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The Quantum cell expansion system manufactured by Terumo-BCT is perhaps the most widely reported Good Manufacturing Practice-compliant bioreactor used for the expansion of adherent cell populations, both for research purposes and clinical cell-based therapies/trials. Although the system was originally designed for adherent cell expansion, more recently suspension cultures and extracellular vesicle manufacturing protocols have been published using the Quantum system. Cell therapy research and regenerative medicine in general is a rapidly expanding field and as such it is likely that the use of this system will become even more widespread and perhaps mandatory, for both research and development and in the clinic. The purpose of this review is to describe, compare and discuss the diverse range of research and clinical applications currently using the Quantum system, which to our knowledge has not previously been reviewed. In addition, current and future challenges will also be discussed.
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Técnicas de Cultivo de Célula , Células Madre Mesenquimatosas , Técnicas de Cultivo de Célula/métodos , Reactores Biológicos , Tratamiento Basado en Trasplante de Células y Tejidos , Proliferación CelularRESUMEN
The immunomodulatory properties of MSCs can be recreated using their extracellular vesicles (EVs). Yet, the true capabilities of the MSC EVs cannot be distinguished from contaminating bovine EVs and protein derived from supplemental foetal bovine serum (FBS). FBS EV depletion protocols can minimise this, but vary in terms of depletion efficiency, which can negatively impact the cell phenotype. We explore the impact of FBS EV depletion strategies, including ultracentrifugation, ultrafiltration, and serum-free, on umbilical cord MSC characteristics. Whilst a greater depletion efficiency, seen in the ultrafiltration and serum-free strategies, did not impact the MSC markers or viability, the MSCs did become more fibroblastic, had slower proliferation, and showed inferior immunomodulatory capabilities. Upon MSC EV enrichment, more particles, with a greater particle/protein ratio, were isolated upon increasing the FBS depletion efficiency, except for serum-free, which showed a decreased particle number. Whilst all conditions showed the presence of EV-associated markers (CD9, CD63, and CD81), serum-free was shown to represent a higher proportion of these markers when normalised by total protein. Thus, we caution MSC EV researchers on the use of highly efficient EV depletion protocols, showing that it can impact the MSC phenotype, including their immunomodulatory properties, and stress the importance of testing in consideration to downstream objectives.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , Albúmina Sérica Bovina/metabolismo , Cordón Umbilical , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , InmunomodulaciónRESUMEN
The extent of ageing in the musculoskeletal system during the life course affects the quality and length of life. Loss of bone, degraded articular cartilage, and degenerate, narrowed intervertebral discs are primary features of an ageing skeleton, and together they contribute to pain and loss of mobility. This review covers the cellular constituents that make up some key components of the musculoskeletal system and summarizes discussion from the 2015 Aarhus Regenerative Orthopaedic Symposium (AROS) (Regeneration in the Ageing Population) about how each particular cell type alters within the ageing skeletal microenvironment.
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Envejecimiento/fisiología , Sistema Musculoesquelético/fisiopatología , Envejecimiento/patología , Huesos/patología , Huesos/fisiopatología , Cartílago Articular/patología , Cartílago Articular/fisiopatología , Senescencia Celular/fisiología , Condrocitos/fisiología , Humanos , Disco Intervertebral/patología , Disco Intervertebral/fisiopatología , Sistema Musculoesquelético/irrigación sanguínea , Sistema Musculoesquelético/patologíaRESUMEN
Autoimmune conditions, such as rheumatoid arthritis, are characterised by a loss of immune tolerance, whereby the immune cells attack self-antigens causing pain and inflammation. These conditions can be brought into remission using pharmaceutical treatments, but often have adverse side effects and some patients do not respond favourably to them. Human umbilical cord mesenchymal stromal cells (UCMSCs) present a promising alternative therapeutic due to their innate anti-inflammatory properties which can be strengthened using pro-inflammatory conditions. Their therapeutic mechanism of action has been attributed to paracrine signalling, by which nanosized acellular particles called 'extracellular vesicles' (EVs) are one of the essential components. Therefore, this research analysed the anti-inflammatory properties of UCMSC-EVs 'primed' with pro-inflammatory cytokines and at baseline with no inflammatory cytokines (control). Both control and primed EVs were co-cultured with un-pooled peripheral blood mononuclear cells (PBMCs; n = 6) from healthy donors. Neither control nor primed EVs exerted a pro-inflammatory effect on PBMCs. Instead, the primed EVs showed the immunosuppressive potential by increasing the expression of the anti-inflammatory protein FoxP3 in PBMCs. This may be attributed to the upregulated miRNAs identified in primed EVs in comparison to control EVs (miR-139-5p, miR-140-5p, miR-214-5p). These findings aid in understanding how UCMSC-EVs mediate immunosuppression and support their potential use in treating autoimmune conditions.
