A Prospective, Randomized, Open-Label Trial of Early versus Late Favipiravir Therapy in Hospitalized Patients with COVID-19.

Favipiravir is an oral broad-spectrum inhibitor of viral RNA-dependent RNA polymerase that is approved for treatment of influenza in Japan. We conducted a prospective, randomized, open-label, multicenter trial of favipiravir for the treatment of COVID-19 at 25 hospitals across Japan. Eligible patients were adolescents and adults admitted with COVID-19 who were asymptomatic or mildly ill and had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. Patients were randomly assigned at a 1:1 ratio to early or late favipiravir therapy (in the latter case, the same regimen starting on day 6 instead of day 1). The primary endpoint was viral clearance by day 6. The secondary endpoint was change in viral load by day 6. Exploratory endpoints included time to defervescence and resolution of symptoms. Eighty-nine patients were enrolled, of whom 69 were virologically evaluable. Viral clearance occurred within 6 days in 66.7% and 56.1% of the early and late treatment groups (adjusted hazard ratio [aHR], 1.42; 95% confidence interval [95% CI], 0.76 to 2.62). Of 30 patients who had a fever (≥37.5°C) on day 1, times to defervescence were 2.1 days and 3.2 days in the early and late treatment groups (aHR, 1.88; 95% CI, 0.81 to 4.35). During therapy, 84.1% developed transient hyperuricemia. Favipiravir did not significantly improve viral clearance as measured by reverse transcription-PCR (RT-PCR) by day 6 but was associated with numerical reduction in time to defervescence. Neither disease progression nor death occurred in any of the patients in either treatment group during the 28-day participation. (This study has been registered with the Japan Registry of Clinical Trials under number jRCTs041190120.).

Cell-Surface MMP-9 Protein Is a Novel Functional Marker to Identify and Separate Proangiogenic Cells from Early Endothelial Progenitor Cells Derived from CD133(+) Cells.

To develop cell therapies for ischemic diseases, endothelial progenitor cells (EPCs) have been expected to play a pivotal role in vascular regeneration. It is desirable to use a molecular marker that is related to the function of the cells. Here, a quantitative polymerase chain reaction array revealed that early EPCs derived from CD133(+) cells exhibited significant expression of MMP-9. Some populations of early EPCs expressed MMP-9 on the cell surface and others did not. We also attempted to separate the proangiogenic fraction from early EPCs derived from CD133(+) cells using a functional cell surface marker, and we then analyzed the MMP-9(+) and MMP-9(-) cell fractions. The MMP-9(+) cells not only revealed higher invasion ability but also produced a high amount of IL-8. Moreover, the stimulative effect of MMP-9(+) cells on angiogenesis in vitro and in vivo was prohibited by anti-IL-8 antibody. These data indicate that MMP-9 is one of the useful cell surface markers for the separation of angiogenic cells. Our treatment of early EPCs with hyaluronidase caused not only a downregulation of cell-surface MMP-9 but also a decrease in invasion ability, indicating that membrane-bound MMP-9, which is one of the useful markers for early EPCs, plays an important role in angiogenesis. Stem Cells 2016;34:1251-1262.

Angiogenic Role of MMP-2/9 Expressed on the Cell Surface of Early Endothelial Progenitor Cells/Myeloid Angiogenic Cells.

Since the introduction of angiogenic cell therapy using early endothelial progenitor cells (EPCs), myeloid angiogenic cells (MACs) have been expected to be useful in treating ischemic diseases. In order to elucidate the angiogenic properties of MACs/EPCs, we clarified the characteristics of MACs as compared to M2 macrophages (Mϕs). Comparison of the gene expression profiles of MACs and late EPCs revealed that MACs expressed greater amounts of metalloproteinase (MMP)-9. It should be noted that the profile of MMP-2/9 expression on the cell surface of MACs was similar to that of M2 Mϕs, and that cell surface MMP-2/9 might be an active form based on molecular size. In addition, the invasion of MACs was prohibited not only by MMP-2/9 inhibitor, but also by the hyaluronidase treatment that caused the down-regulation of MMP-9 on the cell surface of MACs and inhibited their invasion activity. These results indicate that cell surface MMP-2/9 plays an important role in the high invasion ability of MACs. The conditioned medium of both MACs and M2 Mϕs stimulated tube formation of endothelial cells in vitro. MACs caused an increase in vessel formation in in vivo models through the production of IL-8. We propose that the role of MACs with cell surfaces expressing MMP-2/9 is rapidly invading ischemic tissue.

Therapeutic potential of human adipose tissue-derived multi-lineage progenitor cells in liver fibrosis.

