ß.

Osteoarthritis (OA) is a chronic degenerative disease of the articular joint that involves both bone and cartilage degenerative changes. An engineered osteochondral tissue within physiological conditions will be of significant utility in understanding the pathogenesis of OA and testing the efficacy of potential disease-modifying OA drugs (DMOADs). In this study, a multichamber bioreactor was fabricated and fitted into a microfluidic base. When the osteochondral construct is inserted, two chambers are formed on either side of the construct (top, chondral; bottom, osseous) that is supplied by different medium streams. These medium conduits are critical to create tissue-specific microenvironments in which chondral and osseous tissues will develop and mature. Human bone marrow stem cell (hBMSCs)-derived constructs were fabricated in situ and cultured within the bioreactor and induced to undergo spatially defined chondrogenic and osteogenic differentiation for 4 weeks in tissue-specific media. We observed tissue specific gene expression and matrix production as well as a basophilic interface suggesting a developing tidemark. Introduction of interleukin-1ß (IL-1ß) to either the chondral or osseous medium stream induced stronger degradative responses locally as well as in the opposing tissue type. For example, IL-1ß treatment of the osseous compartment resulted in a strong catabolic response in the chondral layer as indicated by increased matrix metalloproteinase (MMP) expression and activity, and tissue-specific gene expression. This induction was greater than that seen with IL-1ß application to the chondral component directly, indicative of active biochemical communication between the two tissue layers and supporting the osteochondral nature of OA. The microtissue culture system developed here offers novel capabilities for investigating the physiology of osteochondral tissue and pathogenic mechanisms of OA and serving as a high-throughput platform to test potential DMOADS.

Single-cell analysis of lizard blastema fibroblasts reveals phagocyte-dependent activation of Hedgehog-responsive chondrogenesis.

Lizards cannot naturally regenerate limbs but are the closest known relatives of mammals capable of epimorphic tail regrowth. However, the mechanisms regulating lizard blastema formation and chondrogenesis remain unclear. Here, single-cell RNA sequencing analysis of regenerating lizard tails identifies fibroblast and phagocyte populations linked to cartilage formation. Pseudotime trajectory analyses suggest spp1+-activated fibroblasts as blastema cell sources, with subsets exhibiting sulf1 expression and chondrogenic potential. Tail blastema, but not limb, fibroblasts express sulf1 and form cartilage under Hedgehog signaling regulation. Depletion of phagocytes inhibits blastema formation, but treatment with pericytic phagocyte-conditioned media rescues blastema chondrogenesis and cartilage formation in amputated limbs. The results indicate a hierarchy of phagocyte-induced fibroblast gene activations during lizard blastema formation, culminating in sulf1+ pro-chondrogenic populations singularly responsive to Hedgehog signaling. These properties distinguish lizard blastema cells from homeostatic and injury-stimulated fibroblasts and indicate potential actionable targets for inducing regeneration in other species, including humans.

Endothelial cell microparticles act as centers of matrix metalloproteinsase-2 (MMP-2) activation and vascular matrix remodeling.

Endothelial cell (EC)-derived microparticles (MPs) are small membrane vesicles associated with various vascular pathologies. Here, we investigated the role of MPs in matrix remodeling by analyzing their interactions with the extracellular matrix. MPs were shown to bind preferentially to surfaces coated with matrix molecules, and MPs bound fibronectin via integrin α(V) . MPs isolated from EC-conditioned medium (Sup) were significantly enriched for matrix-altering proteases, including matrix metalloproteinases (MMPs). MPs lacked the MMP inhibitors TIMP-1 and TIMP-2 found in the Sup and, while Sup strongly inhibited MMP activities but MPs did not. In fact, MPs were shown to bind and activate both endogenous and exogenous proMMP-2. Taken together, these results indicate that MPs interact with extracellular matrices, where they localize and activate MMP-2 to modify the surrounding matrix molecules. These findings provide insights into the cellular mechanisms of vascular matrix remodeling and identify new targets of vascular pathologies.

