Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors: In Vivo Post-Grafting Safety Characterization in Rats and Adult Pig.

One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.

The combination of nanoscaffolds and stem cell transplantation: Paving a promising road for spinal cord injury regeneration.

Spinal cord injury (SCI), one of the most devastating traumas, has caused long-term disability in millions of people worldwide. The pathophysiology of SCI primarily occurs in two stages classified as primary injury and secondary injury. Due to the rupture of axons and the apoptosis of neurons, patients lose their motor, sensory, and reflex functions, which also imposes a huge burden on families and society. However, traditional surgery does not facilitate neuronal regeneration. Although neural stem cells (NSCs) have the potential for multidirectional differentiation, the probability of differentiation into neurons and survival are still low. Surprisingly, the unique properties of nanotechnologies enable targeted drug delivery while reducing adverse reactions, assisting NSCs in differentiating into neurons. Here, recent studies on promising nanoscaffolds are highlighted, and their strengths and drawbacks are evaluated. Although the treatment of SCI remains fraught with challenges, the combination of nanoscaffolds and NSCs pave a promising road for SCI regeneration.

Spinal Cord Repair: From Cells and Tissue Engineering to Extracellular Vesicles.

Spinal cord injury (SCI) is a debilitating condition, often leading to severe motor, sensory, or autonomic nervous dysfunction. As the holy grail of regenerative medicine, promoting spinal cord tissue regeneration and functional recovery are the fundamental goals. Yet, effective regeneration of injured spinal cord tissues and promotion of functional recovery remain unmet clinical challenges, largely due to the complex pathophysiology of the condition. The transplantation of various cells, either alone or in combination with three-dimensional matrices, has been intensively investigated in preclinical SCI models and clinical trials, holding translational promise. More recently, a new paradigm shift has emerged from cell therapy towards extracellular vesicles as an exciting "cell-free" therapeutic modality. The current review recapitulates recent advances, challenges, and future perspectives of cell-based spinal cord tissue engineering and regeneration strategies.

Sustained subcutaneous delivery of secretome of human cardiac stem cells promotes cardiac repair following myocardial infarction.

AIMS: To establish pre-clinical proof of concept that sustained subcutaneous delivery of the secretome of human cardiac stem cells (CSCs) can be achieved in vivo to produce significant cardioreparative outcomes in the setting of myocardial infarction. METHODS AND RESULTS: Rats were subjected to permanent ligation of left anterior descending coronary artery and randomized to receive subcutaneous implantation of TheraCyte devices containing either culture media as control or 1 × 106 human W8B2+ CSCs, immediately following myocardial ischaemia. At 4 weeks following myocardial infarction, rats treated with W8B2+ CSCs encapsulated within the TheraCyte device showed preserved left ventricular ejection fraction. The preservation of cardiac function was accompanied by reduced fibrotic scar tissue, interstitial fibrosis, cardiomyocyte hypertrophy, as well as increased myocardial vascular density. Histological analysis of the TheraCyte devices harvested at 4 weeks post-implantation demonstrated survival of human W8B2+ CSCs within the devices, and the outer membrane was highly vascularized by host blood vessels. Using CSCs expressing plasma membrane reporters, extracellular vesicles of W8B2+ CSCs were found to be transferred to the heart and other organs at 4 weeks post-implantation. Furthermore, mass spectrometry-based proteomic profiling of extracellular vesicles of W8B2+ CSCs identified proteins implicated in inflammation, immunoregulation, cell survival, angiogenesis, as well as tissue remodelling and fibrosis that could mediate the cardioreparative effects of secretome of human W8B2+ CSCs. CONCLUSIONS: Subcutaneous implantation of TheraCyte devices encapsulating human W8B2+ CSCs attenuated adverse cardiac remodelling and preserved cardiac function following myocardial infarction. The TheraCyte device can be employed to deliver stem cells in a minimally invasive manner for effective secretome-based cardiac therapy.

