TFEB/3 Govern Repair Schwann Cell Generation and Function Following Peripheral Nerve Injury.

TFEB and TFE3 (TFEB/3), key regulators of lysosomal biogenesis and autophagy, play diverse roles depending on cell type. This study highlights a hitherto unrecognized role of TFEB/3 crucial for peripheral nerve repair. Specifically, they promote the generation of progenitor-like repair Schwann cells after axonal injury. In Schwann cell-specific TFEB/3 double knock-out mice of either sex, the TFEB/3 loss disrupts the transcriptomic reprogramming that is essential for the formation of repair Schwann cells. Consequently, mutant mice fail to populate the injured nerve with repair Schwann cells and exhibit defects in axon regrowth, target reinnervation, and functional recovery. TFEB/3 deficiency inhibits the expression of injury-responsive repair Schwann cell genes, despite the continued expression of c-jun, a previously identified regulator of repair Schwann cell function. TFEB/3 binding motifs are enriched in the enhancer regions of injury-responsive genes, suggesting their role in repair gene activation. Autophagy-dependent myelin breakdown is not impaired despite TFEB/3 deficiency. These findings underscore a unique role of TFEB/3 in adult Schwann cells that is required for proper peripheral nerve regeneration.

Injectable Peptide Hydrogels Loaded with Murine Embryonic Stem Cells Relieve Ischemia In Vivo after Myocardial Infarction.

Myocardial infarction (MI) is a major cause of morbidity and mortality worldwide, especially in aging and metabolically unhealthy populations. A major target of regenerative tissue engineering is the restoration of viable cardiomyocytes to preserve cardiac function and circumvent the progression to heart failure post-MI. Amelioration of ischemia is a crucial component of such restorative strategies. Angiogenic ß-sheet peptides can self-assemble into thixotropic nanofibrous hydrogels. These syringe aspiratable cytocompatible gels were loaded with stem cells and showed excellent cytocompatibility and minimal impact on the storage and loss moduli of hydrogels. Gels with and without cells were delivered into the myocardium of a mouse MI model (LAD ligation). Cardiac function and tissue remodeling were evaluated up to 4 weeks in vivo. Injectable peptide hydrogels synergized with loaded murine embryonic stem cells to demonstrate enhanced survival after intracardiac delivery during the acute phase post-MI, especially at 7 days. This approach shows promise for post-MI treatment and potentially functional cardiac tissue regeneration and warrants large-scale animal testing prior to clinical translation.

Necl-4/Cadm4 recruits Par-3 to the Schwann cell adaxonal membrane.

Interactions between axons and Schwann cells are essential for the acquisition of Schwann cell radial and longitudinal polarity and myelin sheath assembly. In the internode, the largest of these longitudinal domains, axon-Schwann cell interactions are mediated by the Nectin-like (Necl) cell adhesion proteins, also known as SynCAMs or Cadms. In particular, Necl-1/Cadm3 expressed on the axon surface binds to Necl-4/Cadm4 expressed along the adaxonal membrane of myelinating Schwann cells. Necl-4 promotes myelination in vitro and is required for the timely onset of myelination and the fidelity of the organization of the myelin sheath and the internode in vivo. A key question is the identity of the downstream effectors of Necl-4 that mediate its effects. The cytoplasmic terminal region (CTR) of Necl-4 contains a PDZ-domain binding motif. Accordingly, we used the CTR of Necl-4 in an unbiased proteomic screen of PDZ-domain proteins. We identify Par-3, a multi-PDZ domain containing protein of the Par-aPKC polarity complex previously implicated in myelination, as an interacting protein. Necl-4 and Par-3 are colocalized along the inner Schwann cell membrane and coprecipitate from Schwann cell lysates. The CTR of Necl-4 binds to the first PDZ domain of Par-3 thereby recruiting Par-3 to sites of Necl-4/Necl-1 interaction. Knockdown of Necl-4 perturbs Par-3 localization to the inner membrane of Schwann cells in myelinating co-cultures. These findings implicate interactions of Necl-1/Necl-4 in the recruitment of Par-3 to the Schwann cell adaxonal membrane and the establishment of Schwann cell radial polarity.

Nectin-like 4 Complexes with Choline Transporter-like Protein-1 and Regulates Schwann Cell Choline Homeostasis and Lipid Biogenesis in Vitro.