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Vesículas Extracelulares , Células Madre Mesenquimatosas , MicroARNs , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Leucocitos Mononucleares/metabolismo , Regulación hacia Arriba/genética , Citocinas/metabolismo , Antiinflamatorios/farmacología , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/metabolismoRESUMEN
Allogeneic chondrocyte therapies need to be developed to allow more individuals to be treated with a cell therapy for cartilage repair and to reduce the burden and cost of the current two-stage autologous procedures. Upscale manufacture of chondrocytes using a bioreactor could help provide an off-the-shelf allogeneic chondrocyte therapy with many doses being produced in a single manufacturing run. In this study, we assess a good manufacturing practice-compliant hollow-fiber bioreactor (Quantum®) for adult chondrocyte manufacture. Chondrocytes were isolated from knee arthroplasty-derived cartilage (n = 5) and expanded in media supplemented with 10% fetal bovine serum (FBS) or 5% human platelet lysate (hPL) on tissue culture plastic (TCP) for a single passage. hPL-supplemented cultures were then expanded in the Quantum bioreactor for a further passage. Matched, parallel cultures in hPL or FBS were maintained on TCP. Chondrocytes from all culture conditions were characterized in terms of growth kinetics, morphology, immunoprofile, chondrogenic potential (chondrocyte pellet assays), and single telomere length analysis. Quantum expansion of chondrocytes resulted in 86.4 ± 38.5 × 106 cells in 8.4 ± 1.5 days, following seeding of 10.2 ± 3.6 × 106 cells. This related to 3.0 ± 1.0 population doublings in the Quantum bioreactor, compared with 2.1 ± 0.6 and 1.3 ± 1.0 on TCP in hPL- and FBS-supplemented media, respectively. Quantum- and TCP-expanded cultures retained equivalent chondropotency and mesenchymal stromal cell marker immunoprofiles, with only the integrin marker, CD49a, decreasing following Quantum expansion. Quantum-expanded chondrocytes demonstrated equivalent chondrogenic potential (as assessed by ability to form and maintain chondrogenic pellets) with matched hPL TCP populations. hPL manufacture, however, led to reduced chondrogenic potential and increased cell surface positivity of integrins CD49b, CD49c, and CD51/61 compared with FBS cultures. Quantum expansion of chondrocytes did not result in shortened 17p telomere length when compared with matched TCP cultures. This study demonstrates that large numbers of adult chondrocytes can be manufactured in the Quantum hollow-fiber bioreactor. This rapid, upscale expansion does not alter chondrocyte phenotype when compared with matched TCP expansion. Therefore, the Quantum provides an attractive method of manufacturing chondrocytes for clinical use. Media supplementation with hPL for chondrocyte expansion may, however, be unfavorable in terms of retaining chondrogenic capacity.
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Condrocitos , Trasplante de Células Madre Hematopoyéticas , Adulto , Humanos , Cartílago , Células Cultivadas , Matriz Extracelular/metabolismo , Diferenciación Celular , Proliferación CelularRESUMEN
Regenerative medicine, using cells as therapeutic agents for the repair or regeneration of tissues and organs, offers great hope for the future of medicine. Cell therapy for treating defects in articular cartilage has been an exemplar of translating this technology to the clinic, but it is not without its challenges. These include applying regulations, which were designed for pharmaceutical agents, to living cells. In addition, using autologous cells as the therapeutic agent brings additional costs and logistical challenges compared with using allogeneic cells. The main cell types used in treating chondral or osteochondral defects in joints to date are chondrocytes and mesenchymal stromal cells derived from various sources such as bone marrow, adipose tissue or umbilical cord. This review discusses some of their biology and pre-clinical studies before describing the most pertinent clinical trials in this area.
