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Review

Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review

by
Doreen Goh
1,2,
Yanmeng Yang
1,2,3,
Eng Hin Lee
1,2,3,
James Hoi Po Hui
1,2 and
Zheng Yang
1,2,3,*
1
Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, 1E Kent Ridge Road, NUHS Tower block Level 11, Singapore 119288, Singapore
2
NUS Tissue Engineering Program, Life Sciences Institute, National University of Singapore, 27 Medical Drive, DSO (Kent Ridge) Building, Level 4, Singapore 11751, Singapore
3
Critical Analytics for Manufacturing Personalised-Medicine, Singapore-MIT Alliance for Research and Technology, Singapore 138602, Singapore
*
Author to whom correspondence should be addressed.
Bioengineering 2023, 10(3), 355; https://doi.org/10.3390/bioengineering10030355
Submission received: 18 February 2023 / Revised: 12 March 2023 / Accepted: 12 March 2023 / Published: 13 March 2023
(This article belongs to the Special Issue Mesenchymal Stem Cells in Regenerative Medicine)
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Abstract

:
Articular cartilage defects commonly result from trauma and are associated with significant morbidity. Since cartilage is an avascular, aneural, and alymphatic tissue with a poor intrinsic healing ability, the regeneration of functional hyaline cartilage remains a difficult clinical problem. Mesenchymal stem cells (MSCs) are multipotent cells with multilineage differentiation potential, including the ability to differentiate into chondrocytes. Due to their availability and ease of ex vivo expansion, clinicians are increasingly applying MSCs in the treatment of cartilage lesions. However, despite encouraging pre-clinical and clinical data, inconsistencies in MSC proliferative and chondrogenic potential depending on donor, tissue source, cell subset, culture conditions, and handling techniques remain a key barrier to widespread clinical application of MSC therapy in cartilage regeneration. In this review, we highlight the strategies to manage the heterogeneity of MSCs ex vivo for more effective cartilage repair, including reducing the MSC culture expansion period, and selecting MSCs with higher chondrogenic potential through specific genetic markers, surface markers, and biophysical attributes. The accomplishment of a less heterogeneous population of culture-expanded MSCs may improve the scalability, reproducibility, and standardisation of MSC therapy for clinical application in cartilage regeneration.

Graphical Abstract

1. Articular Cartilage Injury and Management

Articular cartilage is a highly specialised tissue found in synovial joints at the articulating surfaces of bones. It has a low density of chondrocytes, which produce and maintain a rich extracellular matrix (ECM) comprising collagens, proteoglycans, water, and ions [1]. The dense collagen network contributes to tensile strength and shear stiffness of articular cartilage [2]. The high proteoglycan content in cartilage enables articular cartilage to withstand compressive forces and allows for weight-bearing. With the secretion of lubricin by superficial zone chondrocytes, cartilage is coated by a lubricating layer and has a low coefficient of friction, providing a smooth surface for uninterrupted joint movement [3].
However, repeated high-intensity mechanical overloading and acute trauma can cause cartilage injury. Given its avascular, aneural, and alymphatic nature [4], articular cartilage has a limited intrinsic regenerative capacity and does not recover readily from injury. This is exacerbated by the low density of chondrocytes in articular cartilage [5] and the high density of cartilage ECM, which impedes the migration of local chondrocytes to the injury site [6]. If cartilage injury is allowed to progress, changes in load bearing and the release of inflammatory mediators [7] can result in cumulative cartilage degeneration with secondary synovitis, subchondral bone remodelling, and contractions in the surrounding ligaments, joint capsule, and muscles [8]. This predisposes the patient to developing osteoarthritis.
Current management of cartilage injury includes surgical debridement of the joint, bone marrow stimulation, and transplantation approaches. In surgical debridement, loose bodies and calcified cartilage are removed from the joint to avoid further deterioration and fragmentation of the articular surface [9]. Bone marrow stimulation techniques, such as drilling, microfracture, and abrasion chondroplasty, induce the migration of bone marrow cells into the cartilage defect by mechanically penetrating the underlying subchondral bone. A fibrin clot, containing extravasated bone marrow mesenchymal stem cells (BM-MSCs), forms in the defect. Mesenchymal stem cells (MSCs) then participate in cartilage regeneration [10]. However, repair after bone marrow stimulation mostly results in the formation of biomechanically inferior fibrocartilage containing type I collagen, instead of the desired hyaline cartilage with type II collagen [11]. The fibrocartilage repair tissue is poorly integrated into the native cartilage and deteriorates over time [12], leading to declining treatment effectiveness in long-term studies [13,14]. Bone marrow stimulation techniques also disrupt the integrity of the subchondral bone, leading to instability and the formation of subchondral cysts and osteophytes [15].
Alternatively, surgeons can consider transplantation techniques for the management of articular cartilage defects. Osteochondral autograft transfers, either with the osteochondral autograft transfer system (OATS), or Mosaicplasty, are indicated in smaller defects less than 4 cm2 [16]. They involve the transplantation of healthy osteochondral tissue from non-weight-bearing areas of the joint into the defect. However, these techniques are limited by donor site morbidity and the scarce amount of donor tissue that can be harvested. Non-weight-bearing donor cartilage may also have differences in mechanical properties and depth [17]. In larger defects, osteochondral allografting can be applied instead, which involves the transplantation of osteochondral tissue from a cadaveric source. Osteochondral allografting avoids donor-site morbidity and limitations in tissue size and structure. However, allografts are associated with high cost, and the risk of tissue rejection and disease transmission [18].
Autologous chondrocyte implantation (ACI) is the first application of cell therapy in orthopaedics. ACI is suited for defect sizes larger than 3 to 4 cm2 [19]. First-generation ACI is a two-stage procedure that consists of cartilage harvesting from the joint, in vitro isolation and expansion of chondrocytes, and subsequent reintroduction of chondrocytes into the chondral defect under a periosteal patch [20]. However, the periosteal patch is prone to hypertrophy, with more than 20% of patients requiring repeat arthroscopy with debridement. In second generation ACI, a collagen membrane replaces the periosteal patch for covering the defect. In third generation ACI, tissue engineering principles are applied to incorporate chondrocytes directly onto a three-dimensional (3D) scaffold for implantation. The scaffold allows for better graft stability and reduces dedifferentiation of chondrocytes, generating more hyaline-like cartilage [21,22]. Also known as matrix-induced autologous cartilage implantation (M-ACI), this technique has led to improved clinical outcomes in patients compared to microfracture [23].
Despite the promising clinical outcomes of cell therapy approaches, the use of autologous chondrocytes requires tissue harvesting from the non-weight-bearing areas of knee cartilage, resulting in donor site morbidity. Chondrocytes are also scarce, with only 10 cells available in every 0.22 mm2 of human knee articular cartilage [24], necessitating the extensive ex vivo expansion of limited chondrocytes after isolation from patient tissue. Yet, chondrocytes that are cultured for prolonged periods are prone to dedifferentiation, during which cells become fibroblastic with reduced expression of type II collagen, generating neo-cartilage of lower functional efficacy, with mechanically inferior ECM [25]. Clinicians have, hence, explored the use of stem cells, especially adult MSCs, as an alternative cell source for the treatment of cartilage lesions.

