Isolation of ready-made rat microvessels and its applications in effective in vivo vascularization and in angiogenic studies in vitro.

Despite recent advances in the differentiation of human pluripotent stem cells into multiple cell types for application in replacement therapies, tissue vascularization remains a bottleneck for regenerative medicine. Fragments of primary microvessels (MVs) harvested from adipose tissue retain endothelialized lumens and perivascular cell coverage. We have used these MVs to support the survival and engraftment of transplanted human pluripotent stem cell-derived cardiomyocytes, pancreatic progenitors or primary human islets. MVs connect with host vessels, perfuse with blood and form a hierarchal vascular network in vivo after subcutaneous or intracardiac transplantation. MVs also display the ability to remodel and form stable vascular networks with long-term retention (>3.5 months). MVs can be cultured in 3D hydrogels in vitro, where they retain vessel shape and undergo angiogenic sprouting without the need for exogenous growth factor supplementation. Therefore, MVs offer a robust vascularization strategy for regenerative medicine approaches and a platform for angiogenic studies and drug testing in vitro. Here we describe in detail the protocol for: (1) the isolation of MVs from rat epididymal fat by limited collagenase digestion, followed by size-selective sieving; (2) the incorporation of MVs into 3D collagen hydrogels; (3) the in vitro culture of MVs in 3D gels for angiogenic studies; and (4) the in vivo transplantation of 3D hydrogels containing MVs into the mouse subcutis. The isolation procedure does not require highly specific equipment and can be performed in ~3 h by researchers with experience in rodent handling and cell culture.

GP2-enriched pancreatic progenitors give rise to functional beta cells in vivo and eliminate the risk of teratoma formation.

Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) can be differentiated into beta-like cells in vitro and in vivo and therefore have therapeutic potential for type 1 diabetes (T1D) treatment. However, the purity of PPs varies across different hPSC lines, differentiation protocols, and laboratories. The uncommitted cells may give rise to non-pancreatic endodermal, mesodermal, or ectodermal derivatives in vivo, hampering the safety of hPSC-derived PPs for clinical applications and their differentiation efficiency in research settings. Recently, proteomics and transcriptomics analyses identified glycoprotein 2 (GP2) as a PP-specific cell surface marker. The GP2-enriched PPs generate higher percentages of beta-like cells in vitro, but their potential in vivo remains to be elucidated. Here, we demonstrate that the GP2-enriched-PPs give rise to all pancreatic cells in vivo, including functional beta-like cells. Remarkably, GP2 enrichment eliminates the risk of teratomas, which establishes GP2 sorting as an effective method for PP purification and safe pancreatic differentiation.

Cell-based therapies for vascular regeneration: Past, present and future.

Tissue vascularization remains one of the outstanding challenges in regenerative medicine. Beyond its role in circulating oxygen and nutrients, the vasculature is critical for organ development, function and homeostasis. Importantly, effective vascular regeneration is key in generating large 3D tissues for regenerative medicine applications to enable the survival of cells post-transplantation, organ growth, and integration into the host system. Therefore, the absence of clinically applicable means of (re)generating vessels is one of the main obstacles in cell replacement therapy. In this review, we highlight cell-based vascularization strategies which demonstrate clinical potential, discuss their strengths and limitations and highlight the main obstacles hindering cell-based therapeutic vascularization.

Microvessels support engraftment and functionality of human islets and hESC-derived pancreatic progenitors in diabetes models.

Islet transplantation is a promising treatment for type 1 diabetes (T1D), yet the low donor pool, poor islet engraftment, and life-long immunosuppression prevent it from becoming the standard of care. Human embryonic stem cell (hESC)-derived pancreatic cells could eliminate donor shortages, but interventions to improve graft survival are needed. Here, we enhanced subcutaneous engraftment by employing a unique vascularization strategy based on ready-made microvessels (MVs) isolated from the adipose tissue. This resulted in improved cell survival and effective glucose response of both human islets and hESC-derived pancreatic cells, which ameliorated preexisting diabetes in three mouse models of T1D.

Identification of Sec23ip, Part of 14-3-3γ Protein Network, as a Regulator of Acute Steroidogenesis in MA-10 Leydig Cells.