INTRODUCTION: Liver fibrosis is characterized by excessive accumulation of extracellular matrix. In a mouse model of liver fibrosis, systemic injection of bone marrow mesenchymal stem cells (BM-MSCs) was considered to rescue the diseased phenotype. The aim of this study was to assess the effectiveness of human adipose tissue-derived multi-lineage progenitor cells (hADMPCs) in improving liver fibrosis. METHODS AND RESULTS: hADMPCs were isolated from subcutaneous adipose tissues of healthy volunteers and expanded. Six week-old male nude mice were treated with carbon tetra-chloride (CCl4) by intraperitoneal injection twice a week for 6 weeks, followed by a tail vein injection of hADMPCs or placebo control. After 6 more weeks of CCl4 injection (12 weeks in all), nude mice with hADMPCs transplants exhibited a significant reduction in liver fibrosis, as evidenced by Sirius Red staining, compared with nude mice treated with CCl4 for 12 weeks without hADMPCs transplants. Moreover, serum glutamic pyruvate transaminase and total bilirubin levels in hADMPCs-treated nude mice were lower levels than those in placebo controls. Production of fibrinolytic enzyme MMPs from hADMPCs were examined by ELISA and compared to that from BM-MSCs. MMP-2 levels in the culture media were not significantly different, whereas those of MMP-3 and -9 of hADMPCs were higher than those by BM-MSCs. CONCLUSION: These results showed the mode of action and proof of concept of systemic injection of hADMPCs, which is a promising therapeutic intervention for the treatment of patients with liver fibrosis.

Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells.

Stem and progenitor cells are currently being investigated for their applicability in cell-based therapy for periodontal tissue regeneration. We recently demonstrated that the transplantation of adipose tissue-derived multi-lineage progenitor cells (ADMPCs) enhances periodontal tissue regeneration in beagle dogs. However, the molecular mechanisms by which transplanted ADMPCs induce periodontal tissue regeneration remain to be elucidated. In this study, trophic factors released by ADMPCs were examined for their paracrine effects on human periodontal ligament cell (HPDL) function. ADMPC conditioned medium (ADMPC-CM) up-regulated osteoblastic gene expression, alkaline phosphatase activity and calcified nodule formation in HPDLs, but did not significantly affect their proliferative response. ADMPCs secreted a number of growth factors, including insulin-like growth factor binding protein 6 (IGFBP6), hepatocyte growth factor and vascular endothelial growth factor. Among these, IGFBP6 was most highly expressed. Interestingly, the positive effects of ADMPC-CM on HPDL differentiation were significantly suppressed by transfecting ADMPCs with IGFBP6 siRNA. Our results suggest that ADMPCs transplanted into a defect in periodontal tissue release trophic factors that can stimulate the differentiation of HPDLs to mineralized tissue-forming cells, such as osteoblasts and cementoblasts. IGFBP6 may play crucial roles in ADMPC-induced periodontal regeneration.

Characterization of the cell growth analysis for detection of immortal cellular impurities in human mesenchymal stem cells.

The analysis of in vitro cell senescence/growth after serial passaging can be one of ways to show the absence of immortalized cells, which are frequently tumorigenic, in human cell-processed therapeutic products (hCTPs). However, the performance of the cell growth analysis for detection of the immortalized cellular impurities has never been evaluated. In the present study, we examined the growth rates of human mesenchymal stem cells (hMSCs, passage 5 (P = 5)) contaminated with various doses of HeLa cells, and compared with that of hMSCs alone. The growth rates of the contaminated hMSCs were comparable to that of hMSCs alone at P = 5, but significantly increased at P = 6 (0.1% and 0.01% HeLa) or P = 7 (0.001% HeLa) within 30 days. These findings suggest that the cell growth analysis is a simple and sensitive method to detect immortalized cellular impurities in hCTPs derived from human somatic cells.

Report of the international conference on regulatory endeavors towards the sound development of human cell therapy products.

The regulation of human cell therapy products is a key factor in their development and use to treat human diseases. In that regard, there is a recognized need for a global effort to develop a set of common principles that may serve to facilitate a convergence of regulatory approaches to ensure the smooth and efficient evaluation of products. This conference, with experts from regulatory agencies, industry, and academia, contributed to the process of developing such a document. Elements that could form a minimum consensus package of requirements for evaluating human cell therapy products were the overall focus of the conference. The important regulatory considerations that are unique to human cell therapy products were highlighted. Sessions addressed specific points that are different from those of traditional biological/biotechnological protein products. Panel discussions complemented the presentations. The conference concluded that most of the current regulatory framework is appropriate for cell therapy, but there are some areas where the application of the requirements for traditional biologicals is inappropriate. In addition, it was agreed that there is a need for international consensus on core regulatory elements, and that one of the major international organizations should take the lead in formulating such a consensus document.

ROR2 expression predicts human induced pluripotent stem cell differentiation into neural stem/progenitor cells and GABAergic neurons.

