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.

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.

Acceleration of chondrogenic differentiation of human mesenchymal stem cells by sustained growth factor release in 3D graphene oxide incorporated hydrogels.

Damaged articular cartilage has limited self-healing capabilities, leading to degeneration that affects millions of people. Although cartilage tissue engineering is considered a promising approach for treatment, robust and long-term chondrogenesis within a 3-dimensional (3D) scaffold remains a major challenge for complete regeneration. Most current approaches involve incorporation of transforming growth factor-ß (TGF-ß) into the scaffold, but have limited utility owing to the short functional half-life and/or rapid clearance of TGF-ß. In this study, we have tested the incorporation of graphene oxide nanosheets (GO) within a photopolymerizable poly-D, l-lactic acid/polyethylene glycol (PDLLA) hydrogel, for its applicability in sustained release of the chondroinductive growth factor TGF-ß3. We found that with GO incorporation, the hydrogel scaffold (GO/PDLLA) exhibited enhanced initial mechanical strength, i.e., increased compressive modulus, and supported long-term, sustained release of TGF-ß3 for up to 4 weeks. In addition, human bone marrow-derived mesenchymal stem cells (hBMSCs) seeded within TGF-ß3 loaded GO/PDLLA hydrogels displayed high cell viability and improved chondrogenesis in a TGF-ß3 concentration-dependent manner. hBMSCs cultured in GO/PDLLA also demonstrated significantly higher chondrogenic gene expression, including aggrecan, collagen type II and SOX9, and cartilage matrix production when compared to cultures maintained in GO-free scaffolds containing equivalent amounts of TGF-ß3. Upon subcutaneous implantation in vivo, hBMSC-seeded TGF-ß3-GO/PDLLA hydrogel constructs displayed considerably greater cartilage matrix than their TGF-ß3/PDLLA counterparts without GO. Taken together, these findings support the potential application of GO in optimizing TGF-ß3 induced hBMSC chondrogenesis for cartilage tissue engineering. STATEMENT OF SIGNIFICANCE: In this work, we have developed a graphene oxide (GO) incorporated, photocrosslinked PDLLA hybrid hydrogel for localized delivery and sustained release of loaded TGF-ß3 to seeded cells. The incorporation of GO in PDLLA hydrogel suppressed the burst release of TGF-ß3, and significantly prolonged the retention time of the TGF-ß3 initially loaded in the hydrogel. Additionally, the GO improved the initial compressive strength of the hydrogel. Both in vitro analyses and in vivo implantation results showed that the GO/PDLLA constructs seeded with human mesenchymal stem cells (hMSCs) showed significantly higher cartilage formation, compared to GO-free scaffolds containing equivalent amount of TGF-ß3. Findings from this work suggest the potential application of the GO-TGF/PDLLA hydrogel as a functional scaffold for hMSC-based cartilage tissue engineering.

Enhancing chondrogenesis and mechanical strength retention in physiologically relevant hydrogels with incorporation of hyaluronic acid and direct loading of TGF-ß.

Cell-loaded hydrogels are frequently applied in cartilage tissue engineering for their biocompatibility, ease of application, and ability to conform to various defect sites. As a bioactive adjunct to the biomaterial, transforming growth factor beta (TGF-ß) has been shown to be essential for cell differentiation into a chondrocyte phenotype and maintenance thereof, but the low amounts of endogenous TGF-ß in the in vivo joint microenvironment necessitate a mechanism for controlled delivery and release of this growth factor. In this study, TGF-ß3 was directly loaded with human bone marrow-derived mesenchymal stem cells (MSCs) into poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG) hydrogel, or PDLLA-PEG with the addition of hyaluronic acid (PDLLA/HA), and cultured in vitro. We hypothesize that the inclusion of HA within PDLLA-PEG would result in a controlled release of the loaded TGF-ß3 and lead to a robust cartilage formation without the use of TGF-ß3 in the culture medium. ELISA analysis showed that TGF-ß3 release was effectively slowed by HA incorporation, and retention of TGF-ß3 in the PDLLA/HA scaffold was detected by immunohistochemistry for up to 3 weeks. By means of both in vitro culture and in vivo implantation, we found that sulfated glycosaminoglycan production was higher in PDLLA/HA groups with homogenous distribution throughout the scaffold than PDLLA groups. Finally, with an optimal loading of TGF-ß3 at 10 µg/mL, as determined by RT-PCR and glycosaminoglycan production, an almost twofold increase in Young's modulus of the construct was seen over a 4-week period compared to TGF-ß3 delivery in the culture medium. Taken together, our results indicate that the direct loading of TGF-ß3 and stem cells in PDLLA/HA has the potential to be a one-step point-of-care treatment for cartilage injury. STATEMENT OF SIGNIFICANCE: Stem cell-seeded hydrogels are commonly used in cell-based cartilage tissue engineering, but they generally fail to possess physiologically relevant mechanical properties suitable for loading. Moreover, degradation of the hydrogel in vivo with time further decreases mechanical suitability of the hydrogel due in part to the lack of TGF-ß3 signaling. In this study, we demonstrated that incorporation of hyaluronic acid (HA) into a physiologically stiff PDLLA-PEG hydrogel allowed for slow release of one-time preloaded TGF-ß3, and when loaded with adult mesenchymal stem cells and cultured in vitro, it resulted in higher chondrogenic gene expression and constructs of significantly higher mechanical strength than constructs cultured in conventional TGF-ß3-supplemented medium. Similar effects were also observed in constructs implanted in vivo. Our results indicate that direct loading of TGF-ß3 combined with HA in the physiologically stiff PDLLA-PEG hydrogel has the potential to be used for one-step point-of-care treatment of cartilage injury.

