Applications of Plant-Made Fibroblast Growth Factor for Human Pluripotent Stem Cells.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine.

The sinoatrial node extracellular matrix promotes pacemaker phenotype and protects automaticity in engineered heart tissues from cyclic strain.

The composite material-like extracellular matrix (ECM) in the sinoatrial node (SAN) supports the native pacemaking cardiomyocytes (PCMs). To test the roles of SAN ECM in the PCM phenotype and function, we engineered reconstructed-SAN heart tissues (rSANHTs) by recellularizing porcine SAN ECMs with hiPSC-derived PCMs. The hiPSC-PCMs in rSANHTs self-organized into clusters resembling the native SAN and displayed higher expression of pacemaker-specific genes and a faster automaticity compared with PCMs in reconstructed-left ventricular heart tissues (rLVHTs). To test the protective nature of SAN ECMs under strain, rSANHTs and rLVHTs were transplanted onto the murine thoracic diaphragm to undergo constant cyclic strain. All strained-rSANHTs preserved automaticity, whereas 66% of strained-rLVHTs lost their automaticity. In contrast to the strained-rLVHTs, PCMs in strained-rSANHTs maintained high expression of key pacemaker genes (HCN4, TBX3, and TBX18). These findings highlight the promotive and protective roles of the composite SAN ECM and provide valuable insights for pacemaking tissue engineering.

An immune deficient mouse model for mucopolysaccharidosis IIIA (Sanfilippo syndrome).

Mucopolysaccharidosis III (MPSIII, Sanfilippo syndrome) is a devastating lysosomal storage disease that primarily affects the central nervous system. MPSIIIA is caused by loss-of-function mutations in the gene coding for sulfamidase (N-sulfoglucosamine sulfohydrolase/SGSH) resulting in SGSH enzyme deficiency, a buildup of heparin sulfate and subsequent neurodegeneration. There is currently no cure or disease modifying treatment for MPSIIIA. A mouse model for MPSIIIA was characterized in 1999 and later backcrossed onto the C57BL/6 background. In the present study, a novel immune deficient MPSIIIA mouse model (MPSIIIA-TKO) was created by backcrossing the immune competent, C57BL/6 MPSIIIA mouse to an immune deficient mouse model lacking Rag2, CD47 and Il2rg genes. The resulting mouse model has undetectable SGSH activity, exhibits histological changes consistent with MPSIIIA and lacks T cells, B cells and NK cells. This new mouse model has the potential to be extremely useful in testing human cellular therapies in an animal model as it retains the MPSIIIA disease phenotype while tolerating xenotransplantation.

Role of MEF2C in the Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) not only provide an abundant source of vascular cells for potential therapeutic applications in vascular disease but also constitute an excellent model for understanding the mechanisms that regulate the differentiation and the functionality of vascular cells. Here, we reported that myocyte enhancer factor 2C (MEF2C) transcription factor, but not any other members of the MEF2 family, was robustly upregulated during the differentiation of vascular progenitors and endothelial cells (ECs) from hiPSCs. Vascular endothelial growth factors (VEGF) strongly induced MEF2C expression in endothelial lineage cells. The specific upregulation of MEF2C during the commitment of endothelial lineage was dependent on the extracellular signal regulated kinase (ERK). Moreover, knockdown of MEF2C with shRNA in hiPSCs did not affect the differentiation of ECs from these hiPSCs, but greatly reduced the migration and tube formation capacity of the hiPSC-derived ECs. Through a chromatin immunoprecipitation-sequencing, genome-wide RNA-sequencing, quantitative RT-PCR, and immunostaining analyses of the hiPSC-derived endothelial lineage cells with MEF2C inhibition or knockdown compared to control hiPSC-derived ECs, we identified TNF-related apoptosis inducing ligand (TRAIL) and transmembrane protein 100 (TMEM100) as novel targets of MEF2C. This study demonstrates an important role for MEF2C in regulating human EC functions and highlights MEF2C and its downstream effectors as potential targets to treat vascular malfunction-associated diseases.

HDACs regulate the differentiation of endothelial cells from human iPSCs.