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Cartílago Articular , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Tratamiento Basado en Trasplante de Células y Tejidos , Ingeniería de TejidosRESUMEN
Human umbilical cord (hUC)- or bone marrow (hBM)-derived mesenchymal stromal cells (MSCs) were evaluated as an allogeneic source of cells for cartilage repair. We aimed to determine if they could enhance healing of chondral defects with or without the recruitment of endogenous cells. hMSCs were applied into a focal joint surface injury in knees of adult mice expressing tdTomato fluorescent protein in cells descending from Gdf5-expressing embryonic joint interzone cells. Three experimental groups were used: (i) hUC-MSCs, (ii) hBM-MSCs and (iii) PBS (vehicle) without cells. Cartilage repair was assessed after 8 weeks and tdTomato-expressing cells were detected by immunostaining. Plasma levels of pro-inflammatory mediators and other markers were measured by electrochemiluminescence. Both hUC-MSC (n = 14, p = 0.009) and hBM-MSC (n = 13, p = 0.006) treatment groups had significantly improved cartilage repair compared to controls (n = 18). While hMSCs were not detectable in the repair tissue at 8 weeks post-implantation, increased endogenous Gdf5-lineage cells were detected in repair tissue of hUC-MSC-treated mice. This xenogeneic study indicates that hMSCs enhance intrinsic cartilage repair mechanisms in mice. Hence, hMSCs, particularly the more proliferative hUC-MSCs, could represent an attractive allogeneic cell population for treating patients with chondral defects and perhaps prevent the onset and progression of osteoarthritis.
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Trasplante de Médula Ósea , Cartílago Articular/patología , Condrogénesis , Artropatías/cirugía , Trasplante de Células Madre Mesenquimatosas , Cicatrización de Heridas , Adulto , Animales , Reactores Biológicos , Cartílago Articular/lesiones , Cartílago Articular/metabolismo , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Humanos , Mediadores de Inflamación/sangre , Artropatías/metabolismo , Artropatías/patología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , Trasplante Heterólogo , Cordón Umbilical/citología , Adulto JovenRESUMEN
Metal phosphate deposited enzymatically on Serratia sp. has been used successfully for the removal of radionuclides from aqueous flows. Previous studies using biogenic hydrogen uranyl phosphate (HUP) on Serratia sp. biofilm showed removal of 100% of (90)Sr, (137)Cs, and (60)Co via their intercalation into biogenic HUP crystals. Zirconium phosphates (ZrP) offer a potential non-toxic and non-radioactive alternative to HUP for water decontamination. A method was developed for biomanufacturing ZrP. Biogenic ZrP removed ca. 100% of Sr(2+) and Co(2+) (0.5 mM) from solutions to a molar ratio at saturation of ca. 1:0.6 for both Zr:Sr and Zr:Co. The potential for drinking water decontamination via bio-ZrP is discussed with respect to bio-HUP and also other commercially available materials.
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Serratia/metabolismo , Contaminantes Radiactivos del Agua/metabolismo , Purificación del Agua/métodos , Circonio/metabolismo , Radioisótopos de Cobalto/metabolismo , Radioisótopos de Estroncio/metabolismoRESUMEN
Umbilical cord mesenchymal stromal cells (UCMSCs) have shown an ability to modulate the immune system through the secretion of paracrine mediators, such as extracellular vesicles (EVs). However, the culture conditions that UCMSCs are grown in can alter their secretome and thereby affect their immunomodulatory potential. UCMSCs are commonly cultured at 21% O2 in vitro, but recent research is exploring their growth at lower oxygen conditions to emulate circulating oxygen levels in vivo. Additionally, a pro-inflammatory culture environment is known to enhance UCMSC anti-inflammatory potential. Therefore, this paper examined EVs from UCMSCs grown in normal oxygen (21% O2), low oxygen (5% O2) and pro-inflammatory conditions to see the impact of culture conditions on the EV profile. EVs were isolated from UCMSC conditioned media and characterised based on size, morphology and surface marker expression. EV protein cargo was analysed using a proximity-based extension assay. Results showed that EVs had a similar size and morphology. Differences were found in EV protein cargo, with pro-inflammatory primed EVs showing an increase in proteins associated with chemotaxis and angiogenesis. This showed that the UCMSC culture environment could alter the EV protein profile and might have downstream implications for their functions in immunomodulation.