Conduits harnessing spatially controlled cell-secreted neurotrophic factors improve peripheral nerve regeneration.

An essential structure in nerve regeneration within engineered conduits is the "nerve bridge" initiated by centrally migrating Schwann cells in response to chemokine gradients. Introducing exogenous cells secreting neurotrophic factors aims to augment this repair process, but conventional cell-seeding methods fail to produce a directional chemokine gradient. We report a versatile method to encapsulate cells within conduit walls, allowing for reproducible control of spatial distribution along the conduit. Conduits with stem cells encapsulated within the central third possessed markedly different cell distribution and retention over 6 weeks in vivo, compared to standard cell lumen injection. Such a construct promoted Schwann cell migration centrally, and at 16 weeks rats presented with significantly enhanced function and axonal myelination. The method of utilizing a spatially restricted cell secretome departs from traditional homogeneous cell loading, and presents new approaches for studying and maximizing the potential of cell application in peripheral nerve repair.

Neurotrophically Induced Mesenchymal Progenitor Cells Derived from Induced Pluripotent Stem Cells Enhance Neuritogenesis via Neurotrophin and Cytokine Production.

Adult tissue-derived mesenchymal stem cells (MSCs) are known to produce a number of bioactive factors, including neurotrophic growth factors, capable of supporting and improving nerve regeneration. However, with a finite culture expansion capacity, MSCs are inherently limited in their lifespan and use. We examined here the potential utility of an alternative, mesenchymal-like cell source, derived from induced pluripotent stem cells, termed induced mesenchymal progenitor cells (MiMPCs). We found that several genes were upregulated and proteins were produced in MiMPCs that matched those previously reported for MSCs. Like MSCs, the MiMPCs secreted various neurotrophic and neuroprotective factors, including brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), osteopontin, and osteonectin, and promoted neurite outgrowth in chick embryonic dorsal root ganglia (DRG) cultures compared with control cultures. Cotreatment with a pharmacological Trk-receptor inhibitor did not result in significant decrease in MiMPC-induced neurite outgrowth, which was however inhibited upon Jak/STAT3 blockade. These findings suggest that the MiMPC induction of DRG neurite outgrowth is unlikely to be solely dependent on BDNF, but instead Jak/STAT3 activation by IL-6 and/or LIF is likely to be critical neurotrophic signaling pathways of the MiMPC secretome. Taken together, these findings suggest MiMPCs as a renewable, candidate source of therapeutic cells and a potential alternative to MSCs for peripheral nerve repair, in view of their ability to promote nerve growth by producing many of the same growth factors and cytokines as Schwann cells and signaling through critical neurotrophic pathways. Stem Cells Translational Medicine 2018;7:45-58.

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.

Chondroinductive factor-free chondrogenic differentiation of human mesenchymal stem cells in graphene oxide-incorporated hydrogels.

