Mesenchymal Stem Cells Manufacturing Research Articles

Transglutaminase 2 Prevents Premature Senescence and Promotes Osteoblastic Differentiation of Mesenchymal Stem Cells through NRF2 Activation.

Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, kinase, and protein disulfide isomerase (PDI) activities. Of these, transamidase-mediated modification of proteins regulates apoptosis, differentiation, inflammation, and fibrosis. TG2 is highly expressed in mesenchymal stem cells (MSCs) compared with differentiated cells, suggesting a role of TG2 specific for MSC characteristics. In this study, we report a new function of TG2 in the regulation of MSC redox homeostasis. During in vitro MSC expansion, TG2 is required for cell proliferation and self-renewal by preventing premature senescence but has no effect on the expression of surface antigens and oxidative stress-induced cell death. Moreover, induction of differentiation upregulates TG2 that promotes osteoblastic differentiation. Molecular analyses revealed that TG2 mediates tert-butylhydroquinone, but not sulforaphane, -induced nuclear factor erythroid 2-related factor 2 (NRF2) activation in a transamidase activity-independent manner. Differences in the mechanism of action between two NRF2 activators suggest that PDI activity of TG2 may be implicated in the stabilization of NRF2. The role of TG2 in the regulation of antioxidant response was further supported by transcriptomic analysis of MSC. These results indicate that TG2 is a critical enzyme in eliciting antioxidant response in MSC through NRF2 activation, providing a target for optimizing MSC manufacturing processes to prevent premature senescence.

Development and characterization of islet-derived mesenchymal stem cells from clinical grade neonatal porcine cryopreserved islets.

Porcine tissues display a great potential as donor tissues in xenotransplantation, including cell therapy. Cryopreserving clinical grade porcine tissue and using it as a source for establishing therapeutic cells should be advantageous for transportation and scheduled manufacturing of MSCs. Of note, we previously performed encapsulated porcine islet transplantation for the treatment of unstable type 1 diabetes mellitus in the clinical setting. It has been reported that co-transplantation of islets and Mesenchymal stem cells (MSCs) enhanced efficacy. We assume that co-transplantation of porcine islets and porcine islet-derived MSCs could improve the efficacy of clinical islet xenotransplantation. MSCs were established from fresh and cryopreserved non-clinical grade neonatal porcine islets and bone marrow (termed non-clinical grade npISLET-MSCs and npBM-MSCs, respectively), as well as from cryopreserved clinical grade neonatal porcine islets (termed clinical grade npISLET-MSCs). Subsequently, the cell proliferation rate and diameter, surface marker expression, adipogenesis, osteogenesis, and colony-forming efficiency of the MSCs were assessed. Cell proliferation rate and diameter did not differ between clinical grade and non-clinical grade npISLET-MSCs. However, non-clinical grade npBM-MSCs were significantly shorter and smaller than both npISLET-MSCs (p<0.05). MSC markers (CD29, CD44, and CD90) were strongly expressed in clinical grade npISLET-MSCs and non-clinical grade npISLET-MSCs and npBM-MSCs. The expression of MSC-negative markers CD31, CD34, and SLA-DR was low in all MSCs. Clinical grade npISLET-MSCs derived from adipose and osteoid tissues were positive for Oil Red and alkaline phosphatase staining. The results of colony-forming assay were not significantly different between clinical grade npISLET-MSCs and non-clinical grade npBM-MSCs. The method described herein was successful in of developing clinical grade npISLET-MSCs from cryopreserved islets. Cryopreserved clinical grade porcine islets could be an excellent stable source of MSCs for cell therapy.

TRAIL-Armed ER Nanosomes Induce Drastically Enhanced Apoptosis in Resistant Tumor in Combination with the Antagonist of IAPs (AZD5582).

Although mesenchymal stem cells (MSCs) can be engineered to deliver the TNF-related apoptosis-inducing ligand (TRAIL) as an effective anticancer therapy, the clinical application is hampered by the costly manufacturing of therapeutic MSCs. Therefore, it is needed to find an alternative cell-free therapy. In this study, TRAIL-armed endoplasmic reticulum (ER)-derived nanosomes (ERN-T) are successfully prepared with an average size of 70.6nm in diameter from TRAIL transduced MSCs. It is demonstrated that the ERN-T is significantly more efficient for cancer cell killing than the soluble recombinant TRAIL (rTRAIL). AZD5582 is an antagonist of the inhibitors of apoptosis proteins (IAPs), and its combination with ERN-T induces strikingly enhanced apoptosis in cancerous but not normal cells. AZD5582 sensitizes resistant cancer cells to TRAIL through concomitant downregulation of IAP members like XIAP and the Bcl2 family member Mcl-1. Intravenously infused ERN-Ts accumulate in tumors for over 48 h indicating good tumor tropism and retention. The combination of ERN-T and AZD5582 drastically promotes therapeutic efficacy comparing with the cotreatment by rTRAIL and AZD5582 in a subcutaneous MDA-MB-231 xenograft tumor model. The data thus demonstrate that ERN-T can be a novel cell-free alternative to TRAIL-expressing MSC-based anticancer therapy and its efficacy can be drastically enhanced through combination with AZD5582.