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Proliferación Celular/fisiología , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Biomarcadores/metabolismo , Células Cultivadas , Citocinas/metabolismo , HumanosRESUMEN
Objective: The synovial fluid (SF) of patients with focal cartilage defects contains a population of poorly characterised cells that could have pathophysiological implications in early osteoarthritis and joint tissue repair. We have examined the cells within SF of such joints by determining their chondrogenic capacity following culture expansion and establishing the phenotypes of the macrophage subsets in non-cultured cells. Design: Knee SF cells were obtained from 21 patients receiving cell therapy to treat a focal cartilage defect. Cell surface immunoprofiling for stem cell and putative chondrogenic markers, and the expression analysis of key chondrogenic and hypertrophic genes were conducted on culture-expanded SF cells prior to chondrogenesis. Flow cytometry was also used to determine the macrophage subsets in freshly isolated SF cells. Results: Immunoprofiling revealed positivity for the monocyte/macrophage marker (CD14), the haematopoietic/endothelial cell marker (CD34) and mesenchymal stem/stromal cell markers (CD73, CD90, CD105) on culture expanded cells. We found strong correlations between the presence of CD14 and the vascular cell adhesion marker, CD106 (r = 0.81, p = 0.003). Collagen type II expression after culture expansion positively correlated with GAG production (r = 0.73, p = 0.006), whereas CD90 (r = -0.6, p = 0.03) and CD105 (r = -0.55, p = 0.04) immunopositivity were inversely related to GAG production. Freshly isolated SF cells were positive for both pro- (CD86) and anti-inflammatory markers (CD163 and CD206). Conclusions: The cellular content of the SF from patients with focal cartilage injuries is comprised of a heterogeneous population of reparative and inflammatory cells. Additional investigations are needed to understand the role played by these cells in the attempted repair and inflammatory process in diseased joints.
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Objective: This UK-wide OATech Network + consensus study utilised a Delphi approach to discern levels of awareness across an expert panel regarding the role of existing and novel technologies in osteoarthritis research. To direct future cross-disciplinary research it aimed to identify which could be adopted to subcategorise patients with osteoarthritis (OA). Design: An online questionnaire was formulated based on technologies which might aid OA research and subcategorisation. During a two-day face-to-face meeting concordance of expert opinion was established with surveys (23 questions) before, during and at the end of the meeting (Rounds 1, 2 and 3, respectively). Experts spoke on current evidence for imaging, genomics, epigenomics, proteomics, metabolomics, biomarkers, activity monitoring, clinical engineering and machine learning relating to subcategorisation. For each round of voting, ≥80% votes led to consensus and ≤20% to exclusion of a statement. Results: Panel members were unanimous that a combination of novel technological advances have potential to improve OA diagnostics and treatment through subcategorisation, agreeing in Rounds 1 and 2 that epigenetics, genetics, MRI, proteomics, wet biomarkers and machine learning could aid subcategorisation. Expert presentations changed participants' opinions on the value of metabolomics, activity monitoring and clinical engineering, all reaching consensus in Round 2. X-rays lost consensus between Rounds 1 and 2; clinical X-rays reached consensus in Round 3. Conclusion: Consensus identified that 9 of the 11 technologies should be targeted towards OA subcategorisation to address existing OA research technology and knowledge gaps. These novel, rapidly evolving technologies are recommended as a focus for emergent, cross-disciplinary osteoarthritis research programmes.