Fabrication of homotypic neural ribbons as a multiplex platform optimized for spinal cord delivery.

Cell therapy for the injured spinal cord will rely on combined advances in human stem cell technologies and delivery strategies. Here we encapsulate homotypic spinal cord neural stem cells (scNSCs) in an alginate-based neural ribbon delivery platform. We perform a comprehensive in vitro analysis and qualitatively demonstrate graft survival and injury site retention using a rat C4 hemi-contusion model. Pre-configured neural ribbons are transport-stable modules that enable site-ready injection, and can support scNSC survival and retention in vivo. Neural ribbons offer multifunctionality in vitro including co-encapsulation of the injury site extracellular matrix modifier chondroitinase ABC (chABC), tested here in glial scar models, and ability of cervically-patterned scNSCs to differentiate within neural ribbons and project axons for integration with 3-D external matrices. This is the first extensive in vitro characterization of neural ribbon technology, and constitutes a plausible method for reproducible delivery, placement, and retention of viable neural cells in vivo.

Mechanical emulsification of lipoaspirate by different Luer-Lok connector changes the viability of adipose derived stem cells in Nanofat.

Nanofat grafting is a fat transfer procedure that uses a thin needle to smooth out wrinkles, thereby achieving the goal of skin rejuvenation. The Luer-Lok connector is one of the most common methods for obtaining Nanofat. In the present study, we compared three different Luer-Lok connectors (2.0 mm, 1.5 mm and 1.1 mm in diameter) in terms of their impact on the viability of adipose-derived stem cells (ADSCs) to determine the optimal size of the connector for efficient Nanofat grafting. We observed that a smaller diameter of the Luer-Lok connector created a higher mechanical shear force, which broke more fat cells during the emulsifying procedure, thereby reducing the viability of ADSCs from the stromal vascular fraction (SVF). Nanofat obtained from the 2-mm Luer-Lok connector had a better effect on skin rejuvenation than the 1.5-mm and 1.1-mm connectors. Therefore, this study presents an advance in the simple procedure of preparing Nanofat based on a previous technique and provides evidence that a procedure associated with less trauma may be a better choice.

Adipose-derived stem/progenitor cells from lipoaspirates: A comparison between the Lipivage200-5 liposuction system and the Body-Jet liposuction system.

BACKGROUND: Adipose-derived stem/progenitor cells (ADSPCs) are under investigation in many clinical applications for their regenerative potential in a variety of autoimmune, degenerative, and inflammatory diseases. Adipose tissue, which is mainly harvested by manual liposuction, is the main source of these ADSPCs. OBJECTIVE: In the past years, a variety of different liposuction devices have been commercialized. To ensure a high quality of obtained ADSPCs, it is crucial to show the advantages and disadvantages of frequently used liposuction methods. For this reason, the objective of this study was to compare ADSPCs harvested by either the suction-assisted LipiVage200-5 or the water-assisted Body-Jet system. METHODS: The proliferation potential of ADSPCs, harvested from 20 patients, was assessed by cumulative population doublings (cumPD), population doubling time (PDT), colony-forming units (CFU), and cell metabolism assays. To prove the multipotency of the primary isolated cells, ADSPCs were induced to differentiate into adipogenic, osteogenic, and chondrogenic lineages. RESULTS: Our data show a significantly higher cumPD, as well as a significantly lower PDT for cells obtained by the Body-Jet system. No significant differences were found regarding the CFU efficiency and the cell metabolism. Furthermore, we showed that the adipogenic, osteogenic, and chondrogenic potential of ADSPCs is similar in both groups. DISCUSSION/CONCLUSION: In our study, we provide evidence that the cell characteristics and the functional properties of ADSPCs isolated after liposuction with different techniques are largely similar. However, we observed a significantly higher cumPD and a slightly higher adipogenic potential in cells isolated after liposuction with the Body-Jet system. Different cannula sizes and sheer stresses in the used methods might play a role here.