Testosterone production occurs in the Leydig cells of the testes and is essential for virilization, development, reproduction, and quality of life. Although the steroidogenic proteins involved in cholesterol conversion to testosterone (T) are well characterized, the causes of reduced T during fetal, neonatal, and adult life remain uncertain. It is well established that normal cellular function is achieved through fine-tuning of multiple rather than single protein networks. Our objective was to use mass spectrometry (MS)-based proteomics to identify which cellular pathways, other than the steroidogenic machinery, influence testosterone production in MA-10 mouse tumor Leydig cells. The 14-3-3 family of scaffolds mediate protein-protein interactions facilitating the crosstalk between protein networks. We previously showed that in MA-10 cells, 14-3-3γ is a critical regulator of steroidogenesis. Therefore, identifying proteins that interact with 14-3-3γ during steroidogenesis could provide clues into the other networks involved. Using liquid chromatography (LC)-MS, we identified 688 proteins that interact with 14-3-3γ and thus potentially impact MA-10 cell steroidogenesis. The identified proteins belong to multiple protein networks, including endoplasmic reticulum-Golgi cargo sorting and vesicle biogenesis, micro ribonucleic acid-induced gene silencing, inflammation, and vesicle trafficking, to name a few. We found that silencing one of the candidates, Sec23ip, a protein known to be involved in vesicle trafficking, resulted in decreased steroidogenesis. We further showed that in Sec23ip-silenced MA-10 cells, cholesterol mobilization from the cytoplasmic membrane to mitochondria is impaired. Taken together these data suggest that Sec23ip is involved in cholesterol trafficking to supply cholesterol for acute steroidogenesis through its interactions with 14-3-3γ.

Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation.

INTRODUCTION: The biggest bottleneck for cell-based regenerative therapy is the lack of a functional vasculature to support the grafts. This problem is exacerbated in diabetic patients, where vessel growth is inhibited. To address this issue, we aim to identify the causes of poor vascularization in 3D engineered tissues in diabetes and to reverse its negative effects. METHODS: We used 3D vascularized constructs composed of microvessel fragments containing all cells present in the microcirculation, embedded in collagen type I hydrogels. Constructs were either cultured in vitro or implanted subcutaneously in non-diabetic or in a type I diabetic (streptozotocin-injected) mouse model. We used qPCR, ELISA, immunostaining, FACs and co-culture assays to characterize the effect of diabetes in engineered constructs. RESULTS: We demonstrated in 3D vascularized constructs that perivascular cells secrete hepatocyte growth factor (HGF), driving microvessel sprouting. Blockage of HGF or HGF receptor signaling in 3D constructs prevented vessel sprouting. Moreover, HGF expression in 3D constructs in vivo is downregulated in diabetes; while no differences were found in HGF receptor, VEGF or VEGF receptor expression. Low HGF expression in diabetes delayed the inosculation of graft and host vessels, decreasing blood perfusion and preventing tissue engraftment. Supplementation of HGF in 3D constructs, restored vessel sprouting in a diabetic milieu. CONCLUSION: We show for the first time that diabetes affects HGF secretion in microvessels, which in turn prevents the engraftment of engineered tissues. Exogenous supplementation of HGF, restores angiogenic growth in 3D constructs showing promise for application in cell-based regenerative therapies.

Correction to: Type I Diabetes Delays Perfusion and Engraftment of 3D Constructs by Impinging on Angiogenesis; Which can be Rescued by Hepatocyte Growth Factor Supplementation.

[This corrects the article DOI: 10.1007/s12195-019-00574-3.].

Glycoprotein 2 is a specific cell surface marker of human pancreatic progenitors.

