Vascular endothelial cells derived from transgene-free pig induced pluripotent stem cells for vascular tissue engineering.

The heterogeneous population of patients suffering from heart valve disease requires strategies addressing the individual conditions, health, age and co-morbidities. Personalized tissue substitutes would be an attractive alternative to current bioprosthetic or mechanical heart valve replacements as these are burdened with several disadvantages such as the lack of growth and remodeling as well as increased risks for degeneration, thromboembolism or immunological reactions. This is addressed by cardiovascular tissue engineering where heart valves are generated from autologous cells in vitro prior to implantation. The special needs of congenital patients are addressed by the pediatric tissue engineering concept. Here cells are harvested prenatally to have the tissue engineered construct ready for implantation at or shortly after birth. In a systematic series of studies we have investigated different human cell sources as to their suitability for cardiovascular tissue engineering thereby suggesting amniotic fluid-derived multipotent stem cells as the ideal cell source. Here, we explored the potential of cryopreservation of human amniotic-fluid-derived multipotent stem cells to expand their use beyond congenital applications to elderly patients. It was shown that cells kept their multipotency after cryopreservation and led to functional heart valves in vitro. This indicates that cryopreserved amniotic fluid-derived cells are a promising life-long available autologous multipotent stem cell source. In this context banking of amniotic fluid-derived cells for future therapies might be an interesting concept. A preclinical in vivo study addressed growth, maturation and remodeling of tissue engineered constructs in a growing sheep model. In order not to compromise in vivo safety and feasibility of the tissue engineering process investigations were first performed with established cell sources. The long-term functionality and growth of engineered cardiovascular tissue substitutes could be demonstrated over a period of 240 weeks. Moreover, it was shown that biological age of cells in the substitutes did not differ from native tissues indicating that remodeling processes take place in vivo. In the last study the clinically relevant minimally invasive implantation of the tissue engineered substitutes was explored in sheep. Autologous stem cell based tissue engineered valves integrated in clinically established nitinol stents could be fabricated, crimped and successfully implanted by a minimally invasive procedure. The in vivo functionality of these stem cell based heart valves was shown. Furthermore, the presence of a functional antithrombotic surface was indicated by the lack of thrombus formation. The presented autologous heart valve tissue engineered concept might be a further step towards the realization of personalized minimally invasively implantable heart valves, addressing the individual needs of the heterogeneous population of affected patients.

The goal of "Recent Developments" is to provide a concise but comprehensive overview of new advances in cardiovascular research, which we hope will keep our readers abreast of recent scientific discoveries and facilitate discussion, interpretation, and integration of the findings.This will enable readers who are not experts in a particular field to grasp the significance and effect of work performed in other fields.It is our hope and expectation that these "Recent Development" articles will help readers to gain a broader awareness and a deeper understanding of the status of research across the vast landscape of cardiovascular research-The Editors.

Since the discovery of bone marrow mesenchymal stem cells (BMMSCs), many researchers have focused their attention on new sources of mesenchymal stem cells (MSCs). Consequently, MSCs that display self-renewal capacity, multidifferentiation potential and immunomodulatory properties have been isolated from human oral tissues, including tooth, periodontal ligament, and gingiva. Oral MSCs involve dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), dental follicle stem cells (DFCs), stem cells from apical papilla (SCAP) and gingival stem cells (GMSCs). Current research on oral stem cells is expanding at an unprecedented rate. That being the case, a plethora of in vitro differentiation assays, immunodeficient animal transplantations and preclinical trials have demonstrated that these cells exhibit strong potential for both regenerative dentistry and medicine. Oral MSCs have proved their capability to repair cornea, dental pulp, periodontal, bone, cartilage, tendon, neural, muscle and endothelial tissues without neoplasm formation as well as to treat inflammatory diseases and immune disorders. This article describes the current understanding of oral MSCs and their prospective applications in cell-based therapy, tissue engineering and regenerative medicine. Special attention is placed on GMSCs as they are easily accessible and may be obtained in a convenient and minimally invasive way.