Despite the development of various in vitro differentiation protocols for the efficient derivation of specific cell types, human induced pluripotent stem cell (hiPSC) lines have varing ability to differentiate into specific lineages. Therefore, surrogate markers for accurately predicting the differentiation propensity of hiPSC lines may facilitate cell-based therapeutic product development and manufacture. We attempted to identify marker genes that could predict the differentiation propensity of hiPSCs into neural stem/progenitor cells (NS/PCs). Using Spearman's rank correlation coefficients, we investigated genes in the undifferentiated state, the expression levels of which were significantly correlated with the neuronal differentiation propensity of several hiPSC lines. Among genes significantly correlated with NS/PC differentiation (P < 0.01), we identified ROR2 as a novel predictive marker. ROR2 expression in hiPSCs was negatively correlated with NS/PC differentiation tendency, regardless of the differentiation method, whereas its knockdown enhanced differentiation. ROR2 regulates NS/PC differentiation, suggesting that ROR2 is functionally essential for NS/PC differentiation. Selecting cell lines with relatively low ROR2 expression facilitated identification of hiPSCs that can differentiate into NS/PCs. Cells with ROR2 knockdown showed increased efficiency of differentiation into forebrain GABAergic neurons compared to controls. These findings suggest that ROR2 is a surrogate marker for selecting hiPSC lines appropriate for NS/PC and GABAergic neuronal differentiations.

Poor Result Reporting Rate in Cell Therapy Trials Registered at ClinicalTrials.gov.

As research associates in clinical experiments, we have an obligation to disclose clinical methodologies and findings in full transparency in ethics. However, inadequate disclosure in results reporting clinical trials registered on ClinicalTrials.gov has been revealed, with approximately half the trial results not being reported in an applicable manner. Our recent study in clinical trials of regenerative medicine for four kinds of neurological diseases revealed that the rate of result reporting to ClinicalTrials.gov is inadequate for gene and cell therapy (CT) trials. In this path, further curiosity emerged to see what the findings would be if the analysis was conducted for trials in all disease areas, and outcomes if gene therapy (GT) and CT were distinguished in terms. In this study, the scope of analysis was further expanded to include all disease areas, and the drug classification from the AdisInsight database was used for modality classification, with biologic drug trials classified as controls, CT, ex vivo GT, and in vivo GT. To begin, among all interventional clinical trials with registration in the ClinicalTrials.gov registry and with a primary completion between 2010 and 2019, we created a total of 5539 datasets corresponding to trials classified as GT and CT, while biologics (BLG) as controls in the AdisInsight drug classification. The status of reported results of these trials was identified by surveying posting status of ClinicalTrials.gov and publication in journals (PubMed), respectively. Based on the obtained dataset, multivariate analysis was performed on the data on the reporting rate of clinical trial results, aggregated by sponsor, phase, status, and modality (CT, ex vivo GT, in vivo GT, and BLG), respectively. The result shows that CT was identified as an independent factor restraining result reporting ratio in both ClinicalTrials.gov and total disclosures, whereas ex vivo GT as boosting result reporting ratio. Since the result reporting rate of CT results was notably poor, we discussed the causes and solutions in this regard.

A Pilot Study on Result Reporting Rates from Clinical Trials of Regenerative Medicine.

Sharing the methods and results of clinical trials with full transparency is an ethical obligation for those involved in clinical research. In this regard, ClinicalTrials.gov requires reporting of results to the registry within 1 year of completion of the trial. However, a poor result reporting rate has been pointed out, with approximately half the trial results not been reported. It has been suggested that one of the reasons behind this could be the influence of sponsors who conduct the clinical trials. In the course of our previous trend analysis on regenerative medicine for stroke (STR) using ClinicalTrials.gov and the International Clinical Trials Registry Platform (ICTRP) portal site as data sources, we suspected whether the results of gene and/or cell therapy trials are poorly reported. For this reason, a multivariate analysis using data from ClinicalTrials.gov was performed to identify the factors suppressing the result reporting rate, expanding our study to four different kinds of neurological diseases and regenerative medicine as a treatment modality when small-molecule compounds and biologics were set up as controls, in addition to the sponsor type factor. As a result, we found gene and/or cell therapy (therapeutic modality) in addition to STR (disease area), trials completed in 2005-2007, and clinical phases II and IV as independent factors that suppressed the rate of reporting results to ClinicalTrials.gov. On the other hand, big pharmaceutical companies were identified as a factor that increased the reporting result rate to ClinicalTrials.gov. When we applied result reporting publications through PubMed as an index, our study data revealed that the following factors were not identified as the cause for a decrease in the reporting result rate: STR (as disease area), trials completed between 2005 and 2007, and gene/cell therapy (as treatment modality). In this context, our findings indicate that gene/cell therapy has led to the suppression of the result reporting rate to ClinicalTrials.gov. This confirmed our initial suspicion of the low result reporting rate of gene/cell therapy trials. We believe that further studies are required to elucidate the factors affecting the result reporting rate from the perspective of disease area and treatment modality. Impact Statement Several studies have addressed the poor result reporting rate of clinical trials, which still remains an issue. Regenerative medicine holds great promise for the future and the process of its practical application is expected to be challenging. Although having a limited disease area and small sample size, to the best of our knowledge, this is the first study to point out insufficient result reporting of clinical trials of regenerative medicine from the perspective of treatment modality. This report highlights an issue for discussing the path toward its translation through an overview of various factors in comparison with conventional treatment modalities.