Reversible Thermoresponsive Hydrogel Fabricated from Natural Biopolymer for the Improvement of Critical Limb Ischemia by Controlling Release of Stem Cells.

Stem cells therapy is an effective treatment for critical limb ischemia diseases (CLI), but is limited to low cells retention and poor target release in severe ischemia tissues. Due to the notable feature of CLI, namely, the temperature of ischemia tissues decreases with the severity of the lesions, a thermoresponsive and reversible hydrogel based on methylcellulose-salt system encapsulating stem cells is facilely prepared and successfully achieved the goal of releasing stem cells in lower temperature areas. The investigations show that the thermogel presents notable biocompatibility, thermoresponsiveness, and cytoprotection. Furthermore, the combined transplantation of hydrogel and stem cells system effectively inhibits the fibrosis and muscular atrophy of lower limb ischemia, accelerates the recovery of lower limb blood flow, and promotes angiogenesis, indicating that the reversible thermogel can promote vascular repair by controlling the release of loaded stem cells in the treatment of CLI.

Bringing Stem Cell-Based Therapies for Type 1 Diabetes to the Clinic: Early Insights from Bioprocess Economics and Cost-Effectiveness Analysis.

Differentiation of pluripotent stem cells (PSCs) into ß cells could provide insulin independence for type 1 diabetes (T1D) patients. This approach would reduce the clinical complications that most patients managed on intensive insulin therapy (IIT) face. However, bottlenecks of PSC manufacturing and limited engraftment of encapsulated cells hinder the long-term effectiveness of these therapies. A bioprocess decision-support tool is combined with a disease state-transition model to evaluate the cost-effectiveness of the stem cell-based therapy against IIT. Clinical effectiveness is assessed in quality-adjusted life years (QALYs). Manufacturing costs per patient reduce from $430 000 to $160 000 with optimization of batch size and annual demand. For 96% of the patients, cell therapy improves the quality of life compared to IIT. Cost savings are achieved for 2% of the population through prevention of renal disease. The therapy is cost-effective for 3.4% of patients when a willingness to pay (WTP) of up to $150 000 per QALY is considered. A 75% cost reduction in the cell therapy price increases cost-effectiveness likelihood to 51% at $100 000 per QALY. This study highlights the need for scalable manufacturing platforms for stem cell therapies, as well as to prioritizing access to the therapy to patients with an increased likelihood of costly complications.

Development of a transplant injection device for optimal distribution and retention of human induced pluripotent stem cell‒derived cardiomyocytes.

BACKGROUND: Induced pluripotent stem cell (iPSC)‒based regenerative therapy is a promising strategy for cardiovascular disease treatment; however, the method is limited by the myocardial retention of grafted iPSCs. Thus, an injection protocol that efficiently introduces and retains human iPSC-derived cardiomyocytes (hiPSC-CMs) within the myocardium is urgently needed. The objective of the present study was to develop a method to improve the retention of hiPSCs in the myocardium for cardiac therapy. METHODS: We efficiently produced hiPSC-CM spheroids in 3-dimensional (3D) culture using microwell plates, and developed an injection device for optimal 3D distribution of the spheroids in the myocardial layer. Device biocompatibility was assessed with purified hiPSC-CM spheroids. Device effectiveness was evaluated in 10- to 15-month-old farm pigs (n = 15) and 5- to 24-month-old micro-minipigs (n = 20). The pigs were euthanized after injection, and tissues were harvested for retention and histologic analysis. RESULTS: We demonstrated an injection device for direct intramyocardial transplantation of hiPSC-CM spheroids from large-scale culture. The device had no detrimental effects on cell viability, spheroid shape, or size. Direct epicardial injection of spheroids mixed with gelatin hydrogel into beating porcine hearts using this device resulted in better distribution and retention of transplanted spheroids in a layer within the myocardium than did conventional needle injection procedures. CONCLUSIONS: The combination of the newly developed transplant device and spheroid formation promotes the retention of transplanted CMs. These findings support the clinical application of hiPSC-CM spheroid‒based cardiac regenerative therapy in patients with heart failure.