Engineering Stem and Stromal Cell Therapies for Musculoskeletal Tissue Repair.

Endothelial progenitor cells (EPCs) can be defined as bone marrow-derived precursor cells with the ability to differentiate into endothelial cells and to participate in the formation of new blood vessels. Bone marrow is the primary source of endothelial progenitors, which can be mobilized to the peripheral circulation and may seed in remote organs and tissues such as liver, spleen, heart, muscle, and adipose tissue. EPCs have been shown to express various endothelial surface markers, such as CD34, VEGFR2, and CD133, and to home to sites of ischemia1 (Figure 1).Figure 1: Phenotype of cultured endothelial progenitor cells (EPCs) can be characterized according to their ability to uptake Dil-labeled acetylated low-density lipoprotein (Dil-Ac-LDL) to confirm their endothelial lineage. Image demonstrates Dil-Ac-LDL-stained EPCs under fluorescent microscope (×40).Vascular ingrowth at the fracture site has a cardinal role in the healing process and regeneration of the bone following fracture. However, segmental bone defects after severe trauma, infection, and surgical removal of tumors remain a major clinical problem. These facts, together with reports documenting the remarkable therapeutic potential of EPCs to improve neovascularization and tissue perfusion in other disciplines, have been the major drivers recently to investigate the effects of EPC therapy on fracture healing. Matsumoto et al2 investigated the therapeutic potential of systemically administered CD34+ cells on fracture healing in a rodent model. The authors transplanted human peripheral blood CD34+ cells, mononuclear cells, or saline into immunodeficient rats with a non-healing femoral fracture. Fracture healing was significantly enhanced in the CD34+ group compared with the mononuclear cell and saline groups. Laser Doppler imaging demonstrated that fracture-induced ischemia was significantly reduced in the CD34+ cell-transplanted group compared with the other groups. The authors also noted that approximately 20% of human peripheral blood CD34+ cells expressed mRNA for osteocalcin after transplantation to a fracture site; this outcome may indicate the potential of CD34+ cells for osteogenic and endothelial differentiation. Atesok et al3 evaluated the effects of the local use of ex vivo-expanded EPCs on the stimulation of angiogenesis and the promotion of bone healing at a fracture site in a rat femur osteotomy model. Based on the results of radiographic, histologic, and micro-CT comparisons of the EPC-treated group with a control group, the authors stated that “local EPC therapy significantly enhanced bone regeneration in a segmental bone defect in rat femur.” In a similar study, the same research group reported that local EPC therapy favorably affects biomechanical stability.4 Recent reports also suggest that EPCs derived from peripheral blood contribute to osteogenic differentiation by mesenchymal stem cells (MSCs) in vitro and that MSCs support the proliferation of EPCs.5 Aguirre et al6 investigated the interactions between bone marrow EPCs (BM-EPCs) and MSCs in an in vitro co-culture system. Their data suggested that cross-talk occurs between BM-EPCs and MSCs through paracrine and direct cell contact mechanisms, leading to modulation of the angiogenic response. In a rat model study, Seebach et al7 observed a synergistic effect between EPCs and MSCs and suggested that the initial stage of neovascularization by EPCs be considered crucial for complete bone regeneration. Positive effects of EPC therapy have been demonstrated in ligament tissue regeneration, as well. Matsumoto et al8 recently demonstrated that CD34- and CD146-expressing vascular cells exist in human anterior cruciate ligament tissues, have a potential for multilineage differentiation, and are recruited to the rupture site to participate in the intrinsic healing of injured anterior cruciate ligament. In a rodent model, Tei et al9 studied the effects of locally transplanted human peripheral blood CD34+ cells on the healing of medial collateral ligament injury. Macroscopic, histologic, and biomechanical assessments showed significantly enhanced ligament healing in a CD34+ cell transplantation group compared with a control group. The authors suggested that “local transplantation of circulating human CD34+ cells may augment the ligament healing process by promoting a favorable environment through neovascularization.” Based on the promising results from ex vivo and animal model EPC studies, clinical trials have been started. As a pilot case from a clinical trial, Kuroda et al10 reported the results of transplantation of autologous peripheral blood CD34+ cells—the hematopoietic/EPC-enriched population—into a patient with nonunion of a tibia fracture. Clinical and radiologic healing of the fracture was achieved at 12 weeks after the cell therapy with bone grafting, and no serious short-term complications were encountered. EPCs, with their unique features, such as the ability to differentiate into endothelial cells and to participate in the establishment of neovasculature, in addition to their high plasticity, may offer therapeutic alternatives for both bone and ligament tissue engineering. It is likely that more investigators will be attracted in the near future to exploring the potential of these cells in orthopaedic surgery.11