High content analysis reveals distinct morphological profiles of human MSCs under different culture conditions

Background & Aim The number of clinical trials employing Mesenchymal Stem Cells (MSC) for the treatment of human diseases has grown substantially in the last decade. In this context new characterisation tools for cell and media manufacturing process are required. With this in mind, we sought to develop an automated high-content imaging assay to characterize the morphological profile of MSC in response to different media formulations. Methods, Results & Conclusion High content analysis of high resolution images indicated that MSC growing in absence of serum displayed distinct morphological features compared to SC MSC. Specifically, in the absence of serum cells showed a smaller and more circular morphology, with less protrusions compared to SC MSC, which displayed an elongated shape, with numerous cellular protrusions. These phenotypic differences were expressed for instance by a significant higher circularity index and lower form factor by SF MSC, which measure respectively the overall roundness and spreading of a cell. Both automated workflows were able to successfully cluster the two experimental groups based on several morphological descriptors, allowing the generation of a morphological profile. Of note, the assay produced similar morphological profiles using MSC from two donors. The development of serum-free (SF) and chemically defined media (CDM) for the clinical production of human MSCs is a high priority within the cell therapy community. Small molecules as media supplements are an attractive tool as they are generally easily available, chemically defined and tunable, and chemical media design presents an attractive opportunity for improving MSC manufacturing. In this context, we are developing a novel analytical technology aimed at designing chemically defined media (CDM) tailored to the MSC production process. For this purpose, MSC Morphology Profile will be used as one of the read out assays to screen small molecules, with the goal of identifying molecules that sustain proliferation of MSC while retaining their potency. Taken together, we report the development of a high-content assay to readily capture and analyse the morphological profile of MSC grown in different culture conditions and quantitatively showed that serum influences MSC morphology. Importantly, the assay may represents a straightforward strategy to benchmark MSC preparation and media formulations during production processes for therapeutic application.

Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes.

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.

Characterization and cost-benefit analysis of automated bioreactor-expanded mesenchymal stem cells for clinical applications.

Expanding quantities of mesenchymal stem cells (MSCs) sufficient to treat large numbers of patients in cellular therapy and regenerative medicine clinical trials is an ongoing challenge for cell manufacturing facilities. We evaluated options for scaling up large quantities of bone marrow-derived MSCs (BM-MSCs) using methods that can be performed in compliance with Good Manufacturing Practices (GMP). We expanded BM-MSCs from fresh marrow aspirate in αMEM supplemented with 5% human platelet lysate using both an automated cell expansion system (Quantum, Terumo BCT) and a manual flask-based method using multilayer flasks. We compared MSCs expanded using both methods and assessed their differentiation to adipogenic and osteogenic tissue, capacity to suppress T-cell proliferation, cytokines, and growth factor secretion profile and cost-effectiveness of manufacturing enough BM-MSCs to administer a single dose of 100 × 106 cells per subject in a clinical trial of 100 subjects. We have established that large quantities of clinical-grade BM-MSCs manufactured with an automated hollow-fiber bioreactor were phenotypically (CD73, CD90, CD105) and functionally (adipogenic and osteogenic differentiation and cytokine and growth factor secretion) similar to manually expanded BM-MSCs. In addition, MSC manufacturing costs significantly less and required less time and effort when using the Quantum automated cell expansion system over the manual multilayer flasks method. MSCs manufactured by an automated bioreactor are physically and functionally equivalent to the MSCs manufactured by the manual flask method and have met the standards required for clinical application.

Optimization of human mesenchymal stem cell manufacturing: the effects of animal/xeno-free media.

