In this commentary article, we take the view in support of the continued promise of pluripotent stem cell (PSC) research and the potential for curative PSC-derived treatments to be realized within our lifetimes. We address a prominent critique of the PSC field: the failure to-date to successfully transplant lab-grown organs into human patients or to reproducibly demonstrate curative PSC-derived therapies in the clinic.We also discuss the key biological concept of cellular maturation, which is relevant to the eventual widespread clinical application of PSC-therapies. We then examine how an exciting development in the field of genomic engineering has opened up new avenues for discovery in PSC research. Finally, we will comment on possibilities for the future, informed by our experience of actively participating in this research for more than a decade.
The final United Kingdom Regenerative Medicine Platform (UKRMP) conference held in Edinburgh's iconic McEwan Hall between 8th and November 10, 2023 saw a gathering of nearly 200 international delegates presenting exceptional science and celebrating a decade of this initiative. The UKRMP had the core mission to break down the major barriers to clinical translation of regenerative medicine products. UKRMP2 was established as three hubs that worked closely with industry and regulators: 1) Pluripotent Stem Cells and Engineered Cells, 2) Engineered Cell Environments, and 3) Smart Materials. In this meeting report, we outline the original aims of UKRMP, examine how it achieved critical mass, summarise the major developments that the UKRMP hubs delivered, and examine some unresolved challenges that still lie ahead in the field of regenerative medicine.
Calcium phosphates with submicron surface features have demonstrated superior performance to conventional calcium phosphates and equivalence to autologous bone in pre-clinical bone healing models. This is related to their ability to form bone in soft tissues, without the addition of cells and growth factors. It is hypothesized that a specific innate immune response to submicron topography contributes to the enhanced bone healing by these materials. Upregulation of pro-healing, anti-inflammatory ‘M2’ macrophages versus pro-inflammatory ‘M1’ macrophages on submicron-structured calcium phosphates may be involved. In this in vitro study, the response of primary human macrophages to different calcium phosphate bone graft substitutes was assessed. Primary CD14+ monocytes were isolated from human buffy coats and were seeded on two different calcium phosphate materials. The first material had a submicron topography of needle-shaped crystals (BCP<μm) while the second material had no submicron topography (TCP). Macrophage M1/M2 phenotype characterization by protein and gene expression markers at 24 h and 72 h indicated overall stronger macrophage activation and subtle phenotypic skewing towards the M2 phenotype on BCP<μm vs TCP. Moreover, macrophages exhibited an elongated morphology on BCP<μm, which is associated with the M2 phenotype, while macrophages on TCP primarily exhibited a spherical morphology. Conditioned medium of macrophages cultured on BCP<μm resulted in enhanced in vitro angiogenic tube formation and osteogenic differentiation of mesenchymal stromal cells, compared to conditioned medium from macrophages on TCP. Altogether, these findings suggest a potential role of M2 macrophage upregulation in the bone-induction mechanism of calcium phosphates with submicron surface topography.
Chronic inflammation is a major concern after total joint replacements (TJRs), as it is associated with bone loss, limited bone-implant integration (osseointegration), implant loosening and failure. Inflammation around implants could be directed away from adverse outcomes and toward enhanced osseointegration and improved surgical outcome. Activated macrophages exposed to polyethylene particles play a dominant inflammatory role, and exhibit elevated mitochondrial oxidative phosphorylation (OXPHOS) whose role is unclear. By probing the contribution of the electron transport chain (ETC), we show that increased oxygen consumption does not contribute to bioenergetic (ATP) levels in fibroblasts and primary bone marrow-derived macrophages activated by polyethylene particles. Rather, it generates reactive oxygen species (ROS) at complex I by increasing mitochondrial membrane potential in macrophages. Inhibition of OXPHOS in a dosedependent manner without affecting glycolysis was accomplished by targeting complex I of the ETC using either rotenone or metformin. Metformin decreased mitochondrial ROS and, subsequently, expression of proinflammatory cytokines, including IL-1β, IL-6 and MCP-1 but not TNF-a in macrophages. These results highlight the contribution of mitochondrial bioenergetics to activation of immune cells by polyethylene wear particles, offering new opportunities to modulate macrophage states toward desired clinical outcomes.