PDX1+/NKX6-1+ pancreatic progenitors (PPs) give rise to endocrine cells both in vitro and in vivo. This cell population can be successfully differentiated from human pluripotent stem cells (hPSCs) and hold the potential to generate an unlimited supply of ß cells for diabetes treatment. However, the efficiency of PP generation in vitro is highly variable, negatively impacting reproducibility and validation of in vitro and in vivo studies, and consequently, translation to the clinic. Here, we report the use of a proteomics approach to phenotypically characterize hPSC-derived PPs and distinguish these cells from non-PP populations during differentiation. Our analysis identifies the pancreatic secretory granule membrane major glycoprotein 2 (GP2) as a PP-specific cell surface marker. Remarkably, GP2 is co-expressed with NKX6-1 and PTF1A in human developing pancreata, indicating that it marks the multipotent pancreatic progenitors in vivo. Finally, we show that isolated hPSC-derived GP2+ cells generate ß-like cells (C-PEPTIDE+/NKX6-1+) more efficiently compared to GP2- and unsorted populations, underlining the potential therapeutic applications of GP2.Pancreatic progenitors (PPs) can be derived from human pluripotent stem cells in vitro but efficiency of differentiation varies, making it hard to sort for insulin-producing cells. Here, the authors use a proteomic approach to identify the secretory granule membrane glycoprotein 2 as a marker for PDX1+/NKX6-1+ PPs.

Cell Therapy for Type 1 Diabetes: Current and Future Strategies.

PURPOSE OF THE REVIEW: Type 1 diabetes (T1D) is defined by an autoimmune destruction of insulin producing ß-cells located in the endocrine part of the pancreas, the islets of Langerhans. As exogenous insulin administration fails at preventing severe complications associated with this disease, cell replacement therapies are being considered as a means to treat T1D. The purpose of this manuscript is to review the challenges associated with current strategies and discuss the potential of stem cell therapy for the treatment of T1D. RECENT FINDINGS: The most prominent therapy offered to T1D patients is exogenous insulin administration which, despite formulations improvement, remains a suboptimal treatment, due to the frequency of injections and the issues associated with precise dosing. As immunotherapy approaches have remained unsuccessful, the only cure for T1D is transplantation of donor-derived pancreas or islets. However, donor scarcity, graft loss, and immune response to the foreign tissue are issues challenging this approach and limiting the number of patients who can benefit from such treatments. In this review, we discuss the causes of T1D and the shortcomings of the current treatments. Furthermore, we summarize the cutting edge research that aims to tackle the current challenges in reaching a quality-controlled product with long-term effects, with a focus on regenerative medicine approaches using human pluripotent stem cells.

The role of the 14-3-3 protein family in health, disease, and drug development.

14-3-3 proteins regulate intracellular signaling pathways, such as signal transduction, protein trafficking, cell cycle, and apoptosis. In addition to the ubiquitous roles of 14-3-3 isoforms, unique tissue-specific functions are also described for each isoform. Owing to their role in regulating cell cycle, protein trafficking, and steroidogenesis, 14-3-3 proteins are prevalent in human diseases, such as cancer, neurodegeneration, and reproductive disorders, and, therefore, serve as valuable drug targets. In this review, we summarize the role of 14-3-3 proteins in normal and disease states, with a focus on 14-3-3γ and ɛ. We also discuss drug compounds targeting 14-3-3 proteins and their potential therapeutic uses.

Translocator protein-mediated pharmacology of cholesterol transport and steroidogenesis.

Steroidogenesis begins with cholesterol transfer into mitochondria through the transduceosome, a complex composed of cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor proteins, and the outer mitochondrial membrane proteins Translocator Protein (TSPO) and Voltage-Dependent Anion Channel (VDAC). TSPO is a drug- and cholesterol-binding protein found at particularly high levels in steroid synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration, neuropsychiatric disorders and primary hypogonadism. Brain steroids serve as local regulators of neural development and excitability. Reduced levels of these steroids have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is common among subfertile young men and aging men, and is associated with depression, metabolic syndrome and reduced sexual function. Although testosterone-replacement therapy is available, there are undesired side-effects. TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain and testis.

Pharmacological regulation of the cholesterol transport machinery in steroidogenic cells of the testis.

