Generation of a human induced pluripotent stem cell line from a patient with hypomyelinating leukodystrophy 22 (HLD22).

Hypomyelinating Leukodystrophy 22 (HLD22) is caused by a stoploss mutation in CLDN11. To study the molecular mechanisms underlying HLD22, human induced pluripotent stem cells (hiPSCs) were generated from patient fibroblasts carrying the stop-loss mutation in CLDN11.

Development of iPSC-based clinical trial selection platform for patients with ultrarare diseases.

A "Leap-of-Faith" approach is used to treat patients with previously unknown ultrarare pathogenic mutations, often based on evidence from patients having dissimilar but more prevalent mutations. This uncertainty reflects the need to develop personalized prescreening platforms for these patients to assess drug efficacy before considering clinical trial enrollment. In this study, we report an 18-year-old patient with ultrarare Leigh-like syndrome. This patient had previously participated in two clinical trials with unfavorable responses. We established an induced pluripotent stem cell (iPSC)-based platform for this patient, and assessed the efficacy of a panel of drugs. The iPSC platform validated the safety and efficacy of the screened drugs. The efficacy of three of the screened drugs was also investigated in the patient. After 3 years of treatment, the drugs were effective in shifting the metabolic profile of this patient toward healthy control. Therefore, this personalized iPSC-based platform can act as a prescreening tool to help in decision-making with respect to patient's participation in future clinical trials.

Human induced pluripotent stem cell (hiPSC) line UOMi006-A derived from PBMCs of a patient with Kearns-Sayre syndrome.

Induced pluripotent stem cell (iPSC) line was derived from peripheral blood mononuclear cells (PBMCs) of a Kearns-Sayre syndrome (KSS) patient with mtDNA deletion of 4.8 kilobase fragment. KSS is an ultrarare multi-organ disorder and is characterized with (1.1 to 10 kilobase) deletion of mitochondrial DNA (mtDNA) with a frequency of ~1 in 100,000 individuals. Heteroplasmy in PBMCs allowed us to generate an iPSC line with normal mitochondrial DNA that can be used to study therapeutic prospective of iPSC and their derivatives and design future cell replacement therapies.

Development of Fluorine-Free Tantalum Carbide MXene Hybrid Structure as a Biocompatible Material for Supercapacitor Electrodes.

The application of nontoxic 2D transition-metal carbides (MXenes) has recently gained ground in bioelectronics. In group-4 transition metals, tantalum possesses enhanced biological and physical properties compared to other MXene counterparts. However, the application of tantalum carbide for bioelectrodes has not yet been explored. Here, fluorine-free exfoliation and functionalization of tantalum carbide MAX-phase to synthesize a novel Ta4C3Tx MXene-tantalum oxide (TTO) hybrid structure through an innovative, facile, and inexpensive protocol is demonstrated. Additionally, the application of TTO composite as an efficient biocompatible material for supercapacitor electrodes is reported. The TTO electrode displays long-term stability over 10 000 cycles with capacitance retention of over 90% and volumetric capacitance of 447 F cm-3 (194 F g-1) at 1 mV s-1. Furthermore, TTO shows excellent biocompatibility with human-induced pluripotent stem cells-derived cardiomyocytes, neural progenitor cells, fibroblasts, and mesenchymal stem cells. More importantly, the electrochemical data show that TTO outperforms most of the previously reported biomaterials-based supercapacitors in terms of gravimetric/volumetric energy and power densities. Therefore, TTO hybrid structure may open a gateway as a bioelectrode material with high energy-storage performance for size-sensitive applications.

Induced pluripotent stem cell line UOMi002-A from a patient with Leigh syndrome with compound heterozygous mutations in the NDUFV1 gene.

Within the umbrella of mitochondrial disorders, Leigh's disease is characterised as a rarer form with more than 80 genetic and mitochondrial DNA aberration variants. Here we report establishment of an induced pluripotent stem cell (iPSC) line from a 2.5 years old deceased female child, harbouring mutations in the NDUFV1 gene. One of the variants reported here is novel. The establishment of iPSC line allows development of a stable disease model for the specific variations, as there are no other cell/animal disease models for the same.

Hypoxia-induced downregulation of cyclooxygenase 2 leads to the loss of immunoprivilege of allogeneic mesenchymal stem cells.