Immunotherapy is a powerful technique where immune cells are modified to improve cytotoxicity against cancerous cells to treat cancers that do not respond to surgery, chemotherapy, or radiotherapy. Expressing chimeric antigen receptor (CAR) in immune cells, typically T lymphocytes, is a practical modification that drives an immune response against cancerous tissue. CAR-T efficacy is suboptimal in solid tumors due to the tumor microenvironment (TME) that limits T lymphocyte cytotoxicity. In this study, we demonstrate that neutrophils differentiated from human pluripotent stem cells modified with AAVS1-inserted CAR constructs showed a robust cytotoxic effect against prostate-specific membrane antigen (PSMA) expressing LNCaP cells as a model for prostate cancer in vitro. Our results suggest that engineered CARs can significantly enhance the neutrophil anti-tumor effect, providing a new avenue in treating prostate cancers.
End-stage or chronic esophageal disease may eventually lead to surgical intervention and could potentially result in an esophagectomy, followed by a gastric pull-up or colon interposition procedure. Biostage's Cellspan™ Esophageal Implant (CEI) is designed to repair and replace full-circumferential esophageal surgical resections (≤6 cm) using autologous adipose derived mesenchymal stromal cells (Ad-MSCs) seeded on a retrievable polyurethane scaffold. The use of a segmental implant has the advantage of preserving the native non-diseased esophageal tissue as well as the stomach or intestinal tissue following esophagectomy. The mechanism of action, the fate of the Ad-MSC component (biodistribution) and the process of early tissue regrowth/wound repair following implantation remains to be elucidated. CEIs seeded with Ad-MSCs transduced with green fluorescent protein (GFP) were implanted into a pig model of esophageal segmental resection. A 5 cm full-circumferential esophageal resection was performed followed by CEI implantation using an end-to-end anastomoses to bridge the gap between the 2 native esophageal ends. Cell fate and tissue development were assessed at 14 (N = 3), 21 (N = 3), and 28 days (N = 3), post-CEI implantation. All animals in all groups exhibited a contiguous biologic esophageal conduit with a denuded patent lumen at necropsy. Epithelial cell proliferation/regrowth was evident from both anastomotic margins toward the implant center. Morphometric analysis indicated an increase in epithelial regrowth and a concomitant reduction in denuded tissue from day 14 to day 28. Histological evaluation revealed fibrovascular tissue and neovascularization on the adventitial side, with no discernible differences in tissue organization between 14 and 28 day implants. The majority of GFP + cells were on the abluminal esophageal surface and localized around vascular structures. No GFP + cells were detected in lymph nodes or on retrieved scaffolds. These findings support luminal continuity by day 14 post-implantation with Ad-MSC derived pericytes contributing to the neo-fibrovascular tissue. Morphometric analysis of the lumenal surface indicates that the process of lumenal re-epithelialization results from epithelial cell proliferation from the implant margins towards the center of the implant.
•Gene-edited pigs offer an unlimited supply of organs for clinical transplantation.•The innate immune response has largely been overcome by gene-editing of the pigs.•Blockade of the CD40/CD154 co-stimulation pathway prevents T cell rejection.•Pig kidneys have supported life in nonhuman primates for >1 year.•The potential risks of pig pathogen-derived infections have been minimized.
Described herein are findings in 16 patients who had their dysfunctioning mechanical prostheses in the mitral valve position replaced. The mechanical prostheses had been in place from 2 to 157 months (mean 80). All but 1 patient had the mechanical prosthesis excised because of prosthetic stenosis or regurgitation or both secondary to prosthetic thrombus (despite warfarin therapy) on cloth-ring "pannus" overlaying the orifice or parabasilar detachment ("leak"). The dysfunction was the result of non-infected causes in 13 patients and to infective causes in 3. Three patients (19%) died in the early post-operative period; the other 13 patients survived >1 year. The prostheses were excised in a variety of methods by the explanting surgeons. The best procedure to excise the mechanical prosthesis appears to be mainly operator dependent.
The gastroesophageal junction (GEJ) plays a critical role in preventing reflux of stomach contents into the esophagus and airways, but currently there are no options for replacement. Surgical resection of the GEJ or lower esophagus requires reanastomosis through a gastric conduit or intestinal interposition, but these surgeries are associated with significant morbidity and mortality related to loss of the one-way valve function. Despite significant progress made in tissue-engineering for the body of the esophagus, little has been done for the stomach and no attempts have been made to reconstruct a GEJ. A detailed understanding of the GEJ anatomy and physiology complemented by knowledge of approaches used in tissue engineering of the esophagus and stomach is foundational to future attempts to address this gap in the field. These topics, as well as anticipated obstacles, are addressed in this review.