Graphene-based nanomaterials have been applied as biomaterials to enhance stem cell adhesion, growth and differentiation by serving as nanocarriers for growth factors or other small molecules. However, the direct effect of graphene oxide (GO) itself on stem cells, in the absence of exogenous differentiation inductive factors, has not been tested. In this study, we loaded GO nanosheets and human bone marrow-derived mesenchymal stem cells (hBMSC) into a photopolymerizable poly-d,l-lactic acid/polyethylene glycol (PDLLA) hydrogel, a robust chondrosupportive scaffold recently developed in our laboratory, and assessed hBMSC differentiation along the chondrogenic lineage without supplemental chondroinductive factors. We first examined the effect of GO incorporation on the mechanical properties of constructs, and observed that the GO-containing constructs (GO/PDLLA) exhibited enhanced compressive modulus in a GO concentration dependent manner. hBMSCs cultured in GO/PDLLA maintained high cell viability (>95%), indicating minimal cytotoxicity of GO. Importantly, compared to those encapsulated in PDLLA hydrogel, hBMSCs within GO/PDLLA showed significantly higher level of gene expression of the cartilage matrix genes, aggrecan and collagen type II, and produced more cartilage matrix. In addition, the pro-chondrogenesis effect of GO increased with increasing GO concentration. Immunohistochemical results suggested that GO-enhanced hBMSC chondrogenesis was correlated with enriched sequestration of insulin, a necessary supplement known to have pro-chondrogenesis effects on hBMSC. Taken together, these findings demonstrate the utility of using GO to improve the mechanical properties and chondrogenic differentiation state of MSC-laden, engineered hydrogel constructs, without the use of exogenous growth factors, thus representing a potentially promising, biologics-free approach for cartilage tissue engineering.

Chondrogenesis of human bone marrow mesenchymal stem cells in 3-dimensional, photocrosslinked hydrogel constructs: Effect of cell seeding density and material stiffness.

Three-dimensional hydrogel constructs incorporated with live stem cells that support chondrogenic differentiation and maintenance offer a promising regenerative route towards addressing the limited self-repair capabilities of articular cartilage. In particular, hydrogel scaffolds that augment chondrogenesis and recapitulate the native physical properties of cartilage, such as compressive strength, can potentially be applied in point-of-care procedures. We report here the synthesis of two new materials, [poly-l-lactic acid/polyethylene glycol/poly-l-lactic acid] (PLLA-PEG 1000) and [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid] (PDLLA-PEG 1000), that are biodegradable, biocompatible (>80% viability post fabrication), and possess high, physiologically relevant mechanical strength (∼1500 to 1800kPa). This study examined the effects of physiologically relevant cell densities (4, 8, 20, and 50×106/mL) and hydrogel stiffnesses (∼150kPa to∼1500kPa Young's moduli) on chondrogenesis of human bone marrow stem cells incorporated in hydrogel constructs fabricated with these materials and a previously characterized PDLLA-PEG 4000. Results showed that 20×106cells/mL, under a static culture condition, was the most efficient cell seeding density for extracellular matrix (ECM) production on the basis of hydroxyproline and glycosaminoglycan content. Interestingly, material stiffness did not significantly affect chondrogenesis, but rather material concentration was correlated to chondrogenesis with increasing levels at lower concentrations based on ECM production, chondrogenic gene expression, and histological analysis. These findings establish optimal cell densities for chondrogenesis within three-dimensional cell-incorporated hydrogels, inform hydrogel material development for cartilage tissue engineering, and demonstrate the efficacy and potential utility of PDLLA-PEG 1000 for point-of-care treatment of cartilage defects. STATEMENT OF SIGNIFICANCE: Engineering cartilage with physiologically relevant mechanical properties for point-of-care applications represents a major challenge in orthopedics, given the generally low mechanical strengths of traditional hydrogels used in cartilage tissue engineering. In this study, we characterized a new material that possesses high mechanical strength similar to native cartilage, and determined the optimal cell density and scaffold stiffness to achieve the most efficient chondrogenic response from seeded human bone marrow stem cells. Results show robust chondrogenesis and strongly suggest the potential of this material to be applied clinically for point-of-care repair of cartilage defects.

Projection Stereolithographic Fabrication of Human Adipose Stem Cell-Incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering.

The poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL) offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light-based PSL (VL-PSL) system to encapsulate human adipose-derived stem cells (hASCs) into a biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG)]/hyaluronic acid (HA) matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84%) and were uniformly distributed throughout the constructs, which possessed high mechanical properties with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in control or TGF-ß3-containing chondrogenic medium for up to 28 days. In chondrogenic medium-treated group (TGF-ß3 group), hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 × 10(5) fold increases, respectively compared to levels at day 0 in non-chondrogenic medium. The TGF-ß3 group also produced a collagen type II and glycosaminoglycan-rich extracellular matrix, detected by immunohistochemistry, Alcian blue staining, and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group), cell viability decreased with time (65% at 28 days) and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-ß3 group remained high at 240 kPa. Thus, the PSL and PDLLA-PEG/HA-based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage resurfacing.