Lizard Blastema Organoid Model Recapitulates Regenerated Tail Chondrogenesis.

(1) Background: Lizard tail regeneration provides a unique model of blastema-based tissue regeneration for large-scale appendage replacement in amniotes. Green anole lizard (Anolis carolinensis) blastemas contain fibroblastic connective tissue cells (FCTCs), which respond to hedgehog signaling to create cartilage in vivo. However, an in vitro model of the blastema has not previously been achieved in culture. (2) Methods: By testing two adapted tissue dissociation protocols and two optimized media formulations, lizard tail FCTCs were pelleted in vitro and grown in a micromass blastema organoid culture. Pellets were analyzed by histology and in situ hybridization for FCTC and cartilage markers alongside staged original and regenerating lizard tails. (3) Results: Using an optimized serum-free media and a trypsin- and collagenase II-based dissociation protocol, micromass blastema organoids were formed. Organoid cultures expressed FCTC marker CDH11 and produced cartilage in response to hedgehog signaling in vitro, mimicking in vivo blastema and tail regeneration. (4) Conclusions: Lizard tail blastema regeneration can be modeled in vitro using micromass organoid culture, recapitulating in vivo FCTC marker expression patterns and chondrogenic potential.

Differentiation and regeneration potential of mesenchymal progenitor cells derived from traumatized muscle tissue.

Mesenchymal stem cell (MSC) therapy is a promising approach to promote tissue regeneration by either differentiating the MSCs into the desired cell type or by using their trophic functions to promote endogenous tissue repair. These strategies of regenerative medicine are limited by the availability of MSCs at the point of clinical care. Our laboratory has recently identified multipotent mesenchymal progenitor cells (MPCs) in traumatically injured muscle tissue, and the objective of this study was to compare these cells to a typical population of bone marrow derived MSCs. Our hypothesis was that the MPCs exhibit multilineage differentiation and expression of trophic properties that make functionally them equivalent to bone marrow derived MSCs for tissue regeneration therapies. Quantitative evaluation of their proliferation, metabolic activity, expression of characteristic cell-surface markers and baseline gene expression profile demonstrate substantial similarity between the two cell types. The MPCs were capable of differentiation into osteoblasts, adipocytes and chondrocytes, but they appeared to demonstrate limited lineage commitment compared to the bone marrow derived MSCs. The MPCs also exhibited trophic (i.e. immunoregulatory and pro-angiogenic) properties that were comparable to those of MSCs. These results suggest that the traumatized muscle derived MPCs may not be a direct substitute for bone marrow derived MSCs. However, because of their availability and abundance, particularly following orthopaedic injuries when traumatized muscle is available to harvest autologous cells, MPCs are a promising cell source for regenerative medicine therapies designed to take advantage of their trophic properties.

Mesenchymal stem cells inhibit both endogenous and exogenous MMPs via secreted TIMPs.

Mesenchymal stem cells (MSCs) have been shown to be perivascular, occupying a prime location for regulating vessel stability. Here, we focused on the MSC-contribution of key regulators of the perivascular niche, the matrix metalloproteinases (MMPs) and their inhibitors, the TIMPs. Despite secretion of active forms of MMPs by MSCs, MMP enzyme activity was not detected in MSC-conditioned medium (MSC-CM) due to TIMP-mediated inhibition. By means of bifunctional-crosslinking to probe endogenous MMP:TIMP interactions, we showed MMP-2-inhibition by TIMP-2. MSCs also inhibited high levels of exogenous MMP-2 and MMP-9 through TIMP-2 and TIMP-1, respectively. Furthermore, MSC-CM protected vascular matrix molecules and endothelial cell structures from MMP-induced disruption. MSCs remained matrix-protective when exposed to pro-inflammatory cytokines and hypoxia, countering these stresses with increased TIMP-1 expression and augmented MMP-inhibition. Thus, MSCs are revealed as robust sources of TIMP-mediated MMP-inhibition, capable of protecting the perivascular niche from high levels of MMPs even under pathological conditions.

