Attitudes Towards Standardization of Mesenchymal Stromal Cells-A Qualitative Exploration of Expert Views.

Pharmacopoeial standards ensure quality control of established medicines. It is widely believed that translation of cell therapy medicines will be facilitated by defining and adopting relevant standards. Mesenchymal stromal cells (MSCs) are used extensively for multiple indications in regenerative medicine. They are highly heterogeneous in terms of their biological characteristics and their mechanisms of action, making standardization a challenging undertaking. Furthermore, the use of MSCs in therapy appears to attract diverse views, ranging from concern and caution to enthusiastic positivity. We conducted semi-structured interviews with 20 expert stakeholders from academia, industry, regulatory agencies, non-governmental organizations and clinicians to explore their views, experiences, recommendations, and concerns regarding standardization of MSCs. Qualitative thematic analysis of transcribed records led to development of a consensus framework, which identified 5 key themes to facilitate exploration of the interviews' content. On the basis of our findings, we conclude that (1) there is undoubtedly an appetite for standardization, particularly in development of assays that enable comparison or benchmarking across manufacturers, processes, and cell sources; (2) stakeholder groups are not homogeneous in their concerns and attitudes; (3) careful consideration must be given to the points along the development timeline at which different standardization approaches could be beneficial; and (4) the roles of standards could be promoted further for specific aspects of advanced therapy medicinal product (ATMP) development and regulation such as qualification of decentralized manufacturing sites. A unified cross-stakeholder approach will help to advance MSC therapeutics and other cell therapy medicines.

Selectivity and stability of N-terminal targeting protein modification chemistries.

Protein N-termini provide uniquely reactive motifs for single site protein modification. Though a number of reactions have been developed to target this site, the selectivity, generality, and stability of the conjugates formed has not been studied. We have therefore undertaken a comprehensive comparative study of the most promising methods for N-terminal protein modification, and find that there is no 'one size fits all' approach, necessitating reagent screening for a particular protein or application. Moreover, we observed limited stability in all cases, leading to a need for continued innovation and development in the bioconjugation field.

Corrigendum: CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

[This corrects the article DOI: 10.3389/fimmu.2022.903796.].

CD317-Positive Immune Stromal Cells in Human "Mesenchymal Stem Cell" Populations.

Heterogeneity of bone marrow mesenchymal stromal cells (MSCs, frequently referred to as "mesenchymal stem cells") clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterized by CD317 expression (CD317pos (29.77 ± 3.00% of the total MSC population), comprising CD317dim (28.10 ± 4.60%) and CD317bright (1.67 ± 0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarization towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Shaping and Patterning Supramolecular Materials─Stem Cell-Compatible Dual-Network Hybrid Gels Loaded with Silver Nanoparticles.

Hydrogels with spatio-temporally controlled properties are appealing materials for biological and pharmaceutical applications. We make use of mild acidification protocols to fabricate hybrid gels using calcium alginate in the presence of a preformed thermally triggered gel based on a low-molecular-weight gelator (LMWG) 1,3:2:4-di(4-acylhydrazide)-benzylidene sorbitol (DBS-CONHNH2). Nonwater-soluble calcium carbonate slowly releases calcium ions over time when exposed to an acidic pH, triggering the assembly of the calcium alginate gel network. We combined the gelators in different ways: (i) the LMWG was used as a template to spatially control slow calcium alginate gelation within preformed gel beads, using glucono-δ-lactone (GdL) to lower the pH; (ii) the LMWG was used as a template to spatially control slow calcium alginate gelation within preformed gel trays, using diphenyliodonium nitrate (DPIN) as a photoacid to lower the pH, and spatial resolution was achieved by masking. The dual-network hybrid gels display highly tunable properties, and the beads are compatible with stem cell growth. Furthermore, they preserve the LMWG function of inducing in situ silver nanoparticle (AgNP) formation, which provides the gels with antibacterial activity. These gels have potential for eventual regenerative medicine applications in (e.g.) bone tissue engineering.

Self-assembled gel tubes, filaments and 3D-printing with in situ metal nanoparticle formation and enhanced stem cell growth.