Human induced pluripotent stem cells (hiPSCs) possess the potential to differentiate toward vascular cells including endothelial cells (ECs), pericytes, and smooth muscle cells. Epigenetic mechanisms including DNA methylation and histone modification play a crucial role in regulating lineage differentiation and specification. Herein, we utilized a three-stage protocol to induce differentiation of mesoderm, vascular progenitors, and ECs from hiPSCs and investigated the regulatory effects of histone acetylation on the differentiation processes. We found that the expression of several histone deacetylases (HDACs), including HDAC1, HDAC5, and HDAC7, were greatly upregulated at the second stage and downregulated at the third stage. Interestingly, although HDAC1 remained in the nucleus during the EC differentiation, HDAC5 and HDAC7 displayed cytosol/nuclear translocation during the differentiation process. Inhibition of HDACs with sodium butyrate (NaBt) or BML210 could hinder the differentiation of vascular progenitors at the second stage and facilitate EC induction at the third stage. Further investigation revealed that HDAC may modulate the stepwise EC differentiation via regulating the expression of endothelial transcription factors ERG, ETS1, and MEF2C. Opposite to the expression of EC markers, the smooth muscle/pericyte marker ACTA2 was upregulated at the second stage and downregulated at the third stage by NaBt. The stage-specific regulation of ACTA2 by HDAC inhibition was likely through regulating the expression of TGFß2 and PDGFB. This study suggests that HDACs play different roles at different stages of EC induction by promoting the commitment of vascular progenitors and impeding the later stage differentiation of ECs.

Improved MSC Minimal Criteria to Maximize Patient Safety: A Call to Embrace Tissue Factor and Hemocompatibility Assessment of MSC Products.

The number of mesenchymal stromal/stem cell (MSC) therapeutics and types of clinical applications have greatly diversified during the past decade, including rapid growth of poorly regulated "Stem Cell Clinics" offering diverse "Unproven Stem Cell Interventions." This product diversification necessitates a critical evaluation of the reliance on the 2006 MSC minimal criteria to not only define MSC identity but characterize MSC suitability for intravascular administration. While high-quality MSC therapeutics have been safely administered intravascularly in well-controlled clinical trials, repeated case reports of mild-to-more-severe adverse events have been reported. These are most commonly related to thromboembolic complications upon infusion of highly procoagulant tissue factor (TF/CD142)-expressing MSC products. As TF/CD142 expression varies widely depending on the source and manufacturing process of the MSC product, additional clinical cell product characterization and guidelines are needed to ensure the safe use of MSC products. To minimize risk to patients receiving MSC therapy, we here propose to supplement the minimal criteria used for characterization of MSCs, to include criteria that assess the suitability of MSC products for intravascular use. If cell products are intended for intravascular delivery, which is true for half of all clinical applications involving MSCs, the effects of MSC on coagulation and hemocompatibility should be assessed and expression of TF/CD142 should be included as a phenotypic safety marker. This adjunct criterion will ensure both the identity of the MSCs as well as the safety of the MSCs has been vetted prior to intravascular delivery of MSC products.

The oromaxillofacial region as a model for a one-health approach in regenerative medicine.

The concept of a one-health approach in regenerative medicine has gained tremendous momentum in the scientific and public communities in recent years. Knowledge derived from this approach informs innovative biomedical research, clinical trials, and practice. The ultimate goal is to translate regenerative strategies for curing diseases and improving the quality of life in animals and people. Building and fostering strong and enthusiastic interdisciplinary and transdisciplinary collaboration between teams with a wide range of expertise and backgrounds is the cornerstone to the success of the one-health approach and translational sciences. The veterinarian's role in conducting clinical trials in client-owned animals with naturally occurring diseases is critical and unique as it may potentially inform human clinical trials. The veterinary regenerative medicine and surgery field is on a steep trajectory of discoveries and innovations. This manuscript focuses on oromaxillofacial-region regeneration to exemplify how the concept of interdisciplinary and transdisciplinary collaboration and the one-health approach influenced the authors' work experience at the University of California-Davis.

Combination product of dermal matrix, preconditioned human mesenchymal stem cells and timolol promotes wound healing in the porcine wound model.