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BACKGROUND: The manufacture of mesenchymal stem/stromal cells (MSCs) for clinical use needs to be cost effective, safe and scaled up. Current methods of expansion on tissue culture plastic are labour-intensive and involve several 'open' procedures. We have used the closed Quantum® hollow fibre bioreactor to expand four cultures each of MSCs derived from bone marrow (BM) and, for the first time, umbilical cords (UCs) and assessed extensive characterisation profiles for each, compared to parallel cultures grown on tissue culture plastic. METHODS: Bone marrow aspirate was directly loaded into the Quantum®, and cells were harvested and characterised at passage (P) 0. Bone marrow cells were re-seeded into the Quantum®, harvested and further characterised at P1. UC-MSCs were isolated enzymatically and cultured once on tissue culture plastic, before loading cells into the Quantum®, harvesting and characterising at P1. Quantum®-derived cultures were phenotyped in terms of immunoprofile, tri-lineage differentiation, response to inflammatory stimulus and telomere length, as were parallel cultures expanded on tissue culture plastic. RESULTS: Bone marrow cell harvests from the Quantum® were 23.1 ± 16.2 × 106 in 14 ± 2 days (P0) and 131 ± 84 × 106 BM-MSCs in 13 ± 1 days (P1), whereas UC-MSC harvests from the Quantum® were 168 ± 52 × 106 UC-MSCs after 7 ± 2 days (P1). Quantum®- and tissue culture plastic-expanded cultures at P1 adhered to criteria for MSCs in terms of cell surface markers, multipotency and plastic adherence, whereas the integrins, CD29, CD49c and CD51/61, were found to be elevated on Quantum®-expanded BM-MSCs. Rapid culture expansion in the Quantum® did not cause shortened telomeres when compared to cultures on tissue culture plastic. Immunomodulatory gene expression was variable between donors but showed that all MSCs upregulated indoleamine 2, 3-dioxygenase (IDO). CONCLUSIONS: The results presented here demonstrate that the Quantum® can be used to expand large numbers of MSCs from bone marrow and umbilical cord tissues for next-generation large-scale manufacturing, without impacting on many of the properties that are characteristic of MSCs or potentially therapeutic. Using the Quantum®, we can obtain multiple MSC doses from a single manufacturing run to treat many patients. Together, our findings support the development of cheaper cell-based treatments.
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Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Separación Celular , Células Madre Mesenquimatosas/citología , Cordón Umbilical/citología , Adulto , Células de la Médula Ósea/metabolismo , Humanos , Recién Nacido , Masculino , Células Madre Mesenquimatosas/metabolismo , Cordón Umbilical/metabolismoRESUMEN
OBJECTIVE: To assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2). DESIGN: Chondrocytes were extracted from patients undergoing total knee replacement (n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture. RESULTS: NC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity (n = 4) and FC (n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein (FRZB), fibroblast growth factor receptor 3 (FGFR3), and collagen type II (COL2A1) and downregulated activin receptor-like kinase 1 (ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells. CONCLUSIONS: Hypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.