This paper reports simple strategies to fabricate self-assembled artificial tubular and filamentous systems from a low molecular weight gelator (LMWG). In the first strategy, tubular 'core-shell' gel structures based on the dibenzylidenesorbitol-based LMWG DBS-CONHNH2 were made in combination with the polymer gelator (PG) calcium alginate. In the second approach, gel filaments based on DBS-CONHNH2 alone were prepared by wet spinning at elevated concentrations using a 'solvent-switch' approach. The higher concentrations used in wet-spinning prevent the need for a supporting PG. Furthermore, this can be extended into a 3D-printing method, with the printed LMWG objects showing excellent stability for at least a week in water. The LMWG retains its unique ability for in situ precious metal reduction, yielding Au nanoparticles (AuNPs) within the tubes and filaments when they are exposed to AuCl3 solutions. Since the gel filaments have a higher loading of DBS-CONHNH2, they can be loaded with significantly more AuNPs. Cytotoxicity and viability studies on human mesenchymal stem cells show that the DBS-CONHNH2 and DBS-CONHNH2/alginate hybrid gels loaded with AuNPs are biocompatible, with the presence of AuNPs enhancing stem cell metabolism. Taken together, these results indicate that DBS-CONHNH2 can be shaped and 3D-printed, and has considerable potential for use in tissue engineering applications.

Bone marrow remodeling supports hematopoiesis in response to immune thrombocytopenia progression in mice.

Immune thrombocytopenia (ITP) is an acquired autoimmune condition characterized by both reduced platelet production and the destruction of functionally normal platelets by sustained attack from the immune system. However, the effect of prolonged ITP on the more immature hematopoietic progenitors remains an open area of investigation. By using a murine in vivo model of extended ITP, we revealed that ITP progression drives considerable progenitor expansion and bone marrow (BM) remodeling. Single-cell assays using Lin-Sca1+c-Kit+CD48-CD150+ long-term hematopoietic stem cells (LT-HSCs) revealed elevated LT-HSC activation and proliferation in vitro. However, the increased activation did not come at the expense of LT-HSC functionality as measured by in vivo serial transplantations. ITP progression was associated with considerable BM vasodilation and angiogenesis, as well as a twofold increase in the local production of CXCL12, a cytokine essential for LT-HSC function and BM homing expressed at high levels by LepR+ BM stromal cells. This was associated with a 1.5-fold increase in LepR+ BM stromal cells and a 5.5-fold improvement in progenitor homing to the BM. The increase in stromal cells was transient and reverted back to baseline after platelet count returned to normal, but the vasculature changes in the BM persisted. Together, our data demonstrate that LT-HSCs expand in response to ITP and that LT-HSC functionality during sustained hematopoietic stress is maintained through an adapting BM microenvironment.

Characterisation of mesenchymal stromal cells in clinical trial reports: analysis of published descriptors.

BACKGROUND: Mesenchymal stem or stromal cells are the most widely used cell therapy to date. They are heterogeneous, with variations in growth potential, differentiation capacity and protein expression profile depending on tissue source and production process. Nomenclature and defining characteristics have been debated for almost 20 years, yet the generic term 'MSC' is used to cover a wide range of cellular phenotypes. Against a documented lack of definition of cellular populations used in clinical trials, our study evaluated the extent of characterisation of the cellular population or study drug. METHODS: A literature search of clinical trials involving mesenchymal stem/stromal cells was refined to 84 papers upon application of pre-defined inclusion/exclusion criteria. Data were extracted covering background trial information including location, phase, indication, tissue source and details of clinical cell population characterisation (expression of surface markers, viability, differentiation assays and potency/functionality assays). Descriptive statistics were applied, and tests of association between groups were explored using Fisher's exact test for count data with simulated p value. RESULTS: Twenty-eight studies (33.3%) include no characterisation data. Forty-five (53.6%) reported average values per marker for all cell lots used in the trial, and 11 (13.1%) studies included individual values per cell lot. Viability was reported in 57% of studies. Differentiation was discussed: osteogenesis (29% of papers), adipogenesis (27%), and chondrogenesis (20%) and other functional assays arose in 7 papers (8%). The extent of characterisation was not related to the clinical phase of development. Assessment of functionality was very limited and did not always relate to the likely mechanism of action. CONCLUSIONS: The extent of characterisation was poor and variable. Our findings concur with those in other fields including bone marrow aspirate and platelet-rich plasma therapy. We discuss the potential implications of these findings for the use of mesenchymal stem or stromal cells in regenerative medicine, and the importance of characterisation for transparency and comparability of literature.