Allogeneic mesenchymal stem cells (MSCs) from young and healthy donors are reported to hold the potential to treat several immunological and degenerative disorders. However, recent data from animal studies and clinical trials demonstrate that immunogenicity and poor survival of transplanted MSCs impaired the efficacy of cells for regenerative applications. It is reported that initially immunoprivileged under in vitro conditions, MSCs are targeted by the host immune system after transplantation in the ischemic tissues in vivo. We performed in vitro (in MSCs) and in vivo (in the rat model of myocardial infarction [MI]) studies to elucidate the mechanisms responsible for the change in the immunophenotype of MSCs from immunoprivileged to immunogenic under ischemic conditions. We have recently reported that a soluble factor prostaglandin E2 (PGE2) preserves the immunoprivilege of allogeneic MSCs. In the current study, we found that PGE2 levels, which were elevated during normoxia, decreased in MSCs following exposure to hypoxia. Further, we found that proteasome-mediated degradation of cyclooxygenase-2 (COX2, rate-limiting enzyme in PGE2 biosynthesis) in hypoxic MSCs is responsible for PGE2 decrease and loss of immunoprivilege of MSCs. While investigating the mechanisms of COX2 degradation in hypoxic MSCs, we found that in normoxic MSCs, COP9 signalosome subunit 5 (CSN5) binds to COX2 and prevents its degradation by the proteasome. However, exposure to hypoxia leads to a decrease in CSN5 levels and its binding to COX2, rendering COX2 protein susceptible to proteasome-mediated degradation. This subsequently causes PGE2 downregulation and loss of immunoprivilege of MSCs. Maintaining COX2 levels in MSCs preserves immunoprivilege in vitro and improves the survival of transplanted MSCs in a rat model of MI. These data provide novel mechanistic evidence that PGE2 is downregulated in hypoxic MSCs which is responsible for the post-transplantation rejection of allogeneic MSCs. Therefore, our data suggest that the new strategies that target CSN5-COX2 signaling may improve survival and utility of transplanted allogeneic MSCs in the ischemic heart.

Generation of human induced pluripotent stem cell (hiPSC) line UOMi001-A from a patient with Leigh-like syndrome harbouring compound heterozygous variants in ECHS1 gene.

Leigh syndrome is a rare multi-organ system disorder that affects less than 1 in 5000 births. In cases where clinical heterogeneity makes some presentations difficult to categorize as Leigh syndrome, but are highly suggestive, those are referred to as Leigh-like syndrome. It may present with delay after birth and can be slightly milder than classic Leigh. We have created an iPSC line for the novel variants in the ECHS1 gene that was reported in our patient. This cellular model is being used to determine prospective treatment opportunities for the patient.

Establishment of variant free-iPSC (UOMi003-A) line from patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes.

Heteroplasmy in patients affected with Mitochondrial Encephalopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) offers a chance to harvest specific cells which might have a very low or no mutation load of the mitochondrial DNA. Here we demonstrate establishment of induced pluripotent stem cells (iPSC) cell lines (with normal mitochondrial DNA copies), from unaffected tissues of a male patient with MELAS harbouring m.3243A > G mutations. This platform allowed us to compare specific pathway differences between the cells of the affected tissues of the patients and their isogenic counterparts derived from iPSCs, which do not harbour the mutations.

Sweet-MXene hydrogel with mixed-dimensional components for biomedical applications.

Biodegradable hydrogels are promising extracellular matrix-like materials for biomedical applications due to their high compatibility, ease of administration and minimal invasion. The injectable hydrogels to be considered for regenerative therapies should mimic the intrinsic properties of tissues, i.e. self-healing and swelling. Here, we present facile electrically conductive sweet-MXene (S-MXene) hydrogel with novel mixed-dimensional compositions including natural zero dimensional (0D) fluorescent carbon dots in honey, delaminated 2D fluorescent titanium carbide (Ti3C2) nanosheets and bioinspired 3D crosslinked polymeric chitosan networks. The developed versatile (Ti3C2-MXene-honey-chitosan) heterostructure exhibited excellent porous architecture with desired swelling and controlled degradation. This electrically conductive composite is highly biocompatible, it supported cell attachment and survival.

Application of Ti3 C2 MXene Quantum Dots for Immunomodulation and Regenerative Medicine.

