Tuning the resorption-formation balance in an in vitro 3D osteoblast-osteoclast co-culture model of bone.

The aim of the present study was to further improve an in vitro 3D osteoblast (OB) - osteoclast (OC) co-culture model of bone by tuning it towards states of formation, resorption, and equilibrium for their future applications in fundamental research, drug development and personalized medicine. This was achieved by varying culture medium composition and monocyte seeding density, the two external parameters that affect cell behavior the most. Monocytes were seeded at two seeding densities onto 3D silk-fibroin constructs pre-mineralized by MSC-derived OBs and were co-cultured in one of three different media (OC stimulating, Neutral and OB stimulating medium) for three weeks. Histology showed mineralized matrix after co-culture and OC markers in the OC medium group. Scanning Electron Microscopy showed large OC-like cells in the OC medium group. Micro-computed tomography showed increased formation in the OB medium group, equilibrium in the Neutral medium group and resorption in the OC medium group. Culture supernatant samples showed high early tartrate resistant acid phosphatase (TRAP) release in the OC medium group, a later and lower release in the Neutral medium group, and almost no release in the OB medium group. Increased monocyte seeding density showed a less-than-proportional increase in TRAP release and resorption in OC medium, while it proportionally increased TRAP release in Neutral medium without affecting net resorption. The 3D OB-OC co-culture model was effectively used to show an excess of mineral deposition using OB medium, resorption using OC medium, or an equilibrium using Neutral medium. All three media applied to the model may have their own distinct applications in fundamental research, drug development, and personalized medicine.

Gene Therapy in Orthopaedics: Progress and Challenges in Pre-Clinical Development and Translation.

In orthopaedics, gene-based treatment approaches are being investigated for an array of common -yet medically challenging- pathologic conditions of the skeletal connective tissues and structures (bone, cartilage, ligament, tendon, joints, intervertebral discs etc.). As the skeletal system protects the vital organs and provides weight-bearing structural support, the various tissues are principally composed of dense extracellular matrix (ECM), often with minimal cellularity and vasculature. Due to their functional roles, composition, and distribution throughout the body the skeletal tissues are prone to traumatic injury, and/or structural failure from chronic inflammation and matrix degradation. Due to a mixture of environment and endogenous factors repair processes are often slow and fail to restore the native quality of the ECM and its function. In other cases, large-scale lesions from severe trauma or tumor surgery, exceed the body's healing and regenerative capacity. Although a wide range of exogenous gene products (proteins and RNAs) have the potential to enhance tissue repair/regeneration and inhibit degenerative disease their clinical use is hindered by the absence of practical methods for safe, effective delivery. Cumulatively, a large body of evidence demonstrates the capacity to transfer coding sequences for biologic agents to cells in the skeletal tissues to achieve prolonged delivery at functional levels to augment local repair or inhibit pathologic processes. With an eye toward clinical translation, we discuss the research progress in the primary injury and disease targets in orthopaedic gene therapy. Technical considerations important to the exploration and pre-clinical development are presented, with an emphasis on vector technologies and delivery strategies whose capacity to generate and sustain functional transgene expression in vivo is well-established.

Development of hydroxyapatite-coated nonwovens for efficient isolation of somatic stem cells from adipose tissues.

Adipose-derived stem cells (ASCs) are an attractive cell source for cell therapy. Despite the increasing number of clinical applications, the methodology for ASC isolation is not optimized for every individual. In this study, we developed an effective material to stabilize explant cultures from small-fragment adipose tissues. Methods: Polypropylene/polyethylene nonwoven sheets were coated with hydroxyapatite (HA) particles. Adipose fragments were then placed on these sheets, and their ability to trap tissue was monitored during explant culture. The yield and properties of the cells were compared to those of cells isolated by conventional collagenase digestion. Results: Hydroxyapatite-coated nonwovens immediately trapped adipose fragments when placed on the sheets. The adhesion was stable even in culture media, leading to cell migration and proliferation from the tissue along with the nonwoven fibers. A higher fiber density further enhanced cell growth. Although cells on nonwoven explants could not be fully collected with cell dissociation enzymes, the cell yield was significantly higher than that of conventional monolayer culture without impacting stem cell properties. Conclusions: Hydroxyapatite-coated nonwovens are useful for the effective primary explant culture of connective tissues without enzymatic cell dissociation.

Allogeneic Mesenchymal Stromal Cell Injection to Alleviate Ischemic Heart Failure Following Arterial Switch Operation.