Given the failure of pharmacological interventions in acute respiratory distress syndrome (ARDS), researchers have been actively pursuing novel strategies to treat this devastating, life-threatening condition commonly seen in the intensive care unit. There has been considerable research on harnessing the reparative properties of stem and progenitor cells to develop more effective therapeutic approaches for respiratory diseases with limited treatment options, such as ARDS. This review discusses the preclinical literature on the use of stem and progenitor cell therapy and cell-based gene therapy for the treatment of preclinical animal models of acute lung injury (ALI). A variety of cell types that have been used in preclinical models of ALI, such as mesenchymal stem cells, endothelial progenitor cells, and induced pluripotent stem cells, were evaluated. At present, two phase I trials have been completed and one phase I/II clinical trial is well underway in order to translate the therapeutic benefit gleaned from preclinical studies in complex animal models of ALI to patients with ARDS, paving the way for what could potentially develop into transformative therapy for critically ill patients. As we await the results of these early cell therapy trials, future success of stem cell therapy for ARDS will depend on selection of the most appropriate cell type, route and timing of cell delivery, enhancing effectiveness of cells (i.e., potency), and potentially combining beneficial cells and genes (cell-based gene therapy) to maximize therapeutic efficacy. The experimental models and scientific methods exploited to date have provided researchers with invaluable knowledge that will be leveraged to engineer cells with enhanced therapeutic capabilities for use in the next generation of clinical trials.

Human induced pluripotent stem cells (hiPSCs) are capable of differentiating into an unlimited number of cells of any lineage. Collectively, these properties suggest that hiPSC products may be an ideal source of cells for medical science and therapeutic use. Here, we discuss recent advancements in techniques for differentiating hiPSCs into cardiomyocytes, endothelial cells, and smooth muscle cells and in the use of hiPSC-derived cells for cardiovascular therapy and tissue engineering.

Although pioneering preclinical research on the use of cell therapy for cardiac regeneration was conducted in the last quarter of the 20th century,1,2 a preponderance of advances have occurred in the 21st century, making this a relatively young field. In the first important clinical trial of cardiac cell therapy, begun in 2001, Menasche et al3 implanted autologous skeletal myoblasts into postinfarct scar at the time of coronary artery bypass surgery. Although the transplanted cells remained viable and exhibited contraction, they formed the nidus for serious ventricular tachyarrhythmias, which led to premature discontinuation of the trial. Despite this outcome, the trial energized the field, accelerating both preclinical and clinical research, albeit not with skeletal myoblasts. The extensive progress in cardiac regeneration is reviewed in this Compendium, and as occurs frequently in science, important observations have led to more questions and challenges (Table). View this table: Table. Important Challenges to Cell Therapy for Cardiac Regeneration Many cell types have been evaluated as candidates for cardiac regeneration. Among the earliest clinical trials, Zeiher’s group infused autologous bone marrow-derived progenitor cells into the coronary arteries of patients with acute,4 as well as healed myocardial infarction (MI)5 and reported improvements in left ventricular (LV) function. However, these results have not been fully confirmed by later studies, as pointed out in the review in the Compendium by Banarjee, Bolli, and Hare.6 Pittenger et al7 were among the first to direct attention to bone marrow-derived (stromal) mesenchymal stem cells (MSCs), emphasizing that these cells proliferated extensively in culture and suggesting that they could be attractive candidates for transplantation. In 2004, Chen et al8 reported that intracoronary infusion of autologous bone marrow-derived MSCs improved cardiac function. Zimmet and Hare pointed out that MSCs lack histocompatibility type II markers and elude rejection by …