Inflammation is tightly linked to tissue injury. In regenerative medicine, immune activation plays a key role in rejection of transplanted stem cells and reduces the efficacy of stem cell therapies. Next-generation smart biomaterials are reported to possess multiple biologic properties for tissue repair. Here, the first use of 0D titanium carbide (Ti3 C2 ) MXene quantum dots (MQDs) for immunomodulation is presented with the goal of enhancing material-based tissue repair after injury. MQDs possess intrinsic immunomodulatory properties and selectively reduce activation of human CD4+ IFN-γ+ T-lymphocytes (control 87.1 ± 2.0%, MQDs 68.3 ± 5.4%) while promoting expansion of immunosuppressive CD4+ CD25+ FoxP3+ regulatory T-cells (control 5.5 ± 0.7%, MQDs 8.5 ± 0.8%) in a stimulated lymphocyte population. Furthermore, MQDs are biocompatible with bone marrow-derived mesenchymal stem cells and induced pluripotent stem cell-derived fibroblasts. Finally, Ti3 C2 MQDs are incorporated into a chitosan-based hydrogel to create a 3D platform with enhanced physicochemical properties for stem cell delivery and tissue repair. This composite hydrogel demonstrates increased conductivity while maintaining injectability and thermosensitivity. These findings suggest that this new class of biomaterials may help bridge the translational gap in material and stem cell-based therapies for tissue repair and treatment of inflammatory and degenerative diseases.

Hypoxia-induced 26S proteasome dysfunction increases immunogenicity of mesenchymal stem cells.

Bone marrow-derived allogeneic (donor derived) mesenchymal stem cells (MSCs) are immunoprivileged and are considered to be prominent candidates for regenerative therapy for numerous degenerative diseases. Even though the outcome of initial allogeneic MSCs based clinical trials was encouraging, the overall enthusiasm, of late, has dimmed down. This is due to failure of long-term survival of transplanted cells in the recipient. In fact, recent analyses of allogeneic MSC-based studies demonstrated that cells after transplantation turned immunogenic and were subsequently rejected by host immune system. The current study reveals a novel mechanism of immune switch in MSCs. We demonstrate that hypoxia, a common denominator of ischemic tissues, induces an immune shift in MSCs from immunoprivileged to immunogenic state. The immunoprivilege of MSCs is preserved by downregulation or the absence of major histocompatibility complex class II (MHC-II) molecules. We found that 26S proteasome-mediated intracellular degradation of MHC-II helps maintain the absence of MHC-II expression on cell surface in normoxic MSCs and preserves their immunoprivilege. The exposure to hypoxia leads to dissociation of 19S and 20S subunits, and inactivation of 26S proteasome. This prevented the degradation of MHC-II and, as a result, the MSCs became immunogenic. Furthermore, we found that hypoxia-induced decrease in the levels of a chaperon protein HSP90α is responsible for inactivation of 26S proteasome. Maintaining HSP90α levels in hypoxic MSCs preserved the immunoprivilege of MSCs. Therefore, hypoxia-induced inactivation of 26S proteasome assembly instigates loss of immunoprivilege of allogeneic mesenchymal stem cells. Maintaining 26S proteasome activity in mesenchymal stem cells preserves their immunoprivilege.

Elimination or neutralization of endogenous high-molecular-weight FGF2 mitigates doxorubicin-induced cardiotoxicity.

Cardiac fibroblast growth factor 2 (FGF2) exerts multiple paracrine activities related to cardiac response to injury. Endogenous FGF2 is composed of a mixture of 70% high- and 30% low-molecular-weight isoforms (Hi-FGF2 and Lo-FGF2, respectivley); although exogenously added Lo-FGF2 is cardioprotective, the roles of endogenous Hi-FGF2 or Lo-FGF2 have not been well defined. Therefore, we investigated the effect of elimination of Hi-FGF2 expression on susceptibility to acute cardiac damage in vivo caused by an injection of the genotoxic drug doxorubicin (Dox). Mice genetically depleted of endogenous Hi-FGF2 and expressing only Lo-FGF2 [FGF2(Lo) mice] were protected from the Dox-induced decline in ejection fraction displayed by their wild-type FGF2 [FGF2(WT)] mouse counterparts, regardless of sex, as assessed by echocardiography for up to 10 days post-Dox treatment. Because cardiac FGF2 is produced mainly by nonmyocytes, we next addressed potential contribution of fibroblast-produced FGF2 on myocyte vulnerability to Dox. In cocultures of neonatal rat cardiomyocytes (r-cardiomyocytes) with mouse fibroblasts from FGF2(WT) or FGF2(Lo) mice, only the FGF2(Lo)-fibroblast cocultures protected r-cardiomyocytes from Dox-induced mitochondrial and cellular damage. When r-cardiomyocytes were cocultured with or exposed to conditioned medium from human fibroblasts, neutralizing antibodies for human Hi-FGF-2, but not total FGF2, mitigated Dox-induced injury of cardiomyocytes. We conclude that endogenous Hi-FGF2 reduces cardioprotection by endogenous Lo-FGF2. Antibody-based neutralization of endogenous Hi-FGF2 may offer a prophylactic treatment against agents causing acute cardiac damage. NEW & NOTEWORTHY Cardiomyocytes, in vivo and in vitro, were protected from the deleterious effects of the anticancer drug doxorubicin by the genetic elimination or antibody-based neutralization of endogenous paracrine high-molecular-weight fibroblast growth factor 2 isoforms. These findings have a translational potential for mitigating doxorubicin-induced cardiac damage in patients with cancer by an antibody-based treatment.