We reported the short-term outcomes of a regenerative medical treatment to prevent esophageal stricture after endoscopic submucosal dissection (ESD) using cultured tis oral mucosal epithelial cell sheets. This study investigated the long-term outcomes of this treatment. Epithelial cells, isolated from the patient's own oral mucosal tissue, were cultured for 16 days using temperature-responsive culture dishes. Then, the autologous cell sheets were endoscopically transplanted onto the bed of the esophageal ulcer after endoscopic mucosal resection (EMR) and ESD. Results of 10 patients who underwent endoscopic transplantation of oral mucosal epithelial cell sheets from April 2008 through February 2022 were recorded. We analyzed the outcome, the cause, and the endoscopic findings. The median period of observation was 3761 days. No stricture was detected in any of the patients long-term. Two patients died because of pancreatic cancer and brainstem hemorrhage. One patient underwent chemo-radiotherapy for further treatment. The patients underwent surgery due to metastasis to the lymph nodes. Only the lymph nodes were dissected, and the esophagus remained intact. From the endoscopic findings: Melanosis was found at the transplanted site in a patient. Strong iodine staining was shown at the transplanted site in a patient. Transplantation of cultured oral mucosal epithelial cell sheets to prevent esophageal stricture has been proven to be a safe treatment. All patients showed no controlled esophageal stricture in the long term. • Esophageal strictures after ESD require frequent endoscopic balloon dilatation. • Cultured oral mucosal epithelial cell sheet transplantation prevents this. • Long-term follow up of 10 patients for a median of 3761 days showed no strictures.
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.
As a treatment for type I diabetes, clinical islet transplantation (CIT) in which donor islets of Langerhans are transplanted intrahepatically has become a viable option for patients. However, the success of this procedure is limited by factors including ischemia, host immunological factors, and delayed vascularization of the hypoxia-sensitive islets. One solution would be to use a synthetic polymer scaffold as a carrier for the transplanted islets, as it would allow for their transplantation into a more favorable environment and could protect the cells from host immune reactions. To realize this potential solution, it is important that the synthetic polymer used does not interfere with the functionality and survival of the islets. In order to determine which synthetic polymers best meet this requirement, we examined the interactions of human islets from six donors with four clinically approved materials: polyetheretherketone (PEEK), polyvinylidene fluoride (PVDF), polyphenylsulfone (PPSU) and polysulfone (PSU) in vitro. Human islet morphology, viability, insulin secretion, functionality and gene expression were investigated to assess the suitability of these synthetic polymers as a carrier for transplanted islets. We found three of the synthetic synthetic polymers (PEEK, PPSU and PVDF) showed promise based on their overall performance, while the glucose responsiveness of islets cultured on PSU resulted in significantly reduced insulin secretion from five of six donors. Our findings demonstrate that close examination of human islets and their interaction with synthetic polymers is an important factor to consider when selecting synthetic polymers for engineering islet replacement devices.
ObjectivesFor nearly a century, the therapeutic use of exogenous insulin remains the gold standard treatment strategy for patients living with Type 1 Diabetes Mellitus (T1DM). While lifesaving, insulin can fail to prevent the secondary vascular disease and complications inherited with T1DM diagnosis, and for some, this may increase their risk of life-threatening hypoglycemic unawareness. In recent decades transplantation has been demonstrated as the only means (a replacement gold standard therapy) to effectively restore physiologically relevant glycemic control. Moreover, significant advancements in clinical islet transplantation have yielded greater incidences of durable insulin-independence, rescindment of critical hypoglycemia and prevention of comorbidities. Yet, the requisite of life-long immunosuppression and scarcity of a universal and potent cell supply, in addition to the challenges faced with such therapy in vivo, restricts the broad-spectrum application of cell-based therapies.Key findingsHerein, this review presents the history, current status and persisting challenges confronting cell-based replacement therapies for T1DM. Lastly, we examine modern and future research opportunities designed to enhance the efficacy of cellular transplantation, thereby offering a potential functional cure to all those affected by T1DM.ConclusionsGiven the rapid progress in β-cell replacement therapy, it is hopeful that through multidisciplinary innovations, first-in-human stem cell trials, innocuous immunosuppression and efficacious extrahepatic transplant sites, β-cell replacement therapies will become the cornerstone treatment for the millions worldwide afflicated with T1DM.