Nectin-like 4 (NECL4, CADM4) is a Schwann cell-specific cell adhesion molecule that promotes axo-glial interactions. In vitro and in vivo studies have shown that NECL4 is necessary for proper peripheral nerve myelination. However, the molecular mechanisms that are regulated by NECL4 and affect peripheral myelination currently remain unclear. We used an in vitro approach to begin identifying some of the mechanisms that could explain NECL4 function. Using mass spectrometry and Western blotting techniques, we have identified choline transporter-like 1 (CTL1) as a putative complexing partner with NECL4. We show that intracellular choline levels are significantly elevated in NECL4-deficient Schwann cells. The analysis of extracellular d9-choline uptake revealed a deficit in the amount of d9-choline found inside NECL4-deficient Schwann cells, suggestive of either reduced transport capabilities or increased metabolization of transported choline. An extensive lipidomic screen of choline derivatives showed that total phosphatidylcholine and phosphatidylinositol (but not diacylglycerol or sphingomyelin) are significantly elevated in NECL4-deficient Schwann cells, particularly specific subspecies of phosphatidylcholine carrying very long polyunsaturated fatty acid chains. Finally, CTL1-deficient Schwann cells are significantly impaired in their ability to myelinate neurites in vitro To our knowledge, this is the first demonstration of a bona fide cell adhesion molecule, NECL4, regulating choline homeostasis and lipid biogenesis. Phosphatidylcholines are major myelin phospholipids, and several phosphorylated phosphatidylinositol species are known to regulate key aspects of peripheral myelination. Furthermore, the biophysical properties imparted to plasma membranes are regulated by fatty acid chain profiles. Therefore, it will be important to translate these in vitro observations to in vivo studies of NECL4 and CTL1-deficient mice.

Self-Assembling Peptides with Insulin-Like Growth Factor Mimicry.

Growth factor (GF) mimicry involves recapitulating the signaling of larger molecules or cells. Although GF mimicry holds considerable promise in tissue engineering and drug design applications, difficulties in targeting the signaling molecule to the site of delivery and dissociation of mimicking peptides from their target receptors continue to limit its clinical application. To address these challenges, we utilized a self-assembling peptide (SAP) platform to generate synthetic insulin-like growth factor (IGF)-signaling, self-assembling GFs. Our peptide hydrogels are biocompatible and bind target IGF receptors in a dose-dependent fashion, activate proangiogenic signaling, and facilitate formation of angiogenic microtubules in vitro. Furthermore, infiltrated hydrogels are stable for weeks to months. We conclude that the enhanced targeting and long-term stability of our SAP/GF mimicry implants may improve the efficacy and safety of future GF mimic therapeutics.

Antiviral fibrils of self-assembled peptides with tunable compositions.

The lasting threat of viral pandemics necessitates the development of tailorable first-response antivirals with specific but adaptive architectures for treatment of novel viral infections. Here, such an antiviral platform has been developed based on a mixture of hetero-peptides self-assembled into functionalized ß-sheets capable of specific multivalent binding to viral protein complexes. One domain of each hetero-peptide is designed to specifically bind to certain viral proteins, while another domain self-assembles into fibrils with epitope binding characteristics determined by the types of peptides and their molar fractions. The self-assembled fibrils maintain enhanced binding to viral protein complexes and retain high resilience to viral mutations. This method is experimentally and computationally tested using short peptides that specifically bind to Spike proteins of SARS-CoV-2. This platform is efficacious, inexpensive, and stable with excellent tolerability.

Induction of pluripotency.

The molecular and phenotypic irreversibility of mammalian cell differentiation was a fundamental principle of developmental biology at least until the 1980s, despite numerous reports dating back to the 1950s of the induction of pluripotency in amphibian cells by nuclear transfer (NT). Landmark reports in the 1980s and 1990s in sheep progressively challenged this dogmatic assumption; firstly, embryonic development of reconstructed embryos comprising whole (donor) blastomeres fused to enucleated oocytes, and famously, the cloning of Dolly from a terminally differentiated cell. Thus, the intrinsic ability of oocyte-derived factors to reverse the differentiated phenotype was confirmed. The concomitant elucidation of methods for human embryonic stem cell isolation and cultivation presented opportunities for therapeutic cell replacement strategies, particularly through NT of patient nuclei to enucleated oocytes for subsequent isolation of patient-specific (autologous), pluripotent cells from the resulting blastocysts. Associated logistical limitations of working with human oocytes, in addition to ethical and moral objections prompted exploration of alternative approaches to generate autologous stem cells for therapy, utilizing the full repertoire of factors characteristic of pluripotency, primarily through cell fusion and use of pluripotent cell extracts. Stunningly, in 2006, Japanese scientists described somatic cell reprogramming through delivery of four key factors (identified through a deductive approach from 24 candidate genes). Although less efficient than previous approaches, much of current stem cell research adopts this focused approach to cell reprogramming and (autologous) cell therapy. This chapter is a quasi-historical commentary of the various aforementioned approaches for the induction of pluripotency in lineage-committed cells, and introduces transcriptional and epigenetic changes occurring during reprogramming.