Self-assembled low-molecular-weight gelator injectable microgel beads for delivery of bioactive agents.

We report the preparation of hybrid self-assembled microgel beads by combining the low molecular weight gelator (LMWG) DBS-CONHNH2 and the natural polysaccharide calcium alginate polymer gelator (PG). Microgel formulations based on LMWGs are extremely rare due to the fragility of the self-assembled networks and the difficulty of retaining any imposed shape. Our hybrid beads contain interpenetrated LMWG and PG networks, and are obtained by an emulsion method, allowing the preparation of spherical gel particles of controllable sizes with diameters in the mm or µm range. Microgels based on LMWG/alginate can be easily prepared with reproducible diameters <1 µm (ca. 800 nm). They are stable in water at room temperature for many months, and survive injection through a syringe. The rapid assembly of the LMWG on cooling plays an active role in helping control the diameter of the microgel beads. These LMWG microbeads retained the ability of the parent gel to deliver the bioactive molecule heparin, and in cell culture medium this enhanced the growth of human mesenchymal stem cells. Such microgels may therefore have future applications in tissue repair. This approach to fabricating LMWG microgels is a platform technology, which could potentially be applied to a variety of different functional LMWGs, and hence has wide-ranging potential.

The variable toxicity of silver ions in cell culture media.

The elevated interest in silver ions (Ag+) as a broad spectrum antimicrobial for use on medical devices has increased the number and importance of in vitro biocompatibility testing, however little consideration is given to the culture environment in which the assessments are performed. The current investigation assessed the viability of mouse fibroblasts (L929) exposed to different concentrations of Ag+ in both Dulbecco's modified Eagle's medium (DMEM) and minimal essential medium Eagle, alpha modification (αMEM). We identified a significant increase in the EC50 of L929 cells exposed to Ag+ in αMEM compared to DMEM, which was matched by a corresponding decrease in Ag+ availability in αMEM at concentrations ≤400 µM, as detected by inductively coupled plasma mass spectrometry (ICP-MS). The reduced availability was not observed for Ag+ > 400 µM, the concentration above which caused in vitro cytotoxicity in L929 cells in αMEM; while linear quantification of Ag+ was observed in DMEM. Equilibration of the chloride and glucose components between media did not affect cytotoxicity on primary test cells; mesenchymal stromal cells (MSCs). Overall, our results present evidence of the importance of culture conditions on the in vitro evaluation of silver, with DMEM providing a reliable basal media in which to conduct assessments.

How cell culture conditions affect the microstructure and nanomechanical properties of extracellular matrix formed by immortalized human mesenchymal stem cells: An experimental and modelling study.

This paper presents an investigation of how different culture media (i.e. basal and osteogenic media) affect the nanomechanical properties and microstructure of the mineralized matrix produced by the human mesenchymal stem cell line Y201, from both an experimental and theoretical approach. A bone nodule (i.e. mineralized matrix) cultured from basal medium shows a more anisotropic microstructure compared to its counterpart cultured from an osteogenic medium. As confirmed by finite element simulations, this anisotropic microstructure explains the bimodal distribution of the corresponding mechanical properties very well. The overall nanomechanical response of the bone nodule from the osteogenic medium is poorer compared to its counterpart from the basal medium. The bone nodules, from both basal and osteogenic media, have shown reverse aging effects in terms of mechanical properties. These are possibly due to the fact that cell proliferation outcompetes the mineralization process.

Glycan Profiling Shows Unvaried N-Glycomes in MSC Clones with Distinct Differentiation Potentials.