Reduced serum testosterone (T), or hypogonadism, is estimated to affect about 5 million American men, including both aging and young men. Low serum T has been linked to mood changes, worsening cognition, fatigue, depression, decreased lean body mass and bone mineral density, increased visceral fat, metabolic syndrome, decreased libido, and sexual dysfunction. Administering exogenous T, known as T-replacement therapy (TRT), reverses many of the symptoms of low T levels. However, this treatment can result in luteinizing hormone suppression which, in turn, can lead to reduced sperm numbers and infertility, making TRT inappropriate for men who wish to father children. Additionally, TRT may result in supraphysiologic T levels, skin irritation, and T transfer to others upon contact; and there may be increased risk of prostate cancer and cardiovascular disease, particularly in aging men. Therefore, the development of alternate therapies for treating hypogonadism would be highly desirable. To do so requires greater understanding of the series of steps leading to T formation and how they are regulated, and the identification of key steps that are amenable to pharmacological modulation so as to induce T production. We review herein our current understanding of mechanisms underlying the pharmacological induction of T formation in hypogonadal testis.

Protein modifications regulate the role of 14-3-3γ adaptor protein in cAMP-induced steroidogenesis in MA-10 Leydig cells.

The 14-3-3 protein family comprises adaptors and scaffolds that regulate intracellular signaling pathways. The 14-3-3γ isoform is a negative regulator of steroidogenesis that is hormonally induced and transiently functions at the initiation of steroidogenesis by delaying maximal steroidogenesis in MA-10 mouse tumor Leydig cells. Treatment of MA-10 cells with the cAMP analog 8-bromo-cAMP (8-Br-cAMP), which stimulates steroidogenesis, triggers the interaction of 14-3-3γ with the steroidogenic acute regulatory protein (STAR) in the cytosol, limiting STAR activity to basal levels. Over time, this interaction ceases, allowing for a 2-fold induction in STAR activity and maximal increase in the rate of steroid formation. The 14-3-3γ/STAR pattern of interaction was found to be opposite that of the 14-3-3γ homodimerization pattern. Phosphorylation and acetylation of 14-3-3γ showed similar patterns to homodimerization and STAR binding, respectively. 14-3-3γ Ser(58) phosphorylation and 14-3-3γ Lys(49) acetylation were blocked using trans-activator of HIV transcription factor 1 peptides coupled to 14-3-3γ sequences containing Ser(58) or Lys(49). Blocking either one of these modifications further induced 8-Br-cAMP-induced steroidogenesis while reducing lipid storage, suggesting that the stored cholesterol is used for steroid formation. Taken together, these results indicate that Ser(58) phosphorylation and Lys(49) acetylation of 14-3-3γ occur in a coordinated time-dependent manner to regulate 14-3-3γ homodimerization. 14-3-3γ Ser(58) phosphorylation is required for STAR interactions under control conditions, and 14-3-3γ Lys(49) acetylation is important for the cAMP-dependent induction of these interactions.

Induction of androgen formation in the male by a TAT-VDAC1 fusion peptide blocking 14-3-3ɛ protein adaptor and mitochondrial VDAC1 interactions.

Low testosterone (T), a major cause of male hypogonadism and infertility, is linked to mood changes, fatigue, osteoporosis, reduced bone-mass index, and aging. The treatment of choice, T replacement therapy, has been linked with increased risk for prostate cancer and luteinizing hormone (LH) suppression, and shown to lead to infertility, cardiovascular diseases, and obesity. Alternate methods to induce T with lower side effects are desirable. In search of the mechanisms regulating T synthesis in the testes, we identified the 14-3-3ɛ protein adaptor as a negative regulator of steroidogenesis. Steroidogenesis begins in mitochondria. 14-3-3ɛ interacts with the outer mitochondrial membrane voltage-dependent anion channel (VDAC1) protein, forming a scaffold that limits the availability of cholesterol for steroidogenesis. We report the development of a tool able to induce endogenous T formation. Peptides able to penetrate testes conjugated to 14-3-3ɛ site of interaction with VDAC1 blocked 14-3-3ɛ-VDAC1 interactions while at the same time increased VDAC1-translocator protein (18 kDa) interactions that induced steroid formation in rat testes, leading to increased serum T levels. These peptides rescued intratesticular and serum T formation in adult male rats treated with gonadotropin-releasing hormone antagonist, which dampened LH and T production.

Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells.

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.