Cell therapy is a promising tool to prevent and treat heart failure in congenital heart disease. We report the first case of intramyocardial injection of allogeneic mesenchymal stromal cells as rescue therapy in a neonate with ischemic heart failure following arterial switch procedure for isolated transposition of the great arteries. (Level of Difficulty: Advanced.).

Non-clinical assessment of safety, biodistribution and tumorigenicity of human mesenchymal stromal cells.

Guidelines regulating the development of advanced therapy medicinal products (ATMPs) request nonclinical data for toxicity, biodistribution and tumorigenicity before mesenchymal stromal cell (MSC) products can be administered in large clinical trials. We assessed the biodistribution/persistence, safety and tumorigenicity of MC0518, a human allogeneic MSC product from pooled bone marrow mononuclear cells of eight healthy, adult, unrelated donors, which is currently investigated for the treatment of steroid-refractory acute Graft-versus-Host Disease (aGvHD) after hematopoietic stem cell transplantation. In our GLP studies, immuno-deficient mice were administered repeat doses of MC0518 (once weekly for 6 weeks, i.v.) at doses exceeding the proposed human clinical dose 20-60-fold. No signs of toxicity were observed in the combined biodistribution/toxicity study. Human MSCs in mouse tissues were detected by quantitative PCR (qPCR) and in situ hybridization (ISH). MC0518 showed initial trapping in the lung, occasional distribution into other organs and low tissue persistence beyond 24 h after application. No MSC-induced tumors of human origin were identified after a follow-up of six months. Additionally, we found that the combination of different detection methods (qPCR and ISH) is crucial for a reliable interpretation of biodistribution results. Our data suggest that MC0518 is safe for use in human.

Gaps between Asian regulations for eligibility of human mesenchymal stromal cells as starting materials of cell therapy products and comparability of mesenchymal stromal cell-based products subject to changes in their manufacturing process.

Working group 2 (WG2) of the Asia Partnership Conference of Regenerative Medicine has discussed eligibility of mesenchymal stromal cells (MSCs) as starting cells for the manufacture of cell therapy products, and comparability before and after changes in their manufacturing process. Asian countries and regions have their own regulations on the quality of starting cells, and these regulations are not harmonized. As cell therapy products are being developed across countries and regions, we propose a risk-based approach based on donor location, window period of virus test, and additional virus tests on the master cell bank to fill the gaps in regulation while controlling the risk of viral contamination. Moreover, a standard procedure of comparability assessment after changes in the manufacturing process of MSC-based products does not exist. The WG2 discussed points of comparability assessment specifically for MSC-based products considering the similarities and differences with parallel assessments for protein and polypeptide products, which are within the scope of the International Council for Harmonization Q5E guideline. We also summarize possible characterization procedures for MSC-based products and report our discussion on stability evaluations under accelerated and stress conditions for comparability assessment of cell therapy products.

Rejuvenation of Senescent Endothelial Progenitor Cells by Extracellular Vesicles Derived From Mesenchymal Stromal Cells.

Mesenchymal stromal cell (MSC) transplantation is a form of the stem-cell therapy that has shown beneficial effects for many diseases. The use of stem-cell therapy, including MSC transplantation, however, has limitations such as the tumorigenic potential of stem cells and the lack of efficacy of aged autologous cells. An ideal therapeutic approach would keep the beneficial effects of MSC transplantation while circumventing the limitations associated with the use of intact stem cells. This study provides proof-of-concept evidence that MSC-derived extracellular vesicles represent a promising platform to develop an acellular therapeutic approach that would just do that. Extracellular vesicles are membranous vesicles secreted by MSCs and contain bioactive molecules to mediate communication between different cells. Extracellular vesicles can be taken up by recipient cells, and once inside the recipient cells, the bioactive molecules are released to exert the beneficial effects on the recipient cells. This study, for the first time to our knowledge, shows that extracellular vesicles secreted by MSCs recapitulate the beneficial effects of MSCs on vascular repair and promote blood vessel regeneration after ischemic events. Furthermore, MSCs from aged donors can be engineered to produce extracellular vesicles with improved regenerative potential, comparable to MSCs from young donors, thus eliminating the need for allogenic young donors for elderly patients.

Increased error-free DNA repair gene expression through reprogramming in human iPS cells.