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Cartílago Articular/citología , Hipoxia de la Célula/fisiología , Condrocitos/citología , Inmunomodulación/fisiología , Anciano , Cartílago Articular/inmunología , Cartílago Articular/metabolismo , Técnicas de Cultivo de Célula/métodos , Desdiferenciación Celular/fisiología , Hipoxia de la Célula/inmunología , Separación Celular/métodos , Células Cultivadas , Condrocitos/inmunología , Condrocitos/metabolismo , Condrogénesis/genética , Condrogénesis/fisiología , Colágeno Tipo II/metabolismo , Femenino , Citometría de Flujo/métodos , Expresión Génica/fisiología , Humanos , Inmunofenotipificación/métodos , Interferón gamma/inmunología , Masculino , Células Madre Mesenquimatosas/citología , FenotipoRESUMEN
BACKGROUND: The ability to predict the long-term success of surgical treatment in orthopaedics is invaluable, particularly in clinical trials. The quality of repair tissue formed 1 year after autologous chondrocyte implantation (ACI) in the knee was analyzed and compared with clinical outcomes over time. HYPOTHESIS: Better quality repair tissue and a better appearance on magnetic resonance imaging (MRI) 1 year after ACI lead to improved longer-term clinical outcomes. STUDY DESIGN: Cohort study; Level of evidence, 3. METHODS: Repair tissue quality was assessed using either MRI (11.5 ± 1.4 [n = 91] or 39.2 ± 18.5 [n = 76] months after ACI) or histology (16.3 ± 11.0 months [n = 102] after ACI). MRI scans were scored using the whole-organ magnetic resonance imaging score (WORMS) and the magnetic resonance observation of cartilage repair tissue (MOCART) score, with additional assessments of subchondral bone marrow and cysts. Histology of repair tissue was performed using the Oswestry cartilage score (OsScore) and the International Cartilage Repair Society (ICRS) II score. Clinical outcomes were assessed using the modified Lysholm score preoperatively, at the time of MRI or biopsy, and at a mean 8.4 ± 3.7 years (maximum, 17.8 years) after ACI. RESULTS: At 12 months, the total MOCART score and some of its individual parameters correlated significantly with clinical outcomes. The degree of defect fill, overall signal intensity, and surface of repair tissue at 12 months also significantly correlated with longer-term outcomes. The presence of cysts or effusion (WORMS) significantly correlated with clinical outcomes at 12 months, while the presence of synovial cysts/bursae preoperatively or the absence of loose bodies at 12 months correlated significantly with long-term clinical outcomes. Thirty percent of repair tissue biopsies contained hyaline cartilage, 65% contained fibrocartilage, and 5% contained fibrous tissue. Despite no correlation between the histological scores and clinical outcomes at the time of biopsy, a lack of hyaline cartilage or poor basal integration was associated with increased pain; adhesions visible on MRI also correlated with significantly better histological scores. CONCLUSION: These results demonstrate that MRI at 12 months can predict longer-term clinical outcomes after ACI. Further investigation regarding the presence of cysts, effusion, and adhesions and their relationship with histological and clinical outcomes may yield new insights into the mechanisms of cartilage repair and potential sources of pain.
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The infrapatellar fat pad (FP) and synovial fluid (SF) in the knee serve as reservoirs of mesenchymal stromal cells (MSCs) with potential therapeutic benefit. We determined the influence of the donor on the phenotype of donor matched FP and SF derived MSCs and examined their immunogenic and immunomodulatory properties before and after stimulation with the pro-inflammatory cytokine interferon-gamma (IFN-γ). Both cell populations were positive for MSC markers CD73, CD90 and CD105, and displayed multipotency. FP-MSCs had a significantly faster proliferation rate than SF-MSCs. CD14 positivity was seen in both FP-MSCs and SF-MSCs, and was positively correlated to donor age but only for SF-MSCs. Neither cell population was positive for the co-stimulatory markers CD40, CD80 and CD86, but both demonstrated increased levels of human leukocyte antigen-DR (HLA-DR) following IFN-γ stimulation. HLA-DR production was positively correlated with donor age for FP-MSCs but not SF-MSCs. The immunomodulatory molecule, HLA-G, was constitutively produced by both cell populations, unlike indoleamine 2, 3-dioxygenase which was only produced following IFN-γ stimulation. FP and SF are accessible cell sources which could be utilised in the treatment of cartilage injuries, either by transplantation following ex-vivo expansion or endogenous targeting and mobilisation of cells close to the site of injury.
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Tejido Adiposo/citología , Inflamación/patología , Células Madre Mesenquimatosas/citología , Rótula/citología , Líquido Sinovial , Adulto , Anciano , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Autologous chondrocyte implantation (ACI) is a cell-based therapy that has been used clinically for over 20 years to treat cartilage injuries more efficiently in order to negate or delay the need for joint replacement surgery. In this time, very little has changed in the ACI procedure, but now many centres are considering or using alternative cell sources for cartilage repair, in particular mesenchymal stem cells (MSCs). In this study, we have tested the chondrogenic potential of donor-matched MSCs derived from bone marrow (BM), infrapatellar fat pad (FP), and subcutaneous fat (SCF), compared to chondrocytes. We have confirmed that there is a chondrogenic potency hierarchy ranging across these cell types, with the most potent being chondrocytes, followed by FP-MSCs, BM-MSCs, and lastly SCF-MSCs. We have also examined gene expression and surface marker profiles in a predictive model to identify cells with enhanced chondrogenic potential. In doing so, we have shown that Sox-9, Alk-1, and Coll X expressions, as well as immunopositivity for CD49c and CD39, have predictive value for all of the cell types tested in indicating chondrogenic potency. The findings from this study have significant clinical implications for the refinement and development of novel cell-based cartilage repair strategies.