Due to their immunosuppressive properties, mesenchymal stem cells (MSC) have been evaluated for the treatment of immunological diseases. However, the animal-derived growth supplements utilized for MSC manufacturing may lead to clinical complications. Characterization of alternative media formulations is imperative for MSC therapeutic application. Human BMMSC and AdMSC were expanded in media supplemented with either human platelet lysates (HPL), serum-free media/xeno-free FDA-approved culture medium (SFM/XF), or fetal bovine serum (FBS) and the effects on their properties were investigated. The immunophenotype of resting and IFN-γ primed BMMSC and AdMSC remained unaltered in all media. Both HPL and SFM/XF increased the proliferation of BMMSC and AdMSC. Expansion of BMMSC and AdMSC in HPL increased their differentiation, compared to SFM/XF and FBS. Resting BMMSC and AdMSC, expanded in FBS or SFM/XF, demonstrated potent immunosuppressive properties in both non-primed and IFN-γ primed conditions, whereas HPL-expanded MSC exhibited diminished immunosuppressive properties. Finally, IFN-γ primed BMMSC and AdMSC expanded in SFM/XF and HPL expressed attenuated levels of IDO-1 compared to FBS. Herein, we provide strong evidence supporting the use of the FDA-approved SFM/XF medium, in contrast to the HPL medium, for the expansion of MSC towards therapeutic applications.

Current concepts: the role of mesenchymal stem cells in the management of knee osteoarthritis.

Context:The number of adults with osteoarthritis in the United States is expected to nearly double from 21.4 million in 2005 to 41.1 million by 2030. As a result, medical costs and associated comorbidity will exponentially increase in the coming decades. In the past decade, mesenchymal stem cells (MSCs) have emerged as a novel treatment for degenerative joint disease.Evidence Acquisition:PubMed (from 1990 to 2013) was searched to identify relevant studies. Reference lists of included studies were also reviewed.Study Design:Clinical review.Level of Evidence:Level 3.Results:We identified 9 animal and 7 human studies investigating the use of MSCs in the treatment of osteoarthritis, with varying levels of support for this therapy.Conclusion:While MSCs have shown potential for improving function and decreasing inflammation in animal studies, translation to patients is still in question. There is a great deal of heterogeneity in treatment methods. Standardizing the manufacturing and characterization of MSCs will allow for better comparisons.

Partnership between platelet-rich plasma and mesenchymal stem cells: in vitro experience

We aim to identify current in vitro research exploring platelet-rich plasma (PRP) effects in human Mesenchymal Stem Cells (MSCs) that may encourage or limit the clinical application of MSCs along with PRP. After a systematic search, we identified 57 in vitro studies, focused on optimization of MSC manufacturing, and expanding knowledge about how PRP modifies MSCs behavior for translational purposes. Influences of PRP on proliferation, migration, stemness, preservation of MSC immune-modulatory properties and appearance of senescence phenotype have been explored. Overall PRP stimulates MSC proliferation, preserves MSCs multipotency and does not interfere with any lineage differentiation. PRP (as platelet lysate or releasate) preserves the immune-privileged potential of MSCs and may delay the appearance of the senescent phenotype. Currently there are few data linking precise molecules and biological mechanisms. Various gaps of knowledge need to be addressed in order to obtain enough useful information for translational purposes.

Mesenchymal stem cells in tissue repairing and regeneration: Progress and future.

The presence of mesenchymal progenitor cells within bone marrow has been known since the late nineteenth century. To date, mesenchymal stem cells (MSCs) have been isolated from several different connective tissues, such as adipose tissue, muscle, placenta, umbilical cord matrix, blood, liver, and dental pulp. Bone marrow, however, is still one of the major sources of MSCs for preclinical and clinical research. MSCs were first evaluated for regenerative applications and have since been shown to directly influence the immune system and to promote neovascularization of ischemic tissues. These observations have prompted a new era of MSC transplantation as a treatment for various diseases. In this review, we summarize the important studies that have investigated the use of MSCs as a therapeutic agent for regenerative medicine, immune disorders, cancer, and gene therapy. Furthermore, we discuss the mechanisms involved in MSC-based therapies and clinical-grade MSC manufacturing.