A combination product of human mesenchymal stem/stromal cells (MSCs) embedded in an extracellular matrix scaffold and preconditioned with hypoxia and the beta-adrenergic receptor antagonist, timolol, combined with sustained timolol application post implantation, has shown promising results for improving wound healing in a diabetic mouse model. In the present study, we extend those findings to the more translatable large animal porcine wound model and show that the combined treatment promotes wound reepithelialization in these excisional wounds by 40.2% and increases the CD31 immunostaining marker of angiogenesis compared with the matrix control, while maintaining an accumulated timolol plasma concentration below the clinically safe level of 0.3 ng/mL after the 15-day course of topical application. Human GAPDH was not elevated in the day 15 wounds treated with MSC-containing device relative to wounds treated with matrix alone, indicating that the xenografted human MSCs in the treatment do not persist in these immune-competent animals after 15 days. The work demonstrates the efficacy and safety of the combined treatment for improving healing in the clinically relevant porcine wound model.

Autologous Muscle-Derived Cell Therapy for Swallowing Impairment in Patients Following Treatment for Head and Neck Cancer.

OBJECTIVES/HYPOTHESIS: To evaluate the safety and potential efficacy of autologous muscle-derived cells (AMDCs) for the treatment of swallowing impairment following treatment for oropharynx cancer. STUDY DESIGN: Prospective, phase I, open label, clinical trial. METHODS: Oropharynx cancer survivors disease free ≥2 years post chemoradiation were recruited. All patients had swallowing impairment but were not feeding tube dependent (Functional Oral Intake Scale [FOIS] ≥ 5). Muscle tissue (50-250 mg) was harvested from the vastus lateralis and 150 × 106 AMDCs were prepared (Cook MyoSite Inc., Pittsburgh, PA). The cells were injected into four sites throughout the intrinsic tongue musculature. Participants were followed for 24 months. The primary outcome measure was safety. Secondary endpoints included objective measures on swallowing fluoroscopy, oral and pharyngeal pressure, and changes in patient-reported outcomes. RESULTS: Ten individuals were enrolled. 100% (10/10) were male. The mean age of the cohort was 65 (±8.87) years. No serious adverse event occurred. Mean tongue pressure increased significantly from 26.3 (±11.1) to 31.8 (±9.5) kPa (P = .017). The mean penetration-aspiration scale did not significantly change from 5.6 (±2.1) to 6.8 (±1.8), and the mean FOIS did not significantly change from 5.4 (±0.5) to 4.6 (±0.7). The incidence of pneumonia was 30% (3/10) and only 10% (1/10) experienced deterioration in swallowing function throughout 2 years of follow-up. The mean eating assessment tool (EAT-10) did not significantly change from 24.1 (±5.57) to 21.3 (±6.3) (P = .12). CONCLUSION: Results of this phase I clinical trial demonstrate that injection of 150 × 106 AMDCs into the tongue is safe and may improve tongue strength, which is durable at 2 years. A blinded placebo-controlled trial is warranted. LEVEL OF EVIDENCE: 3 Laryngoscope, 132:523-527, 2022.

Analysis of the retinal capillary plexus layers in a murine model with diabetic retinopathy: effect of intravitreal injection of human CD34+ bone marrow stem cells.

BACKGROUND: Diabetic retinopathy is a retinal vasculopathy involving all three retinal capillary plexus layers. Since human CD34+ bone marrow stem cells (BMSCs) have the potential to promote revascularization of ischemic tissue, this study tests the hypothesis that intravitreal injection of human CD34+ BMSCs can have protective effects on all layers of the retinal vasculature in eyes with diabetic retinopathy. METHODS: Streptozotocin (STZ)-induced diabetic mice were injected intravitreally with 50,000 human CD34+ BMSCs or phosphate-buffered saline (PBS) into the right eye. Systemic immunosuppression with rapamycin and tacrolimus was started 5 days before the injection and maintained for study duration to prevent rejection of human cells. All mice were euthanized 4 weeks after intravitreal injection; both eyes were enucleated for retinal flat mount immunohistochemistry. The retinal vasculature was stained with Isolectin-GS-IB4. Confocal microscopy was used to image four circular areas of interest of retina, 1-mm diameter around the optic disc. Images of superficial, intermediate, and deep retinal capillary plexus layers within the areas of interest were obtained and analyzed using ImageJ software with the Vessel Analysis plugin to quantitate the retinal vascular density and vascular length density in the three plexus layers. RESULTS: Three distinct retinal capillary plexus layers were visualized and imaged using confocal microscopy. Eyes that received intravitreal injection of CD34+ BMSCs (N=9) had significantly higher vascular density and vascular length density in the superficial retinal capillary plexus when compared to the untreated contralateral eyes (N=9) or PBS treated control eyes (N=12; P values <0.05 using ANOVA followed by post-hoc tests). For the intermediate and deep plexus layers, the difference was not statistically significant. CONCLUSIONS: The protective effect of intravitreal injection of the human CD34+ BMSCs on the superficial retinal capillary plexus layers is demonstrated using confocal microscopy in this murine model of diabetic retinopathy.