Method and Apparatus for the Automated Delivery of Continuous Neural Stem Cell Trails Into the Spinal Cord of Small and Large Animals.

BACKGROUND: Immature neurons can extend processes after transplantation in adult animals. Neuronal relays can form between injected neural stem cells (NSCs) and surviving neurons, possibly improving recovery after spinal cord injury (SCI). Cell delivery methods of single or multiple bolus injections of concentrated cell suspensions thus far tested in preclinical and clinical experiments are suboptimal for new tract formation. Nonuniform injectate dispersal is often seen due to gravitational cell settling and clumping. Multiple injections have additive risks of hemorrhage, parenchymal damage, and cellular reflux and require additional surgical exposure. The deposition of multiply delivered cells boluses may be uneven and discontinuous. OBJECTIVE: To develop an injection apparatus and methodology to deliver continuous cellular trails bridging spinal cord lesions. METHODS: We improved the uniformity of cellular trails by formulating NSCs in hyaluronic acid. The TrailmakerTM stereotaxic injection device was automatized to extend a shape memory needle from a single-entry point in the spinal cord longitudinal axis to "pioneer" a new trail space and then retract while depositing an hyaluronic acid-NSC suspension. We conducted testing in a collagen spinal models, and animal testing using human NSCs (hNSCs) in rats and minipigs. RESULTS: Continuous surviving trails of hNSCs within rat and minipig naive spinal cords were 12 and 40 mm in length. hNSC trails were delivered across semi-acute contusion injuries in rats. Transplanted hNSCs survived and were able to differentiate into neural lineage cells and astrocytes. CONCLUSION: The TrailmakerTM creates longitudinal cellular trails spanning multiple levels from a single-entry point. This may enhance the ability of therapeutics to promote functional relays after SCI.

An exploratory study on the preparation and evaluation of a "same-day" adipose stem cell-based tissue-engineered vascular graft.

OBJECTIVE: Tissue-engineered vascular grafts containing adipose-derived mesenchymal stem cells offer an alternative to small-diameter vascular grafts currently used in cardiac and lower-extremity revascularization procedures. Adipose-derived, mesenchymal stem cell-infused, tissue-engineered vascular grafts have been shown to promote remodeling and vascular homeostasis in vivo and offer a possible treatment solution for those with cardiovascular disease. Unfortunately, the time needed to cultivate adipose-derived mesenchymal stem cells remains a large hurdle for tissue-engineered vascular grafts as a treatment option. The purpose of this study was to determine if stromal vascular fraction (known to contain progenitor cells) seeded tissue-engineered vascular grafts would remain patent in vivo and remodel, allowing for a "same-day" process for tissue-engineered vascular graft fabrication and implantation. METHODS: Stromal vascular fraction, obtained from adult human adipose tissue, was seeded within 4 hours after acquisition from the patient onto poly(ester urethane)urea bilayered scaffolds using a customized rotational vacuum seeding device. Constructs were then surgically implanted as abdominal aortic interposition grafts in Lewis rats. RESULTS: Findings revealed patency in 5 of 7 implanted scaffolds at 8 weeks, along with neotissue formation and remodeling occurring in patent tissue-engineered vascular grafts. Patency was documented using angiography and gross inspection, and remodeling and vascular components were detected using immunofluorescent chemistry. CONCLUSIONS: A "same-day" cell-seeded, tissue-engineered vascular graft can remain patent after implantation in vivo, with neotissue formation and remodeling occurring by 8 weeks.

Ex vivo biomechanical characterization of syringe-needle ejections for intracerebral cell delivery.