Different cell types have different N-glycomes in mammals. This means that cellular differentiation is accompanied by changes in the N-glycan profile. Yet when the N-glycomes of cell types with differing fates diverge is unclear. We have investigated the N-glycan profiles of two different clonal populations of mesenchymal stromal cells (MSCs). One clone (Y101), when differentiated into osteoblasts, showed a marked shift in the glycan profile toward a higher abundance of complex N-glycans and more core fucosylation. Yet chemical inhibition of complex glycan formation during osteogenic differentiation did not prevent the formation of functional osteoblasts. However, the N-glycan profile of another MSC clone (Y202), which cannot differentiate into osteoblasts, was not significantly different from that of the clone that can. Interestingly, incubation of Y202 cells in osteogenic medium caused a similar reduction of oligomannose glycan content in this non-differentiating cell line. Our analysis implies that the N-glycome changes seen upon differentiation do not have direct functional links to the differentiation process. Thus N-glycans may instead be important for self-renewal rather than for cell fate determination.

Collagen-Poly(N-isopropylacrylamide) Hydrogels with Tunable Properties.

There is a lack of hydrogel materials whose properties can be tuned at the point of use. Biological hydrogels, such as collagen, gelate at physiological temperatures; however, they are not always ideal as scaffolds because of their low mechanical strength. Their mechanics can be improved through cross-linking and chemical modification, but these methods still require further synthesis. We have demonstrated that by combining collagen with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM), the mechanical properties can be improved while maintaining cytocompatibility. Furthermore, different concentrations of this polymer led to a range of hydrogels with shear moduli ranging from 10(5) Pa down to less than 10(2) Pa, similar to the soft tissues in the body. In addition to variable mechanical properties, the hydrogel blends have a range of micron-scale structures and porosities, which caused adipose-derived stromal cells (ADSCs) to adopt different morphologies when encapsulated within and may therefore be able to direct cell fate.

A virtual approach to evaluate therapies for management of multiple myeloma induced bone disease.

Multiple myeloma bone disease is devastating for patients and a major cause of morbidity. The disease leads to bone destruction by inhibiting osteoblast activity while stimulating osteoclast activity. Recent advances in multiple myeloma research have improved our understanding of the pathogenesis of multiple myeloma-induced bone disease and suggest several potential therapeutic strategies. However, the effectiveness of some potential therapeutic strategies still requires further investigation and optimization. In this paper, a recently developed mathematical model is extended to mimic and then evaluate three therapies of the disease, namely: bisphosphonates, bortezomib and TGF-ß inhibition. The model suggests that bisphosphonates and bortezomib treatments not only inhibit bone destruction, but also reduce the viability of myeloma cells. This contributes to the current debate as to whether bisphosphonate therapy has an anti-tumour effect. On the other hand, the analyses indicate that treatments designed to inhibit TGF-ß do not reduce bone destruction, although it appears that they might reduce the viability of myeloma cells, which again contributes to the current controversy regarding the efficacy of TGF-ß inhibition in multiple myeloma-induced bone disease.

In vivo biocompatibility of custom-fabricated apatite-wollastonite-mesenchymal stromal cell constructs.

We have used the additive manufacturing technology of selective laser sintering (SLS), together with post SLS heat treatment, to produce porous three dimensional scaffolds from the glass-ceramic apatite-wollastonite (A-W). The A-W scaffolds were custom-designed to incorporate a cylindrical central channel to increase cell penetration and medium flow to the center of the scaffolds under dynamic culture conditions during in vitro testing and subsequent in vivo implantation. The scaffolds were seeded with human bone marrow mesenchymal stromal cells (MSCs) and cultured in spinner flasks. Using confocal and scanning electron microscopy, we demonstrated that MSCs formed and maintained a confluent layer of viable cells on all surfaces of the A-W scaffolds during dynamic culture. MSC-seeded, with and without osteogenic pre-differentiation, and unseeded A-W scaffolds were implanted subcutaneously in MF1 nude mice where osteoid formation and tissue in-growth were observed following histological assessment. The results demonstrate that the in vivo biocompatibility and osteo-supportive capacity of A-W scaffolds can be enhanced by SLS-custom design, without the requirement for osteogenic pre-induction, to advance their potential as patient-specific bone replacement materials.

A predator-prey based mathematical model of the bone remodelling cycle: exploring the relationship between the model parameters and biochemical factors.