Subretinal versus intravitreal administration of human CD34+ bone marrow-derived stem cells in a rat model of inherited retinal degeneration.

BACKGROUND: To evaluate whether subretinal or intravitreal injection of human CD34+ bone marrow-derived stem cells (BMSC) can have protective effects on retinal degeneration that may be enhanced by coadministration of exosomes harvested from human bone marrow mesenchymal stem cells (MSCs). METHODS: Human CD34+ cells were harvested from the mononuclear cell fraction of bone marrow using magnetic beads and labeled with EGFP. Exosomes were harvested from cultured human MSCs under hypoxic conditions. Royal College of Surgeons (RCS) 3-weeks-old rats, immunosuppressed with cyclosporine A, received subretinal or intravitreal injection of CD34+ cells (50,000 cells), CD34+ cells with exosomes (50,000 cells+10 µg), exosomes alone (10 µg), or PBS. Retinal function was examined using electroretinography (ERG), and the eyes were harvested for histologic and immunohistochemical analysis. RESULTS: The b-wave amplitude of ERG at 2 weeks after injection was significantly higher in eyes with subretinal or intravitreal CD34+ BMSC alone or in combination with exosomes when compared to PBS injected eyes or untreated contralateral eyes. At 4 weeks after injection, the ERG signal decreased in all groups but eyes with subretinal CD34+ BMSCs alone or combined with exosomes showed partially preserved ERG signal and preservation of the outer nuclear layer of the retina near the injection site on histology when compared to eyes with PBS injection. Immunohistochemical analysis identified the human cells in the outer retina. Subretinal or intravitreal exosome injection had no effect on retinal degeneration when administered alone or in combination with CD34+ cells. CONCLUSIONS: Both subretinal and intravitreal injection of human CD34+ BMSCs can provide functional rescue of degenerating retina, although the effects were attenuated over time in this rat model. Regional preservation of the outer retina can occur near the subretinal injection site of CD34+ cells. These results suggest that CD34+ cells may have therapeutic potential in retinal degeneration.

Mesenchymal stromal cell variables influencing clinical potency: the impact of viability, fitness, route of administration and host predisposition.

The International Society for Cell & Gene Therapy mesenchymal stromal cell (MSC) committee has been an interested observer of community interests in all matters related to MSC identity, mechanism of action, potency assessment and etymology, and it has regularly contributed to this conversation through a series of MSC pre-conferences and committee publications dealing with these matters. Arising from these reflections, the authors propose that an overlooked and potentially disruptive perspective is the impact of in vivo persistence on potency that is not predicted by surrogate cellular potency assays performed in vitro and how this translates to in vivo outcomes. Systemic delivery or extravascular implantation at sites removed from the affected organ system seems to be adequate in affecting clinical outcomes in many pre-clinical murine models of acute tissue injury and inflammatory pathology, including the recent European Medicines Agency-approved use of MSCs in Crohn-related fistular disease. The authors further propose that MSC viability and metabolic fitness likely dominate as a potency quality attribute, especially in recipients poised for salutary benefits as defined by emerging predictive biomarkers of response.

An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.

NODAL inhibition promotes differentiation of pacemaker-like cardiomyocytes from human induced pluripotent stem cells.

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c, a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18, and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

Cell-based therapies for coronavirus disease 2019: proper clinical investigations are essential.