Combination cell therapy for the treatment of acute myocardial infarction

Over the past 10 years, several studies have been carried out to determine whether the infusion of peripheral blood (PBSC) or bone marrow-derived cell products (mononuclear, CD34, CD133 or ALDH cells) may improve cardiac function, after myocardial infarction (MI). The hypothesis behind these studies has been that infusion of a product containing endothelial progenitor cells (ECs)mayenhance the formation of newblood vessels. Results have shown thatwhile the infusion of these products is safe and feasible, the effect on left ventricular function has been negligible ormodest. The poor responses observed could be related to timing, type andnumber of cells used, route of administration or other variables [1,2]. The formation of blood vessel requires in addition to ECs, other cellular and molecular components, including pericytes, cytokines, chemokines and extracellular matrix molecules (ECM). Pericytes share functional properties and a gene-expression profile with mesenchymal stem cells (MSC). In turn, MSC produce and release angiogenic growth factors and ECM which contribute to the formation of a mature and stable blood vessel [3]. Based on these notions, we hypothesize that a combination, rather a single, cell product consisting of a source of ECs and pericytes could be more effective in promoting the formation of new blood vessels. This phase I clinical study was designed to investigate safety and feasibility after the intracoronary infusion to MI patients of a combination cell product consisting of bone marrow-derived mononuclear cell (BMMNC, a source of ECs) and MSC (a source of pericytes, angiogenic growth factors and ECM). Ten patients were enrolled to be treated with the combination cell product and five patients were used as controls. Table 1 shows main baseline characteristics of study and control patients. Study patients had a primary and secondary iliac crest bone marrow aspiration, 3–4 days after angioplasty and 17±7 days after primary aspiration, respectively. Bone marrow aspirations were not performed to control patients. For preparation of MSC and MNC, the methods previously published were used [4]. The combination cell product for infusion contained 7.5×10 MSC and 7.5×10 MNC (the content of EC-like cells in MNC measured as CD45 cells expressing antigens CD34, CD133 or CD144, was 3.5±1.1%.) After assessment of lot-release criteria (USP sterility, endotoxin, mycoplasma and Gram's stain), the combination cell product was intracoronary infused to study patients. After infusion, coronary angiography, ECG, chemistry panel, complete blood count (CBC) and Troponin I was performed. The average time between onset of MI and angioplasty was 15.7± 14 h. Bone marrow aspiration to initiate the preparation of BM-MSC was performed 3.7±1.8 days after PCI. In turn the cell product or placebo solution was infused 17.2±3.8 and 14.3±1.2 days after MI onset, respectively (Table 1). Intracoronary cell infusion probed to be safe and feasible and did not result in ischemia or infarction in the treated areas. Troponin I remained normal in all subjects at 24 h and 14 days post-infusion. Left Ventricular Ejection Fraction (LVEF)was evaluated in both study and control patients using quantitative gated SPECT at months 1 and 3. Echocardiography was utilized to evaluate ejection fraction and wall motion abnormalities. Table 2A shows data for LVEF at baseline and three months after cell/placebo infusion. No significant changes in LVEF or wall motion (not shown) were detected in either group. Assessment of myocardial ischemiawas performed using non-gated SPECT images myocardial perfusion summed stress score (SSS), summed rest score (SRS) and summed difference score (SDS). As seen in Table 2B, in the group of study patients no differences were observed between SSS and SRS at baseline and month 3 after cell infusion. However, this was not the case for SDS since in all treated patients, baseline scores were considerable higher than those at month 3. In turn, in control patients SSS, SRS and SDS values were comparable at baseline and month 3 after placebo infusion. This phase I studywas designed to assess the feasibility and safety of the intracoronary infusion of a combination cell product to MI patients. Feasibility was demonstrated by the procedural ability to initiate MSC manufacturing 3.7±1.8 days after angioplasty. In turn, the safe preparation and infusion of the cell products were confirmed by the inclusive fulfillment of release criteria and by the absence of adverse clinical events, up to 6 months after cell infusion. These observations corroborate results of our previous animal study showing safe and workable use of the combination cell product [5].

Closed system isolation and scalable expansion of human placental mesenchymal stem cells

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost-effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale-up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single-use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large-scale process.

Banking Human Umbilical Cord-Derived Mesenchymal Stromal Cells for Clinical Use

A great deal of interest has arisen recently with respect to human mesenchymal stem cells (MSCs), due to their broad therapeutic potential. However, the safety and efficacy of MSCs expanded ex vivo for clinical applications remain a concern. In this article, we establish a standardized process for manufacture of human umbilical cord-derived MSCs (UC-MSCs), which encompasses donor screening and testing, recovery, two-stage expansion, and administration. The biological properties and safety of UC-MSCs were then characterized and tested. The safety data from use in human patients have also been reported. After clinical-scale expansion, a yield of 1.03-3.78 × 10(8) MSCs was achieved in 10 batch manufacturing runs. The biological properties, such as plastic adherence, morphology, specific surface antigen (CD105, CD73, CD90, positive ≥ 95%; CD45, CD34, CD31, CD11b, CD19, HLA-DR, negative ≤2%), and multipotent differentiation potential (osteogenesis and adipogenesis) were retained. Bacterial and mycoplasma tests were negative and endotoxin levels were lower than 2 EU/ml. No adverse events were noted in two patients treated with intravenously and/or intrathecally administered MSCs. The data obtained indicate that banking UC-MSCs for clinical use is feasible.