Bone remodelling is a vital process which enables bone to repair, renew and optimize itself. Disorders in the bone remodelling process are inevitably manifested in bone-related diseases, such as hypothyroidism, primary hyperparathyroidism and osteoporosis. In our previous work, a predator-prey based mathematical model was developed to simulate bone remodelling cycles under normal and two pathological conditions, hypothyroidism and primary hyperparathyroidism, for trabecular bone at a fixed point. However, the biochemical meanings of the model parameters were not fully explored. This article first extends the previous work by proposing relationships between the model parameters and biochemical factors involved in the bone remodelling process and by examining whether those relationships do predict the behaviours observed in vivo. The model is then applied to the simulation and investigation of bone remodelling of postmenopausal osteoporosis. The proposed connections are supported by good agreement between the model simulations and published experimental observations for the normal condition and all three pathological variations in bone remodelling.

Investigating the efficacy of bisphosphonates treatment against multiple myeloma induced bone disease using a computational model.

Multiple myeloma (MM)-induced bone disease is mortal for most MM patients. Bisphosphonates are first-line treatment for MM-induced bone disease, since it can inhibit osteoclast activity and the resultant bone resorption by suppressing the differentiation of osteoclast precursors into mature osteoclasts, promoting osteoclast apoptosis and disrupting osteoclast function. However, it is still unclear whether bisphosphonates have an anti-tumour effect. In our previous work, a computational model was built to simulate the pathology of MM-induced bone disease. This paper extends this proposed computational model to investigate the efficacy of bisphosphonates treatment and then clear the controversy of this therapy. The extended model is validated through the good agreement between simulation results and experimental data. The simulation results suggest that bisphosphonates indeed have an anti-tumour effect.

Mathematical modelling of the pathogenesis of multiple myeloma-induced bone disease.

Multiple myeloma (MM) is the second most common haematological malignancy and results in destructive bone lesions. The interaction between MM cells and the bone microenvironment plays an important role in the development of the tumour cells and MM-induced bone disease and forms a 'vicious cycle' of tumour development and bone destruction, intensified by suppression of osteoblast activity and promotion of osteoclast activity. In this paper, a mathematical model is proposed to simulate how the interaction between MM cells and the bone microenvironment facilitates the development of the tumour cells and the resultant bone destruction. It includes both the roles of inhibited osteoblast activity and stimulated osteoclast activity. The model is able to mimic the temporal variation of bone cell concentrations and resultant bone volume after the invasion and then removal of the tumour cells and explains why MM-induced bone lesions rarely heal even after the complete removal of MM cells. The behaviour of the model compares well with published experimental data. The model serves as a first step to understand the development of MM-induced bone disease and could be applied further to evaluate the current therapies against MM-induced bone disease and even suggests new potential therapeutic targets.

Wnt-dependent osteogenic commitment of bone marrow stromal cells using a novel GSK3ß inhibitor.

Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) can differentiate into multiple lineages including osteogenic and adipogenic cells. Wnt signalling has been implicated in controlling BMSC fate, but the mechanisms are unclear and apparently conflicting data exist. Here we show that a novel glycogen synthase kinase 3ß inhibitor, AR28, is a potent activator of canonical Wnt signalling using in vitro ß-catenin translocation studies and TCF-reporter assays. In vivo, AR28 induced characteristic axis duplication and secondary regions of chordin expression in Xenopus laevis embryos. Using human BMSCs grown in adipogenic medium, we confirmed that AR28-mediated Wnt signalling caused a significant (p<0.05) dose-dependent reduction of adipogenic markers. In osteogenic media, including dexamethasone, AR28 caused significant (p<0.05) decreases in alkaline phosphatase (ALP) activity compared to vehicle controls, indicative of a reduced osteogenic response. However, when excluding dexamethasone from the osteogenic media, increases in both ALP and mineralisation were identified following AR28 treatment, which was blocked by mitomycin C. Pre-treatment of BMSCs with AR28 for 7 days before osteogenic induction also increased ALP activity and mineralisation. Furthermore, BMP2-induced osteogenic differentiation was strongly enhanced by AR28 addition within 3 days, but without concomitant changes in cell number, therefore revealing BMP-dependent and independent mechanisms for Wnt-induced osteogenesis.