The serious consequences of the global coronavirus disease 2019 (COVID-19) pandemic have prompted a rapid global response to develop effective therapies that can lessen disease severity in infected patients. Cell-based approaches, primarily using mesenchymal stromal cells (MSCs), have demonstrated a strong safety profile and possible efficacy in patients with acute respiratory distress syndrome (ARDS), but whether these therapies are effective for treating respiratory virus-induced ARDS is unknown. According to the World Health Organization International Clinical Trials Registry Platform and the National Institutes of Health ClinicalTrials.gov databases, 27 clinical investigations of MSC-based cell therapy approaches have begun in China since the onset of the COVID-19 outbreak, with a growing number of academic and industry trials elsewhere as well. Several recent published reports have suggested potential efficacy; however, the available data presented are either anecdotal or from incomplete, poorly controlled investigations. Therefore, although there may be a potential role for MSCs and other cell-based therapies in treatment of COVID-19, these need to be investigated in a rationally designed, controlled approach if safety and efficacy are to be demonstrated accurately. The authors urge that the field proceed by finding a balance between swift experimentation and communication of results and scientifically coherent generation and analysis of clinical data.

Combination product of dermal matrix, human mesenchymal stem cells, and timolol promotes diabetic wound healing in mice.

Diabetic foot ulcers are a major health care concern with limited effective therapies. Mesenchymal stem cell (MSC)-based therapies are promising treatment options due to their beneficial effects of immunomodulation, angiogenesis, and other paracrine effects. We investigated whether a bioengineered scaffold device containing hypoxia-preconditioned, allogeneic human MSCs combined with the beta-adrenergic antagonist timolol could improve impaired wound healing in diabetic mice. Different iterations were tested to optimize the primary wound outcome, which was percent of wound epithelialization. MSC preconditioned in 1 µM timolol at 1% oxygen (hypoxia) seeded at a density of 2.5 × 105 cells/cm2 on Integra Matrix Wound Scaffold (MSC/T/H/S) applied to wounds and combined with daily topical timolol applications at 2.9 mM resulted in optimal wound epithelialization 65.6% (24.9% ± 13.0% with MSC/T/H/S vs 41.2% ± 20.1%, in control). Systemic absorption of timolol was below the HPLC limit of quantification, suggesting that with the 7-day treatment, accumulative steady-state timolol concentration is minimal. In the early inflammation stage of healing, the MSC/T/H/S treatment increased CCL2 expression, lowered the pro-inflammatory cytokines IL-1B and IL6 levels, decreased neutrophils by 44.8%, and shifted the macrophage ratio of M2/M1 to 1.9 in the wound, demonstrating an anti-inflammatory benefit. Importantly, expression of the endothelial marker CD31 was increased by 2.5-fold with this treatment. Overall, the combination device successfully improved wound healing and reduced the wound inflammatory response in the diabetic mouse model, suggesting that it could be translated to a therapy for patients with diabetic chronic wounds.

Improvement of motor and behavioral activity in Sandhoff mice transplanted with human CD34+ cells transduced with a HexA/HexB expressing lentiviral vector.

BACKGROUND: Tay-Sachs and Sandhoff disease are debilitating genetic diseases that affect the central nervous system leading to neurodegeneration through the accumulation of GM2 gangliosides. There are no cures for these diseases and treatments do not alleviate all symptoms. Hematopoietic stem cell gene therapy offers a promising treatment strategy for delivering wild-type enzymes to affected cells. By genetically modifying hematopoietic stem cells to express wild-type HexA and HexB, systemic delivery of functional enzyme can be achieved. METHODS: Primary human hematopoietic stem/progenitor cells and Tay-Sachs affected cells were used to evaluate the functionality of the vector. An immunodeficient and humanized mouse model of Sandhoff disease was used to evaluate whether the HexA/HexB lentiviral vector transduced cells were able to improve the phenotypes associated with Sandhoff disease. An immunodeficient NOD-RAG1-/-IL2-/- (NRG) mouse model was used to evaluate whether the HexA/HexB vector transduced human CD34+ cells were able to engraft and undergo normal multilineage hematopoiesis. RESULTS: HexA/HexB lentiviral vector transduced cells demonstrated strong expression of HexA and HexB and restored enzyme activity in Tay-Sachs affected cells. Upon transplantation into a humanized Sandhoff disease mouse model, improved motor and behavioral skills were observed. Decreased GM2 gangliosides were observed in the brains of HexA/HexB vector transduced cell transplanted mice. Increased peripheral blood levels of HexB was also observed in transplanted mice. Normal hematopoiesis in the peripheral blood and various lymphoid organs was also observed in transplanted NRG mice. CONCLUSIONS: These results highlight the potential use of stem cell gene therapy as a treatment strategy for Tay-Sachs and Sandhoff disease.