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Mesenchymal stromal cells (MSC) can be isolated from several regions of human umbilical cords, including Wharton's jelly (WJ), artery, vein or cord lining. These MSC appear to be immune privileged and are promising candidates for cell therapy. However, isolating MSC from WJ, artery, vein or cord lining requires time-consuming tissue dissection. MSC can be obtained easily via briefly digesting complete segments of the umbilical cord, likely containing heterogenous or mixed populations of MSC (MC-MSC). MC-MSC are generally less well characterized than WJ-MSC, but nevertheless represent a potentially valuable population of MSC. This study aimed to further characterize MC-MSC in comparison to WJ-MSC and also the better-characterized bone marrow-derived MSC (BM-MSC). MC-MSC proliferated faster, with significantly faster doubling times reaching passage one 8.8 days sooner and surviving longer in culture than WJ-MSC. All MSC retained the safety aspect of reducing telomere length with increasing passage number. MSC were also assessed for their ability to suppress T-cell proliferation and for the production of key markers of pluripotency, embryonic stem cells, tolerogenicity (CD40, CD80, CD86 and HLA-DR) and immunomodulation (indoleamine 2,3-dioxygenase [IDO] and HLA-G). The MC-MSC population displayed all of the positive attributes of WJ-MSC and BM-MSC, but they were more efficient to obtain and underwent more population doublings than from WJ, suggesting that MC-MSC are promising candidates for allogeneic cell therapy in regenerative medicine.
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AIM: Autologous chondrocyte implantation (ACI) is used worldwide for the treatment of cartilage defects. This study has aimed to assess for the first time the cells that are contained within human ACI repair tissues several years post-treatment. We have compared the phenotypic properties of cells from within the ACI repair with adjacent chondrocytes and subchondral bone-derived mesenchymal stromal/stem cells (MSCs). MATERIALS & METHODS: Two patients undergoing arthroplasty of their ACI-treated joint were investigated. Tissue and cells were isolated from the repair site, adjacent macroscopically normal cartilage and MSCs from the subchondral bone were characterized for their growth kinetics, morphology, immunoprofile and differentiation capacity. RESULTS: ACI repair tissue appeared fibrocartilaginous, and ACI repair cells were heterogeneous in morphology and size when freshly isolated, becoming more homogeneous, resembling chondrocytes from adjacent cartilage, after culture expansion. The same weight of ACI repair tissue resulted in less cells than macroscopically normal cartilage. During expansion, ACI repair cells proliferated faster than MSCs but slower than chondrocytes. ACI repair cell immunoprofiles resembled chondrocytes, but their differentiation capacity matched MSCs. CONCLUSION: This novel report demonstrates that human ACI repair cell phenotypes resemble both chondrocytes and MSCs but at different stages of their isolation and expansion in vitro.
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Condrocitos/citología , Condrocitos/trasplante , Cicatrización de Heridas , Adulto , Cartílago/patología , Diferenciación Celular , Proliferación Celular , Forma de la Célula , Demografía , Humanos , Cinética , Masculino , Persona de Mediana Edad , Fenotipo , Trasplante AutólogoRESUMEN
Umbilical cords as a source of stem cells are of increasing interest for cell therapies as they present little ethical consideration and are reported to contain immune privileged cells which may be suitable for allogeneic based therapies. Mesenchymal stem cells (MSCs) sourced from several different cord regions, including artery, vein, cord lining, and Wharton's jelly, are described in the literature. However, no one study has yet isolated and characterised MSCs from all regions of the same cord to determine the most suitable cells for cell based therapeutics.