High throughput screening reveals no significant changes in protein synthesis, processing, and degradation machinery during passaging of mesenchymal stem cells 1.

Increasing reports of successful and safe application of bone marrow derived mesenchymal stem cells (BM-MSCs) for cell therapy are pouring in from numerous studies. However poor survival of transplanted cells in the recipient has impaired the benefits of BM-MSCs based therapies. Therefore cell product preparation procedures pertaining to MSC therapy need to be optimized to improve the survival of transplanted cells. One of the important ex vivo procedures in the preparation of cells for therapy is passaging of BM-MSCs to ensure a suitable number of cells for transplantation, which may affect the turnover of proteins involved in regulation of cell survival and (or) death pathways. In the current study, we investigated the effect of an increase in passage number of BM-MSCs in cell culture on the intracellular protein turnover (protein synthesis, processing, and degradation machinery). We performed proteomic analysis of BM-MSCs at different passages. There was no significant difference observed in the ribosomal, protein processing, and proteasomal pathways related proteins in BM-MSCs with an increase in passage number from P3 to P7. Therefore, expansion of MSCs in the cell culture in clinically relevant passages (Passage 3-7) does not affect the quality of MSCs in terms of intracellular protein synthesis and turnover.

Graphene Oxide-Gold Nanosheets Containing Chitosan Scaffold Improves Ventricular Contractility and Function After Implantation into Infarcted Heart.

Abnormal conduction and improper electrical impulse propagation are common in heart after myocardial infarction (MI). The scar tissue is non-conductive therefore the electrical communication between adjacent cardiomyocytes is disrupted. In the current study, we synthesized and characterized a conductive biodegradable scaffold by incorporating graphene oxide gold nanosheets (GO-Au) into a clinically approved natural polymer chitosan (CS). Inclusion of GO-Au nanosheets in CS scaffold displayed two fold increase in electrical conductivity. The scaffold exhibited excellent porous architecture with desired swelling and controlled degradation properties. It also supported cell attachment and growth with no signs of discrete cytotoxicity. In a rat model of MI, in vivo as well as in isolated heart, the scaffold after 5 weeks of implantation showed a significant improvement in QRS interval which was associated with enhanced conduction velocity and contractility in the infarct zone by increasing connexin 43 levels. These results corroborate that implantation of novel conductive polymeric scaffold in the infarcted heart improved the cardiac contractility and restored ventricular function. Therefore, our approach may be useful in planning future strategies to construct clinically relevant conductive polymer patches for cardiac patients with conduction defects.

Early passaging of mesenchymal stem cells does not instigate significant modifications in their immunological behavior.

BACKGROUND: Bone marrow-derived allogeneic mesenchymal stem cells (MSCs) from young healthy donors are immunoprivileged and their clinical application for regenerative medicine is under evaluation. However, data from preclinical and initial clinical trials indicate that allogeneic MSCs after transplantation provoke a host immune response and are rejected. In the current study, we evaluated the effect of an increase in passage number in cell culture on immunoprivilege of the MSCs. Since only limited numbers of MSCs can be sourced at a time from a donor, it is imperative to expand them in culture to meet the necessary numbers required for cell therapy. Presently, the most commonly used passages for transplantation include passages (P)3-7. Therefore, in this study we included clinically relevant passages, i.e., P3, P5, and P7, for evaluation. METHODS: The immunoprivilege of MSCs was assessed with the mixed leukocyte reaction assay, where rat MSCs were cocultured with peripheral blood leukocytes for 72 h. Leukocyte-mediated cytotoxicity, apoptosis (Bax/Bcl-xl ratio), leukocyte proliferation, and alterations in cellular bioenergetics in MSCs were assessed after the coculture. Furthermore, the expression of various oxidized phospholipids (oxidized phosphatidylcholine (ox-PC)) was analyzed in MSCs using a lipidomic platform. To determine if the ox-PCs were acting in tandem with downstream intracellular protein alterations, we performed proteome analysis using a liquid chromatography/mass spectrometry (LC/MS) proteomic platform. RESULTS: Our data demonstrate that MSCs were immunoprivileged at all three passages since coculture with leukocytes did not affect the survival of MSCs at P3, P5, and P7. We also found that, with an increase in the passage number of MSCs, leukocytes did not cause any significant effect on cellular bioenergetics (basal respiration rate, spare respiratory capacity, maximal respiration, and coupling efficiency). Interestingly, in our omics data, we detected alterations in some of the ox-PCs and proteins in MSCs at different passages; however, these changes were not significant enough to affect their immunoprivilege. CONCLUSIONS: The outcome of this study demonstrates that an increase in passage number (from P3 to P7) in the cell culture does not have any significant effect on the immunoprivilege of MSCs.