Comparison of SGLT1, SGLT2, and Dual Inhibitor biological activity in treating Type 2 Diabetes Mellitus.

Diabetes Mellitus Type 2 (T2D) is an emerging health burden in the USand worldwide, impacting approximately 15% of Americans. Current front-line therapeutics for T2D patients include sulfonylureas that act to reduce A1C and/or fasting blood glucose levels, or Metformin that antagonizes the action of glucagon to reduce hepatic glucose production. Next generation glucomodulatory therapeutics target members of the high-affinity glucose transporter Sodium-Glucose-Linked-Transporter (SGLT) family. SGLT1 is primarily expressed in intestinal epithelium, whose inhibition reduces dietary glucose uptake, whilst SGLT2 is highly expressed in kidney - regulating glucose reabsorption. A number of SGLT2 inhibitors are FDA approved whilst SGLT1 and dual SGLT1 & 2 inhibitor are currently in clinical trials. Here, we discuss and compare SGLT2, SGLT1, and dual inhibitors' biochemical mechanism and physiological effects.

Lineage-specific expression of heterochromatin protein 1gamma in post-compaction, in vitro-produced bovine embryos.

Heterochromatin protein 1gamma (HP1gamma) is a highly conserved regulator of euchromatic and heterochromatic gene expression. Mammalian HP1gamma is essential for both successful preimplantation embryo development and maintenance of pluripotency in embryonic stem cells in vitro. Here, we describe HP1gamma protein localisation in matured (MII) bovine oocytes and IVF preimplantation embryos at defined developmental stages. HP1gamma is expressed in post-compaction embryos in a highly lineage-specific pattern. In embryonic stages preceding the maternal to embryonic transition (MET), HP1gamma protein was primarily cytoplasmic, whereas in 8-16-cell embryos (post MET), HP1gamma was primarily nuclear. Lineage-specific patterns of HP1gamma protein localisation become evident from compaction, being restricted to peripheral, extraembryonic cells at the morula and blastocyst stages (Days 7-9). Surprisingly, we detected HP1gamma mRNA in both embryonic and extraembryonic cells in blastocysts by fluorescence in situ hybridisation. In trophectoderm cells, HP1gamma protein was localised in specific patterns at the mitotic and interphase stages of the cell cycle. These results demonstrate lineage- and cell cycle-specific patterns of HP1gamma protein localisation in the post-compaction, preimplantation bovine embryo and raise interesting questions about the role of HP1gamma in early embryo development.

Dynamic changes in localization of Chromobox (Cbx) family members during the maternal to embryonic transition.

The Chromobox domain (Cbx) gene family, consisting of Polycomb and Heterochromatin Protein 1 genes, is involved in transcriptional repression, cell cycle regulation and chromatin remodeling. We report the first study of gene expression and protein localization of the Cbx genes in in vitro produced bovine embryos. All but one gene (Cbx6) were expressed. This was confirmed by immunolocalization for HP1alpha, beta, gamma, and Pc2, 3. HP1beta was found in the nuclei of embryos from the two-cell stage onwards, whereas HP1gamma showed diffuse cytoplasmic/nuclear localization at the two- and eight-cell stages, and predominantly nuclear localization at the four-cell stage and the 16-cell stage onwards. Leptomycin B (LMB), a specific inhibitor of the nuclear export protein CRM-1 (chromosomal regional maintenance-1), was found to increase nuclear localization of HP1gamma at the eight-cell stage, and to prevent progression past this stage of embryogenesis. This indicates that HP1gamma possesses a CRM-1-dependent nuclear export pathway which may represent part of the basis of HP1gamma's ability to shuttle between the nucleus and the cytoplasm in dynamic fashion. HP1alpha was expressed in embryonic nuclei at all stages, but was found to relocalise from euchromatin to heterochromatin during the maternal to embryonic transition (MET). In contrast, Pc2 and Pc3 were evenly distributed between cytoplasm and nucleus until the eight- and sixteen-cell stages or the morula stage, respectively, before relocating preferentially to the cytoplasm. Collectively, the results suggest that dynamic changes of the nuclear-cytoplasmic and subnuclear distribution of members of the Cbx family may be central to the MET.