Hematopoietic stem cells (HSCs) are essential in the production and maintenance of red blood and immune cells. Small molecules that target HSC modulators may aid in the proliferation and expansion of HSCs. To that end, we investigated the effect of two small molecules on HSC expansion: pluripotin (an ERK1 and RasGAP inhibitor) and CHIR-99021 (a GSK-3 inhibitor). After 7 days of treatment, both Pluripotin and CHIR-99021 resulted in a 3-fold increase in the murine pool of HSCs in a dose-dependent manner. Furthermore, we looked into the effect of Pluripotin on the ex vivo expansion of human umbilical cord blood and bone marrow mononuclear cells. Pluripotin treatment, in particular, increased human CD34+ and ALDHbr HSC content up to threefold when compared to the control. Furthermore, Pluripotin treatment increased the number of human CD133+ HSC cells by a factor of five. Intriguingly, Pluripotin treatment reduces bone marrow-derived mesenchymal stem cell (MSC) proliferation and fibroblast growth while having no effect on adipose-derived MSCs. CHIR-99021 treatment had no effect on MSC or fibroblast proliferation. In conclusion, pluripotin-induced stem cell expansion is unique to HSCs and can be used to expand HSCs while suppressing unwanted fibroblast or MSC growth in primary ex vivo cultures.
Islet transplantation is a promising therapy for a subset of people with Type 1 diabetes (T1D). However, beta cell transplant clinical trials have largely failed to maintain long-term normal glycemia in transplant recipients. This is broadly due to immune rejection of the transplanted islets themselves or the devices in which these cells are encapsulated. As an autoimmune condition, the T1D host presents a uniquely challenging immunological niche for the transplant of additional beta cells. An understanding of the autoimmune environment is crucial for the development of successful beta cell transplant therapies. Here, we provide an overview of the immune cell pathways leading to autoimmune T1D, and the resulting immune niche. Next, we examine biomaterial platforms that can be used for cell transplantation, and describe those that seek to modulate the immune environment to mitigate immune rejection. These approaches include delivery of localized immune cues, co-transplantation with immunomodulatory cells, strategies to engineer islets ex-vivo, and antigen-specific immunomodulation to generate operational tolerance. Finally, we describe therapies which seek to prevent T1D progression which could be repurposed to support beta cell transplantation and future immunoengineering design considerations for successful islet transplantation therapies.
Extracellular vesicles (EV) have long been recognized as an important means of cell to cell communication, but current metrics to delineate various subpopulations of EV are limited. Recently, a distinctive subpopulation of EV embedded within the extracellular matrix of soft tissues, termed matrix-bound nanovesicles (MBV), has been described. Although the lipid membrane composition and intravesicular cargo of MBV clearly differ from liquid phase EV (i.e. exosomes), a more comprehensive characterization of the physical and biologic properties of MBV vs. exosomes and those of a separate subpopulation of EV, specifically bone matrix vesicles, would contribute to our understanding of the biogenesis and physiologic role of these three EV subpopulations. The physical characteristics, protein and miRNA cargo profiling, vesicle membrane lipidomics, and immunomodulatory activity were used to compare skeletal muscle-derived MBV, liquid phase plasma exosomes, and mineralization-competent matrix vesicles of provisional bone matrix. We show that despite similar physical characteristics, these three preparations of EV represent distinct entities with different biologic activity. These results inform metrics for the categorization of EV and provide tools for the isolation of EV for potential diagnostic and therapeutic applications.