Derivation of Mesenchymal Stem Cells from Embryonic Stem Cells: A Non-Variable and Inexhaustive Source of Adult Stem Cells.

Mesenchymal stem cells (MSCs) provide an opportunity to bring the field of regenerative medicine to realization. A lot of clinical trials are presently trying to establish their applicability in real-world scenarios. Some of the biggest challenges encountered in bringing MSCs from bench to bedside are the number of MSCs required, their procurement from various sources, and the batch-to-batch variability. This often leads to inconclusive results within and between different studies. Therefore, we have hereby proposed a simple protocol to source mesenchymal stem cells through differentiation of embryonic stem cells.

Human-Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells as an Individual-Specific and Renewable Source of Adult Stem Cells.

This chapter deals with the employment of human-induced pluripotent stem cells (hiPSCs) as a candidate to differentiate into mesenchymal stem cells (MSCs). This would enable to help establish a regular source of human MSCs with the aim of avoiding the problems associated with procuring the MSCs either from different healthy individuals or patients, limited extraction potentials, batch-to-batch variations or from diverse sources such as bone marrow or adipose tissue. The procedures described herein allow for a guided and ensured approach for the regular maintenance of hiPSCs and their subsequent differentiation into MSCs using the prescribed medium. Subsequently, an easy protocol for the successive isolation and purification of the hiPSC-differentiated MSCs is outlined, which is carried out through passaging and can be further sorted through flow cytometry. Further, the maintenance and expansion of the resultant hiPSC-differentiated MSCs using appropriate characterization techniques, i.e., Reverse-transcription PCR and immunostaining is also elaborated. The course of action has been deliberated keeping in mind the awareness and the requisites available to even beginner researchers who mostly have access to regular consumables and medium components found in the general laboratory.

Methods for Long-Term Storage of Murine Bone Marrow-Derived Mesenchymal Stem Cells.

This chapter is based on a simplified method to validate the current preservation procedure of mesenchymal stem cells (MSCs). Currently, there are various media available for freezing and thus preserving the MSCs, making it hard to decide which agent will be apt for cellular requirements. The study describes the effect of two different compositions of freezing media used in regular cell culture experiments, on the morphology, proliferation, and doubling rate of MSCs. Commonly used agents for the cryopreservation of MSCs include DMSO (Dimethyl Sulfoxide) and FBS (Fetal Bovine Serum) and DMEM (Dulbecco's Modified Eagle Medium). To ascertain that the currently used agents do not lead to major changes in the MSC morphology and proliferation, the cells are frozen using the above-mentioned agents in different groups and then their effects analyzed. Thus, the chapter helps to decide what reagents can suit the MSCs, hence minimizing the laboratory to laboratory variability of their characteristics.

A Simple Protocol for the Generation of Cardiomyocytes from Human Pluripotent Stem Cells.

Efficient generation of cardiomyocytes from pluripotent stem cells (PSCs) for multiple downstream applications such as regenerative medicine, disease modeling, and drug screening remains a challenge. Cardiomyogenesis may be regulated in vitro by a controlled differentiation process, which involves various signaling molecules and extracellular environment. Here, we describe a simple method to efficiently generate cardiomyocytes from human embryonic stem cells and human induced pluripotent stem cells.

Stem cell therapy for cardiac regeneration: hits and misses.

Cardiac injury and loss of cardiomyocytes is a causative as well as a resultant condition of cardiovascular disorders, which are the leading cause of death throughout the world. This loss of cardiomyocytes cannot be completely addressed through the currently available drugs being administered, which mainly function only in relieving the symptoms. There is a huge potential being investigated for regenerative and cell replacement therapies through recruiting stem cells of various origins namely embryonic, reprogramming/induction, and adult tissue. These sources are being actively studied for translation to clinical scenarios. In this review, we attempt to discuss some of these promising scenarios, including the clinical trials and the obstacles that need to be overcome, and hope to address the direction in which stem cell therapy is heading.