Introducing dorsoventral patterning in adult regenerating lizard tails with gene-edited embryonic neural stem cells.

Lizards regenerate amputated tails but fail to recapitulate the dorsoventral patterning achieved during embryonic development. Regenerated lizard tails form ependymal tubes (ETs) that, like embryonic tail neural tubes (NTs), induce cartilage differentiation in surrounding cells via sonic hedgehog (Shh) signaling. However, adult ETs lack characteristically roof plate-associated structures and express Shh throughout their circumferences, resulting in the formation of unpatterned cartilage tubes. Both NTs and ETs contain neural stem cells (NSCs), but only embryonic NSC populations differentiate into roof plate identities when protected from endogenous Hedgehog signaling. NSCs were isolated from parthenogenetic lizard embryos, rendered unresponsive to Hedgehog signaling via CRISPR/Cas9 gene knockout of smoothened (Smo), and implanted back into clonally-identical adults to regulate tail regeneration. Here we report that Smo knockout embryonic NSCs oppose cartilage formation when engrafted to adult ETs, representing an important milestone in the creation of regenerated lizard tails with dorsoventrally patterned skeletal tissues.

Single Cell Sequencing Analysis of Lizard Phagocytic Cell Populations and Their Role in Tail Regeneration.

Lizards are the closest relatives of mammals capable of tail regeneration, but the specific determinants of amniote regenerative capabilities are currently unknown. Macrophages are phagocytic immune cells that play a critical role in wound healing and tissue regeneration in a wide range of species. We hypothesize that macrophages regulate the process of lizard tail regeneration, and that comparisons with mammalian cell populations will yield insight into the role phagocytes play in determining an organism's regenerative potential. Single cell RNA sequencing (scRNAseq) was used to profile lizard immune cells and compare with mouse counterparts to contrast cell types between the two species. Treatment with clodronate liposomes effectively inhibited lizard tail stump tissue ablation and subsequent regeneration, and scRNAseq was used to profile changes in lizard immune cell populations resulting from tail amputation as well as identifying specific cell types affected by clodronate treatment. ScRNAseq analysis of lizard bone marrow, peripheral blood, and tissue-resident phagocyte cell populations was used to trace marker progression during macrophage differentiation and activation. These results indicated that lizard macrophages are recruited to tail amputation injuries faster than mouse populations and express high levels of matrix metalloproteinases (MMPs). In turn, treatment with MMP inhibitors inhibited lizard tail regeneration. These results provide single cell sequencing data sets for evaluating and comparing lizard and mammalian immune cell populations, and identifying macrophage populations that are critical regulators of lizard tail regrowth.

Mesenchymal stem cell modification of endothelial matrix regulates their vascular differentiation.

Mesenchymal stem cells (MSCs) respond to a variety of differentiation signal provided by their local environments. A large portion of these signals originate from the extracellular matrix (ECM). At the same time, MSCs secrete various matrix-altering agents, including proteases, that alter ECM-encoded differentiation signals. Here we investigated the interactions between MSC and ECM produced by endothelial cells (EC-matrix), focusing not only on the differentiation signals provided by EC-matrix, but also on MSC-alteration of these signals and the resultant affects on MSC differentiation. MSCs were cultured on EC-matrix modified in one of three distinct ways. First, MSCs cultured on native EC-matrix underwent endothelial cell (EC) differentiation early during the culture period and smooth muscle cell (SMC) differentiation at later time points. Second, MSCs cultured on crosslinked EC-matrix, which is resistant to MSC modification, differentiated towards an EC lineage only. Third, MSCs cultured on EC-matrix pre-modified by MSCs underwent SMC-differentiation only. These MSC-induced matrix alterations were found to deplete the factors responsible for EC-differentiation, yet activate the SMC-differentiation factors. In conclusion, our results demonstrate that the EC-matrix contains factors that support MSC differentiation into both ECs and SMCs, and that these factors are modified by MSC-secreted agents. By analyzing the framework by which EC-matrix regulates differentiation in MSCs, we have uncovered evidence of a feedback system in which MSCs are able to alter the very matrix signals acting upon them.