Intracerebral implantation of cell suspensions is finding its clinical translation with encouraging results in patients with stroke. However, the survival of cells in the brain remains poor. Although the biological potential of neural stem cells (NSCs) is widely documented, the biomechanical effects of delivering cells through a syringe-needle remain poorly understood. We here detailed the biomechanical forces (pressure, shear stress) that cells are exposed to during ejection through different sized needles (20G, 26G, 32G) and syringes (10, 50, 250 µL) at relevant flow rates (1, 5, 10 µL/min). A comparison of 3 vehicles, Phosphate Buffered Saline (PBS), Hypothermosol (HTS), and Pluronic, indicated that less viscous vehicles are favorable for suspension with a high cell volume fraction to minimize sedimentation. Higher suspension viscosity was associated with greater shear stress. Higher flow rates with viscous vehicle, such as HTS reduced viability by ~10% and also produced more apoptotic cells (28%). At 5 µL/min ejection using a 26G needle increased neuronal differentiation for PBS and HTS suspensions. These results reveal the biological impact of biomechanical forces in the cell delivery process. Appropriate engineering strategies can be considered to mitigate these effects to ensure the efficacious translation of this promising therapy.

An oxygen plasma treated poly(dimethylsiloxane) bioscaffold coated with polydopamine for stem cell therapy.

In this study, 3D macroporous bioscaffolds were developed from poly(dimethylsiloxane) (PDMS) which is inert, biocompatible, non-biodegradable, retrievable and easily manufactured at low cost. PDMS bioscaffolds were synthesized using a solvent casting and particulate leaching (SCPL) technique and exhibited a macroporous interconnected architecture with 86 ± 3% porosity and 300 ± 100 µm pore size. As PDMS intrinsically has a hydrophobic surface, mainly due to the existence of methyl groups, its surface was modified by oxygen plasma treatment which, in turn, enabled us to apply a novel polydopamine coating onto the surface of the bioscaffold. The addition of a polydopamine coating to bioscaffolds was confirmed using composition analysis. Characterization of oxygen plasma treated-PDMS bioscaffolds coated with polydopamine (polydopamine coated-PDMS bioscaffolds) showed the presence of hydroxyl and secondary amines on their surface which resulted in a significant decrease in water contact angle when compared to uncoated-PDMS bioscaffolds (35 ± 3%, P < 0.05). Seeding adipose tissue-derived mesenchymal stem cells (AD-MSCs) into polydopamine coated-PDMS bioscaffolds resulted in cells demonstrating a 70 ± 6% increase in viability and 40 ± 5% increase in proliferation when compared to AD-MSCs seeded into uncoated-PDMS bioscaffolds (P < 0.05). In summary, this two-step method of oxygen plasma treatment followed by polydopamine coating improves the biocompatibility of PDMS bioscaffolds and only requires the use of simple reagents and mild reaction conditions. Hence, our novel polydopamine coated-PDMS bioscaffolds can represent an efficient and low-cost bioscaffold platform to support MSC therapies.

An Intelligent Neural Stem Cell Delivery System for Neurodegenerative Diseases Treatment.

Transplanted stem cells constitute a new therapeutic strategy for the treatment of neurological disorders. Emerging evidence indicates that a negative microenvironment, particularly one characterized by the acute inflammation/immune response caused by physical injuries or transplanted stem cells, severely impacts the survival of transplanted stem cells. In this study, to avoid the influence of the increased inflammation following physical injuries, an intelligent, double-layer, alginate hydrogel system is designed. This system fosters the matrix metalloproeinases (MMP) secreted by transplanted stem cell reactions with MMP peptide grafted on the inner layer and destroys the structure of the inner hydrogel layer during the inflammatory storm. Meanwhile, the optimum concentration of the arginine-glycine-aspartate (RGD) peptide is also immobilized to the inner hydrogels to obtain more stem cells before arriving to the outer hydrogel layer. It is found that blocking Cripto-1, which promotes embryonic stem cell differentiation to dopamine neurons, also accelerates this process in neural stem cells. More interesting is the fact that neural stem cell differentiation can be conducted in astrocyte-differentiation medium without other treatments. In addition, the system can be adjusted according to the different parameters of transplanted stem cells and can expand on the clinical application of stem cells in the treatment of this neurological disorder.