The high recurrence and complications associated with severe pressure injuries (PI) necessitate the exploration of advanced treatments, such as cell-based therapies, to facilitate wound healing. Such techniques harness the ability of different cell types to promote angiogenesis, re-epithelialization of the skin, and tissue regeneration. This systematic review explores the efficacy of cell-based therapies and tissue engineering in treating deep PI. We searched for interventional studies using cells in the treatment of PI in adults in four online libraries (PubMed, Embase, Ovid Medline, and Cochrane; latest search 10th June 2023). We found one randomized clinical trial (RCT), two non-RCT, and three pre-post studies, comprising 481 study participants with PI (253 intervention/228 controls). The risk of bias was categorized as moderate due to minimal bias in outcome measurements, or high owing to unclear patient randomization methods, as assessed by the ROBINS-I, NIH, and RoB-2 tools. Four cell types were identified in the context of cell-based therapies of PI: bone marrow mononuclear stem cells (BM-MNCs, n = 2); hematopoietic derived stem cells (HSC, n = 1); macrophages and activated macrophage suspensions (AMS, n = 2); and cryopreserved placental membrane containing viable cells (vCPM, n = 1). Wound healing outcomes were observed in patients undergoing cell-based therapies, including complete wound closure (AMS, vCPM; n = 142), faster healing rate (BM-MNCs, AMS; n = 146), improved granulation tissue formation (HSC, n = 3) and shorter hospitalization time (BM-MNCs; n = 108) compared to standard of care, with no adverse reactions. PI healing rate decreased only in one study with BM-MNC therapy, compared to control (n = 86). Based on the available data, though with limited evidence, it seems that macrophage deployment showed the most favorable outcomes. The results indicate that cell-based therapies offer a potential avenue for enhancing wound healing and tissue repair in PI; however, more extensive research is needed in this domain.

Expanded Opportunities for Methods and Clinical Development

Pluripotent cells have the potential to differentiate into all of the cell types of an animal. This unique cell state is governed by an interconnected network of transcription factors. Among these, Oct4 plays an essential role both in the development of pluripotent cells in the embryo and in the self-renewal of its in vitro counterpart, embryonic stem (ES) cells. Furthermore, Oct4 is one of the four Yamanaka factors and its overexpression alone can generate induced pluripotent stem (iPS) cells. Recent reports underscore Oct4 as an essential regulator of opposing cell state transitions, such as pluripotency establishment and differentiation into embryonic germ lineages. Here we discuss these recent studies and the potential mechanisms underlying these contrasting functions of Oct4.

A vast spectrum of therapeutic interventions is based on stem cell technology, ranging from inert biomaterials to pluripotent, virally-modified cells. On the other hand, the scientific and regulatory community is concerned with the safety of stem cell-based products and therapies and the potential health risks associated with cell-based therapies. Scientific publications have revealed shared genetic similarities and cellular morphologic and phenotypic properties between stem cells obtained from embryonic and adult sources and cancer stem cells, thus creating considerable uncertainties about the long-term safety of stem cell-based products and therapies. Existing regulatory policy and guidelines are generally inadequate to address safety assessment and risk mitigation approaches to stem cell technology-derived products and therapies. Therefore, a need exists to develop core competencies within regulatory and standards-setting institutions to better understand the unique characteristics of stem cell-derived products and to perform safety assessments on a caseby-case basis, taking into account potential hazards and risk mitigation considerations. This article outlines an evidence-based risk mitigation framework that discusses two interrelated pathways in stem cell-based biotechnologies—stem cell-derived product development and stem cell-based therapies. The proposed conceptual framework integrates biohazard considerations for cell-based products with product and facility biosafety issues that are critical to research and product development activities. The stem cell-based clinical therapeutic pathway addresses the need for integrated Institutional Review Board and Institutional Biosafety Committee reviews as a way to develop core competencies to mitigate risks and ensure that institutional policy and safety considerations are adequately addressed in stem cell-based clinical therapy and product development activities.