Macrophages can be found in various tissues and play an important role in organ function by sensing and eradicating pathogens, regulating immune responses and contributing to tissue homeostasis and repair. Nowadays, increasing numbers of macrophage-based cell therapies are entering (pre-) clinical studies e.g. for the treatment of liver cirrhosis. Given limited availability of suitable donors as well as problems with variability in quantities and qualities of human monocytes that can be derived from apheresis, induced pluripotent stem cells (iPSC) offer an attractive source of therapeutic macrophages. However, considering the diverse functions, activation stages and overall plasticity of macrophages, further knowledge about (i) the potential to induce different activation stages in iPSC-derived macrophages (iPSC-Mac) as well as (ii) the stability of these phenotypes upon additional external stimuli is of high relevance. We here demonstrate that iPSC-Mac produced in a scalable differentiation platform can be polarized into defined pro- (M1) and anti-inflammatory (M2) activation stages characterized by specific surface marker expression, cytokine secretion and whole transcriptome analysis, similarly to peripheral blood-derived macrophages. Even more importantly, we show that differentially polarized iPSC-Mac maintained key characteristics of their activation status upon a subsequent inflammatory trigger. Interferon (IFN) γ polarized, M1-iPSC-Mac demonstrated an enhanced inflammatory response after additional lipopolysaccharide (LPS) stimulation, whereas Interleukin (IL)-4 stimulated M2a iPSC-Mac and IL-10/TGFβ primed M2c iPSC-Mac showed a reduced activation upon LPS treatment and maintained expression of anti-inflammatory genes. Together, our data demonstrate that defined polarized iPSC-Mac subsets can be generated. Moreover, these cells maintain key characteristics of their activation profile upon a subsequent inflammatory trigger. Thus, the use of stably polarized iPSC-Mac has the potential to further improve the applicability and efficacy of macrophage-based therapies.
Progress in human pluripotent stem cells has opened up an opportunity to autologous β-cell replacement therapies in patients with diabetes. Such an approach could render immunologically compatible islets from an unconstrained source without requirement for chronic immune suppression. Several proof-of-concept studies have generated stem cell-derived islets (SC-islets) capable of reversing diabetes in rodents and with similar functional characteristics to human donor islets. Autologous SC-islets offer potential to improve the life of patients living with diabetes by enabling cell replacement therapy that provides physiologic glycemic control with less risk to the recipient. Such efforts are impeded from ongoing challenges in scalability, latent potential for teratogenicity, an inability to fully recapitulate metabolic responses observed with primary islets, and protection from autoimmune recurrence in the setting of Type 1 diabetes. In this review, we outline potential opportunities and impediments for successful clinical translation of SC-islets as an effective therapy for patients with all forms of diabetes. We discuss recent advancements in scale-up manufacturing, the promise of gene-editing for optimized cellular protection, and methods to deliver safe and immune shielded cells to improve engraftment and survival. Finally, we discuss in detail goals and challenges in islet bioengineering and emphasize the need for improved methods to overcome the roadblocks in translating autologous SC-islet cell therapies to the clinic.
Type 1 diabetes (T1D) is characterized by hyperglycemia due to autoimmune destruction of the insulin-producing beta-cells in the pancreas. The reason for this occurring is still unknown and effective therapies to halt the autoimmune response are lacking, but several promising concepts are being trialed. Regenerative medicine approaches in expanding the remaining beta cell pool, or transplanting beta cells derived from stem cells are other options trialed. Lessons learned from the study of immune regulation in T1D could be applied to immunosuppression in transplantation of beta cells derived from stem cell sources to avoid recurring autoimmunity. In this review, immunological issues in beta cell replacement therapies are discussed along possible treatment avenues such as genetically modified grafts to evade immune responses, novel immunosuppressive protocols, and the harnessing of endogenous pools of regulatory immune cell subsets. Looking into promising treatments for T1D may lead to effective immunosuppressive regimens also for beta-cell grafts from stem cells where recurring autoimmunity is a major issue to address.
Cytokine manipulation has been widely used to bolster innate healing mechanisms in an array of modern therapeutics. While other anatomical locations have a more definitive analysis of cytokine data, the tendon presents unique challenges to detection that make a complete portrayal of cytokine involvement during injury unattainable thus far. Without this knowledge, the advancement of tendon healing modalities is limited. In this review, we discuss what is known of the cytokine profile within the injured tendinous environment and the unique obstacles facing cytokine detection in the tendon while proposing possible solutions to these challenges. IL-1β, TNF-α, and IL-6 in particular have been identified as key cytokines for initiating tendon healing, but their function and temporal expression are still not well understood. Methods used for cytokine evaluation in the tendon including cell culture, tissue biopsy, and microdialysis have their strengths and limitations, but new methods and approaches are needed to further this research. We conclude that future study design for cytokine detection in the injured tendon should meet set criteria to achieve definitive characterization of cytokine expression to guide future therapeutics.