Human mesenchymal stem cells express vascular cell phenotypes upon interaction with endothelial cell matrix.

Mesenchymal stem cells (MSCs) are thought to occupy a perivascular niche where they are exposed to signals originating from vascular cells. This study focused on the effects of endothelial cell (EC)-derived signals on MSC differentiation toward vascular cell lineages. Upon co-culture with two types of ECs, macrovascular (macro) ECs and microvascular (micro) ECs, the former caused MSCs to increase expression of both EC and smooth muscle cell (SMC) markers, while the latter induced expression of EC markers only. These marker changes in MSCs were linked to the extracellular matrixes secreted by the ECs (EC-matrix) rather than soluble EC-secreted factors. Beyond enhanced marker expression, EC-matrix also induced functional changes in MSCs indicative of development of a genuine vascular cell phenotype. These included enhanced incorporation into vessels and cytoskeletal localization of vascular SMC-specific contractile elements. The bioactivity of EC-matrix was sensitive to EDTA washes and required sulfated glycosaminoglycans. However, neither soluble VEGF nor substrate surfaces coated with fibronectin, collagen type IV, or laminin recreated the effects of EC-matrix on MSC vascular differentiation. In conclusion, these results identified EC-matrix as a critical regulator of vascular cell differentiation of MSCs. Elucidating these MSC-EC-matrix interactions and identifying the specific EC-matrix components involved will shed light on the perivascular signals seen by MSCs in vivo.

Efficient in vivo bone formation by BMP-2 engineered human mesenchymal stem cells encapsulated in a projection stereolithographically fabricated hydrogel scaffold.

BACKGROUND: Stem cell-based bone tissue engineering shows promise for bone repair but faces some challenges, such as insufficient osteogenesis and limited architecture flexibility of the cell-delivery scaffold. METHODS: In this study, we first used lentiviral constructs to transduce ex vivo human bone marrow-derived stem cells with human bone morphogenetic protein-2 (BMP-2) gene (BMP-hBMSCs). We then introduced these cells into a hydrogel scaffold using an advanced visible light-based projection stereolithography (VL-PSL) technology, which is compatible with concomitant cell encapsulation and amenable to computer-aided architectural design, to fabricate scaffolds fitting local physical and structural variations in different bones and defects. RESULTS: The results showed that the BMP-hBMSCs encapsulated within the scaffolds had high viability with sustained BMP-2 gene expression and differentiated toward an osteogenic lineage without the supplement of additional BMP-2 protein. In vivo bone formation efficacy was further assessed using an intramuscular implantation model in severe combined immunodeficiency (SCID) mice. Microcomputed tomography (micro-CT) imaging indicated rapid bone formation by the BMP-hBMSC-laden constructs as early as 14 days post-implantation. Histological examination revealed a mature trabecular bone structure with considerable vascularization. Through tracking of the implanted cells, we also found that BMP-hBMSC were directly involved in the new bone formation. CONCLUSIONS: The robust, self-driven osteogenic capability and computer-designed architecture of the construct developed in this study should have potential applications for customized clinical repair of large bone defects or non-unions.

TISSUE REPAIR AND EPIMORPHIC REGENERATION: AN OVERVIEW.

PURPOSE OF THE REVIEW: This manuscript discusses wound healing as a component of epimorphic regeneration and the role of the immune system in this process. RECENT FINDINGS: Epimorphic regeneration involves formation of a blastema, a mass of undifferentiated cells capable of giving rise to the regenerated tissues. The apical epithelial cap plays an important role in blastemal formation. SUMMARY: True regeneration is rarely observed in mammals. With the exception of transgenic strains, tissue repair in mammals usually leads to non-functional fibrotic tissue formation. In contrast, a number of lower order species including planarians, salamanders, and reptiles, have the ability to overcome the burden of scarring and tissue loss through complex adaptations that allow them to regenerate various anatomic structures through epimorphic regeneration. Blastemal cells have been suggested to originate via various mechanisms including de-differentiation, transdifferentiation, migration of pre-existing adult stem cell niches, and combinations of these.