Tissue engineering

Neurological diseases such as stroke, encephalitis, neurologic tumors, and neurodegenerative diseases are far from alien to us. These diseases are refractory, harmful, and sometimes devastating. The pathogenesis of some nervous system diseases remains elusive, especially the neurodegenerative diseases such as AD and PD. Many therapeutic strategies have been developed to potentially intervene in these progressive neurodegenerative events and minimize damage to the CNS. Despite the promising results that these researches have shown in in vivo and in vitro studies, challenges still remain, particularly when it comes to clinical translation. The causes are various, which may include biologic and physiologic differences between the preclinical models and the human condition and discrepancies in dosing and the timing of drug therapy. The last few years have witnessed an explosion in the use of genes and cells as biomedicines. Gene and cell therapies are researches with the goals of repairing the direct cause of genetic diseases in the DNA or cellular population, respectively. In many diseases, gene and cell therapies are combined in the development of promising therapies, which have helped provide reagents, concepts, and techniques that are elucidating the finer points of gene regulation, stem cell lineage, regenerative capacity, and remodeling. Additionally, increasing studies of novel drug delivery systems bring more considerable future on gene and cell therapies. This special issue mainly focuses on the latest ideas, developments, and applications in the field of gene and cell therapies for neurological disorders, most notably development of highly efficient gene expression and delivery system including viral and nonviral gene vectors for tissue repair in the nervous system; exploration and evaluation of the efficiency of new ideal routes of administration of gene and cells into brain; evaluation of appropriate biomaterials in terms of biocompatibility and biodegradability, biomechanical competency, and functionalization with instructive cues to guide neuroregeneration; tissue engineering that integrates cell therapies and scaffolding technologies to replace damaged neurons and to reestablish the coaxial connection of neuronal circuits for functional recovery; assessment of the safety and toxic side effects with gene and cell therapies; latest technologies of noninvasive in vivo imaging to track the transplanted gene or cells; new insights into the pathogenesis of neurological disorders by using cellular or animal models. In the accepted papers, researches of pathogenesis of some diseases, such as Aβ-induced autophagy in AD and ischemia-reperfusion injury induced apoptosis in stroke, were discussed. Studying specific aptamer for GP73 through systematic evolution of ligands by exponential enrichment and identifying its application in detecting hepatocellular carcinoma might provide a novel molecular probe for cell therapy. In vivo targeted MR imaging of endogenous neural stem cells (NSCs) in adult mouse brain by using certain nanoparticles offered a promising molecular probe in NSCs research. Moreover, the role of Nogo-A in PD and NTRK1 in NSCs differentiation was preliminarily explored. These findings above provided new insights into the pathogenesis of certain diseases and also represented part of the latest ideas, developments, and applications in the field of gene and cell therapies for neurological disorders. Jun Liu William Z. Suo Xing-Mei Zhang Chen Zhang Gongxiong Wu

Recent advances in cell therapy and tissue engineering opened new windows for regenerative medicine, but still necessitate innovative noninvasive imaging technologies. We demonstrate that high-resolution magnetic resonance imaging (MRI) allows combining cellular-scale resolution with the ability to detect two cell types simultaneously at any tissue depth. Two contrast agents, based on iron oxide and gadolinium oxide rigid nanoplatforms, were used to "tattoo" endothelial cells and stem cells, respectively, with no impact on cell functions, including their capacity for differentiation. The labeled cells' contrast properties were optimized for simultaneous MRI detection: endothelial cells and stem cells seeded together in a polysaccharide-based scaffold material for tissue engineering appeared respectively in black and white and could be tracked, at the cellular level, both in vitro and in vivo. In addition, endothelial cells labeled with iron oxide nanoparticles could be remotely manipulated by applying a magnetic field, allowing the creation of vessel substitutes with in-depth detection of individual cellular components.