INTRODUCTION: Many studies have reported that human-induced pluripotent stem (hiPS)/embryonic stem (hES) cells have an exceptional ability to repair damaged DNA. Moreover, unlike differentiated cells, hES cells have features and mechanisms such as apoptosis-prone mitochondria, which prevent any changes in genetic information caused by DNA damage to be transmitted to their descendants. Type-A (dark) spermatogonia and cancer stem cells are thought to be dormant. However, hiPS/hES cells, the so-called stem cells used in regenerative medicine, generally have a high proliferative capacity. This suggests that in these cells, oxidative DNA damage associated with vigorous proliferation and DNA scission associated with replication occur frequently. Although pluripotency according to change of genomic structure is well studied, the change of DNA repair through reprogramming has not been well studied. METHODS: We analyzed the expression of DNA repair-related genes in hiPS cells using microarray and western blotting analyses and assessed changes in PARP activity through reprogramming. RESULTS: Through reprogramming, hiPS cells were found to upregulate poly (ADP-ribose) polymerase (PARP) activity and genes regulating homologous recombination (HR). Simultaneously, the expression level of genes involved in non-homologous end joining (NHEJ) was not high, suggesting that at least at the gene expression level, frequently occurring DNA scission is preferentially dealt with via HR instead of NHEJ. Also, reflecting the high proliferative activity, genes related to mismatch repair (MMR) were upregulated through reprogramming. Conversely, error-prone polymerase was downregulated through reprogramming. These are also likely to be the mechanisms preventing changes in genetic information. CONCLUSIONS: High PARP activity and HR-related gene expression in hiPS cells were achieved through reprogramming and likely facilitate precise genome editing in these cells in exchange for a high possibility of cell death.

Th22 cells increase in poor prognosis multiple myeloma and promote tumor cell growth and survival.

There is increased production of plasmacytoid dendritic cells (pDCs) in the bone marrow (BM) of multiple myeloma (MM) patients and these favor Th22 cell differentiation. Here, we found that the frequency of interleukin (IL)-22+IL-17-IL-13+ T cells is significantly increased in peripheral blood (PB) and BM of stage III and relapsed/refractory MM patients compared with healthy donors and patients with asymptomatic or stage I/II disease. Th22 cells cloned from the BM of MM patients were CCR6+CXCR4+CCR4+CCR10- and produced IL-22 and IL-13 but not IL-17. Furthermore, polyfunctional Th22-Th2 and Th22-Th1 clones were identified based on the co-expression of additional chemokine receptors and cytokines (CRTh2 or CXCR3 and IL-5 or interferon gamma [IFNγ], respectively). A fraction of MM cell lines and primary tumors aberrantly expressed the IL-22RA1 and IL-22 induced STAT-3 phosphorylation, cell growth, and resistance to drug-induced cell death in MM cells. IL-13 treatment of normal BM mesenchymal stromal cells (MSCs) induced STAT-6 phosphorylation, adhesion molecule upregulation, and increased IL-6 production and significantly favored MM cell growth compared with untreated BM MSCs. Collectively, our data show that increased frequency of IL-22+IL-17-IL-13+ T cells correlates with poor prognosis in MM through IL-22 and IL-13 protumor activity and suggest that interference with IL-22 and IL-13 signaling pathways could be exploited for therapeutic intervention.

Global histone deacetylase enzymatic activity is an independent prognostic marker associated with a shorter overall survival in chronic lymphocytic leukemia patients.

Histone deacetylases (HDAC) play a crucial role in transcriptional regulation and are often deregulated in many cancers. However, global HDAC enzymatic activity has never been investigated in Chronic Lymphocytic Leukemia (CLL). We measured HDAC activity in protein extracts from CD19+ B-cells purified from 114 CLL patients with a median follow-up of 91 months (range: 11-376). HDAC activity was equivalent in CLL and normal B-cells but higher in patients who died during the study than in living patients (152.1 vs. 65.04 pmol; P = 0.0060). Furthermore, HDAC activity correlated with treatment-free survival (TFS; P = 0.0156) and overall survival (OS; P < 0.0001): patients with low HDAC activity (n = 75) had a median TFS and OS of 101 and > 376 months, respectively, whereas patients with high HDAC activity (n = 39) had a median TFS and OS of 47 and 137 months, respectively. Multivariate analyses indicated that HDAC activity is an independent predictor of OS (hazard ratio = 7.68; P = 0.0017). Finally, HDAC activity increased after B-cell receptor stimulation using IgM, suggesting a role for microenvironment stimuli (n = 10; P = 0.0371). In conclusion, high HDAC activity in CLL B-cells is associated with shorter TFS and OS and is an independent marker of OS, refining the use of other prognostic factors. This work provides a biological base for the use of HDAC inhibitors in CLL treatment.