Lentiviral Transduction of Rat Schwann Cells and Dorsal Root Ganglia Neurons for In Vitro Myelination Studies.

Lentiviral transduction is a gene delivery method that provides numerous advantages over direct transfection and traditional retroviral or adenoviral delivery methods. It facilitates for the transduction of primary cells inherently difficult to transfect, delivers constructs of interest to nondividing as well as dividing cells, and permits the long-term expression of sizable DNA inserts (e.g., <7 kb). The study of peripheral nerve myelination at the molecular level has long benefited from the Schwann cells/dorsal root ganglia (DRG) neurons myelinating co-culture system. As this culture system takes about a month to develop and perform experiments with, lentiviral-delivered constructs can be used to manipulate gene expression in Schwann cells and DRG neurons, primary cells that are otherwise resilient to direct transfection. Here we present our protocol for lentiviral production and purification and subsequent infection of large numbers of Schwann cells and/or DRG neurons for the molecular study of peripheral nerve myelination in vitro.

Design and screening of a glial cell-specific, cell penetrating peptide for therapeutic applications in multiple sclerosis.

Multiple Sclerosis (MS) is an autoimmune, neurodegenerative disease of the central nervous system (CNS) characterized by demyelination through glial cell loss. Current and proposed therapeutic strategies to arrest demyelination and/or promote further remyelination include: (i) modulation of the host immune system; and/or (ii) transplantation of myelinating/stem or progenitor cells to the circulation or sites of injury. However, significant drawbacks are inherent with both approaches. Cell penetrating peptides (CPP) are short amino acid sequences with an intrinsic ability to translocate across plasma membranes, and theoretically represent an attractive vector for delivery of therapeutic peptides or nanoparticles to glia to promote cell survival or remyelination. The CPPs described to date are commonly non-selective in the cell types they transduce, limiting their therapeutic application in vivo. Here, we describe a theoretical framework for design of a novel CPP sequence that selectively transduces human glial cells (excluding non-glial cell types), and conduct preliminary screens of purified, recombinant CPPs with immature and matured human oligodendrocytes and astrocytes, and two non-glial cell types. A candidate peptide, termed TD2.2, consistently transduced glial cells, was significantly more effective at transducing immature oligodendrocytes than matured progeny, and was virtually incapable of transducing two non-glial cell types: (i) human neural cells and (ii) human dermal fibroblasts. Time-lapse confocal microscopy confirms trafficking of TD2.2 (fused to EGFP) to mature oligodendrocytes 3-6 hours after protein application in vitro. We propose selectivity of TD2.2 for glial cells represents a new therapeutic strategy for the treatment of glial-related disease, such as MS.

Temporal Requirements of cMyc Protein for Reprogramming Mouse Fibroblasts.

Exogenous expression of Oct4, Sox2, Klf4, and cMyc forces mammalian somatic cells to adopt molecular and phenotypic characteristics of embryonic stem cells, commencing with the required suppression of lineage-associated genes (e.g., Thy1 in mouse). Although omitting cMyc from the reprogramming cocktail minimizes risks of uncontrolled proliferation, its exclusion results in fold reductions in reprogramming efficiency. Thus, the feasibility of substituting cMyc transgene with (non-integrative) recombinant "pTAT-mcMyc" protein delivery was assessed, without compromising reprogramming efficiency or the pluripotent phenotype. Purification and delivery of semisoluble/particulate pTAT-mcMyc maintained Oct4-GFP(+) colony formation (i.e., reprogramming efficiency) whilst supporting pluripotency by various criteria. Differential repression of Thy1 by pTAT-mcMyc ± Oct4, Sox2, and Klf4 (OSK) suggested differential (and non-additive) mechanisms of repression. Extending these findings, attempts to enhance reprogramming efficiency through a staggered approach (prerepression of Thy1) failed to improve reprogramming efficiency. We consider protein delivery a useful tool to decipher temporal/molecular events characterizing somatic cell reprogramming.