Stem Cell Smart Technology, where are we now and how far we have to go?

Approximately eight million people in the United States have peripheral arterial disease, which increases exponentially with age. There have been a plethora of available treatments including surgery, angioplasty, atherectomy, laser technology, and cell-based therapies. Cell-based therapies were developed in the hope of translating laboratory-based technology into clinical successes. However, clinical results have been disappointing. Infusion or injection for stem cell therapy is still considered experimental and investigational, and major questions on safety and durability have arisen. In no option patients, how can they be treated safely and successfully? In this article, we review contemporary practice for cell therapy, its pitfalls and breakthroughs, and look at the future ahead. We introduce a novel smart system for minimally invasive delivery of cell therapies, which exemplifies the next generation of endovascular solutions to stem cell technology and promises safety, efficacy, and reliability.

Transplantation of Zebrafish Cells by Conventional Pneumatic Microinjector.

Generating chimeric zebrafish by transplantation is extremely useful for live imaging in developmental, stem cell, and cancer biology, and to answer the questions of how cells acquire, keep, and/or change their fate. However, as it is technically challenging, the use of transplantation approach remains very limited by the zebrafish community. In this study, we show that this cell grafting operation can be easily achieved by using a conventional pneumatic microinjector normally used for microinjections. Compared with previously published protocols, which need additional transplantation apparatus, this alternative transplantation method works well, but needs a simpler experimental setup, and is more accessible to all investigators.

Ultrasound guided percutaneous common carotid artery access in piglets for intracoronary stem cell infusion.

In pigs, the deep location of the common carotid artery and overlying sternomastoideus muscle in the neck has led to the recommendation for a surgical cutdown for common carotid access, as opposed to minimally invasive techniques for vascular access. We sought to determine if direct percutaneous common carotid artery access in piglets is attainable. Seventeen piglets were anesthetized and intubated. Under two-dimensional and color flow Doppler ultrasound guidance, a 21 gauge needle was utilized to access the right common carotid artery. Following arterial puncture, the Seldinger technique was applied to place a 4 or 5 French introducer. Upon completion of cardiac catheterization with intracoronary stem cell infusion the introducer was removed and manual pressure was applied to prevent hematoma development. Successful access with an introducer was achieved in all 17 piglets. The average weight was 8.5 ± 1.7 kg. One piglet developed a hematoma with hemorrhaging from the catheterization site and was euthanized. This piglet was given bivalirudin for the procedure. After this incident, subsequent piglets were not given anticoagulation and no other complications occurred. Ultrasound guided percutaneous common carotid artery access in piglets is attainable in a safe, reliable, and reproducible manner when performed by microvascular experts.

[The emerging technology of tissue engineering : Focus on stem cell niche]. / Zukunftstechnologie Tissue-Engineering : Fokus auf die Stammzellnische.

Limbal stem cells reside in a highly specialized complex microenvironment that is known as the stem cell niche, an anatomically protected region at the bottom of the Palisades of Vogt, where the stem cells are located and where their quiescence, proliferation and differentiation are maintained in balance. Besides the epithelial stem and progenitor cell clusters, the limbal niche comprises several types of supporting niche cells and a specific extracellular matrix mediating biochemical and biophysical signals. Stem cell-based tissue engineering aims to mimic the native stem cell niche and to present appropriate microenvironmental cues in a controlled and reproducible fashion in order to maintain stem cell function within the graft. Current therapeutic approaches for ex vivo expansion of limbal stem cells only take advantage of surrogate niches. However, new insights into the molecular composition of the limbal niche and innovative biosynthetic scaffolds have stimulated novel strategies for niche-driven stem cell cultivation. Promising experimental approaches include collagen-based organotypic coculture systems of limbal epithelial stem cells with their niche cells and biomimetic hydrogel platforms prefunctionalized with appropriate biomolecular and biophysical signals. Future translation of these novel regenerative strategies into clinical application is expected to improve long-term outcomes of limbal stem cell transplantation for ocular surface reconstruction.