Cartilage and Muscle Cell Fate and Origins during Lizard Tail Regeneration.

INTRODUCTION: Human cartilage is an avascular tissue with limited capacity for repair. By contrast, certain lizards are capable of musculoskeletal tissue regeneration following tail loss throughout all stages of their lives. This extraordinary ability is the result of a complex process in which a blastema forms and gives rise to the tissues of the regenerate. Blastemal cells have been shown to originate either from dedifferentiated tissues or from existing progenitor cells in various species, but their origin has not been determined in lizards. As reptiles, lizards are the closest relatives to mammals with enhanced regenerative potential, and the origin of blastemal cells has important implications for the regenerative process. Hence, the aim of this study is to determine the cellular origin of regenerated cartilage and muscle tissues in reptiles using the mourning gecko lizard as the regenerative model. METHODS: To trace the fate and differentiation potential of cartilage during tail regeneration, cartilage cells pre-labeled with the fluorescent tracer Dil were injected into lizard tails, and the contribution of cartilage cells to regenerated tail tissues was assessed by histologic examination at 7, 14, and 21 days post-tail amputation. The contribution of muscle cells to regenerated tail tissues was evaluated using muscle creatine kinase promoter-driven Cre recombinase in conjunction with the Cre-responsive green-to-red fluorescence shift construct CreStoplight. 21 days after amputation, tail tissues were analyzed by histology for red fluorescent protein (RFP)-positive cells. RESULTS: At 7 days post-amputation, Dil-labeled cartilage cells localized to the subapical space contributing to the blastema. At 14 and 21 days post-amputation, Dil-labeled cells remained in the subapical space and colocalized with Collagen type II (Col2) staining in the cartilage tube and myosin heavy chain (MHC) staining in regenerated muscle. Lineage tracing of myocytes showed colocalization of RFP with Col2 and MHC in differentiated tissues at 21 days post-amputation. CONCLUSION: This study demonstrates that differentiated cartilage cells contribute to both regenerated muscle and cartilage tissues following tail loss, and in turn, differentiated muscle cells contribute to both tissue types as well. These findings suggest that dedifferentiation and/or transdifferentiation are at least partially responsible for the regenerative outcome in the mourning gecko.

Projection Stereolithographic Fabrication of BMP-2 Gene-activated Matrix for Bone Tissue Engineering.

Currently, sustained in vivo delivery of active bone morphogenetic protein-2 (BMP-2) protein to responsive target cells, such as bone marrow-derived mesenchymal stem cells (BMSCs), remains challenging. Ex vivo gene transfer method, while efficient, requires additional operation for cell culture and therefore, is not compatible with point-of-care treatment. In this study, two lentiviral gene constructs - (1) Lv-BMP/GFP, containing human BMP-2 and green fluorescent protein (GFP) gene (BMP group); or (2) Lv-GFP, containing GFP gene (GFP group) - were incorporated with human BMSCs into a solution of photocrosslinkable gelatin, which was then subjected to visible light-based projection stereolithographic printing to form a scaffold with desired architectures. Upon in vitro culture, compared to the GFP group, cells from BMP group showed >1,000-fold higher BMP-2 release, and the majority of them stained intensely for alkaline phosphatase activity. Real-time RT-PCR also showed dramatically increased expression of osteogenesis marker genes only in the BMP group. 3.5 months post-implantation into SCID mice, the micro-computed tomography imaging showed detectable mineralized areas only in the BMP group, which was restricted within the scaffolds. Alizarin red staining and immunohistochemistry of GFP and osteocalcin further indicated that the grafted hBMSCs, not host cells, contributed primarily to the newly formed bone.