Generation of clinically relevant "induced pluripotent stem" (iPS) cells.

Proviral expression of early development genes Oct4 and Sox2, in concert with cMyc and Klf4 or Nanog and Lin28, can induce differentiated cells to adopt morphological and functional characteristics of pluripotency indistinguishable from embryonic stem cells. Termed induced pluripotent stem (iPS) cells, in mice the pluripotency of these cells was confirmed by altered gene/surface antigen expression, remodeling of the epigenome, ability to contribute to embryonic lineages following blastocyst injection and commitment to all three germ layers in teratomas and liveborn chimeras. Importantly, in vitro directed differentiation of iPS cells yield cells capable of treating mouse models of humanized disease. Despite these impressive results, iPS cell conversion is frustratingly inefficient. Also, the unpredictable and random mutagenesis imposed on the host cell genome, inherent with integrative viral methodologies, continues to hamper use of these cells in a therapeutic setting. This has initiated exploration of non-integrating strategies for generating iPS cells. Here, we review mechanisms that drive conversion of somatic cells to iPS cells and the strategies adopted to circumvent integrative viral strategies. Finally, we discuss practical, ethical and legal considerations that require addressing before iPS cells can realize their potential as patient-specific cells for treatment of degenerative disease.

Adult cardiac-resident MSC-like stem cells with a proepicardial origin.

Colony-forming units - fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.

Reprogramming of somatic cells after fusion with induced pluripotent stem cells and nuclear transfer embryonic stem cells.

In this study we examine whether a somatic cell, once returned to a pluripotent state, gains the ability to reprogram other somatic cells. We reprogrammed mouse embryonic fibroblasts by viral induction of oct4, sox2, c-myc, and klf-4 genes. Upon fusion of the resulting iPS cells with somatic cells harboring an Oct4-GFP transgene we observed, GFP expression along with activation of Oct4 from the somatic genome, expression of key pluripotency genes, and positive immunostaining for Oct4, SSEA-1, and alkaline phosphatase. The iPS-somatic hybrids had the ability to differentiate into cell types indicative of the three germ layers and were able to localize to the inner cell mass of aggregated embryos. Furthermore, ntES cells were used as fusion partners to generate hybrids, which were also confirmed to be reprogrammed to a pluripotent state. These results demonstrate that once a somatic cell nucleus is reprogrammed, it acquires the capacity and potency to reprogram other somatic cells by cell fusion and shares this functional property with normal embryonic stem (ES) cells.

Somatic cell nuclear transfer: pros and cons.

Even though the technique of mammalian SCNT is just over a decade old it has already resulted in numerous significant advances. Despite the recent advances in the reprogramming field, SCNT remains the bench-mark for the generation of both genetically unmodified autologous pluripotent stem cells for transplantation and for the production of cloned animals. In this review we will discuss the pros and cons of SCNT, drawing comparisons with other reprogramming methods.

Dynamic changes in the localization of five members of the methyl binding domain (MBD) gene family during murine and bovine preimplantation embryo development.

There are five methyl binding domain (MBD) proteins characterized by a methyl CpG-binding domain. Four MBD proteins (MeCP2 and MBDs 1-3) are linked to transcriptional repression and one (MBD4), to DNA repair. During preimplantation development, the embryo undergoes global demethylation following fertilization and selective remethylation following the maternal to zygotic transition (MZT). This study characterized changes in MBD mRNA expression and protein localization during both murine and bovine preimplantation development. These species were selected because they undergo MZT at different developmental stages. Gene expression profiling during preimplantation development detected the presence of all MBDs examined, although stage and species-specific differences were observed. MBD2 was not expressed in murine or bovine oocytes and MeCP2 was not detected in murine blastocysts, subcellular protein localization was found to vary at time points critical in development. Most MBDs showed species-specificity in localization patterns and differences were found between individual MBDs. MBD1 localization is consistent with a novel role during MZT for both species. MBD3, known to play a crucial role in murine embryogenesis, was highly localized to the nucleus before and after, but not during the MZT in the bovine. MBD2, MBD4, and MeCP2 show varying patterns of localization which indicate possible roles in the early cleavage stages and in inner cell mass differentiation. Further experiments are currently underway to define discreet functional roles for specific MBDs during bovine preimplantation embryogenesis.