2. Mesenchymal Stem Cells (MSCs) for Cartilage Regeneration

MSCs are multipotent cells with a multilineage differentiation potential, including the ability to differentiate into chondrocytes and generate ECM containing type II collagen. The use of MSCs as an alternative cell source for cartilage regeneration confers numerous advantages. Firstly, autologous MSCs can be harvested relatively easily through less invasive means, without injuring existing healthy cartilage [26]. Various tissue sources are available for MSC harvest, including bone marrow, adipose tissue, umbilical cord blood, and synovial fluid [27,28]. Unlike chondrocytes, MSCs can undergo expansion in vitro without losing their phenotype through dedifferentiation [29]. Allogenic cells can also be used in tissue engineering, as MSCs have been shown to be immune-evasive [30].
As such, many pre-clinical studies and clinical trials have been conducted on the use of MSCs for cartilage regeneration [31]. In vivo, intra-articular implantation of MSCs has led to enhanced cartilage repair in both small and large animal models [32]. Clinically, seven years of extended follow up, after implantation of MSCs encapsulated within a hyaluronic acid hydrogel, have found MSCs to be safe and efficacious for the treatment of cartilage defects [33]. As an adjuvant treatment, a large multicentre prospective randomised clinical trial has recently found that the intra-articular injection of BM-MSCs after microfracture led to improved repair, and a trend towards improved clinical outcomes in articular cartilage injuries of the knee [34]. A single-stage cartilage repair procedure, employing allogenic MSCs and autologous chondrons in fibrin glue, has similarly reported good five-year safety outcomes and led to clinical improvement [35].
In addition to being capable of chondrogenic differentiation, MSCs exert trophic effects on the surrounding chondrocytes, secreting bioactive molecules that promote ECM formation [36]. The therapeutic potential of the MSC secretome has led to an increased focus on cell-free strategies to facilitate cartilage repair. A systematic review of pre-clinical research reports that MSC exosomes are effective in the treatment of articular cartilage injury, resulting in hyaline-like neocartilage formation, improved integration with native cartilage, and increased ECM deposition in the defect site [37]. Exosomes secreted by MSCs enhance cartilage regeneration through multiple pathways, including the activation of AKT and ERK signalling to promote chondrocyte proliferation [38]. CD73 found in MSC exosomes also initiate tissue repair by hydrolysing the post-injury extracellular pro-apoptotic ATP signal into the pro-survival adenosine signal, while exosomal miRNAs are involved in MSC chondrogenesis and matrix synthesis [39]. MSCs also promote a regenerative milieu in injured tissue, increasing the infiltration of regenerative M2 macrophages and inhibiting pro-inflammatory cytokines [38,40]. The MSC secretome further increases the anti-inflammatory IL-10 and TGF-β1 levels, and induces regulatory T cells through CCL18 signalling [39,41]. miRNAs found in MSC exosomes contribute to the immunomodulatory role of MSCs [42]. In an in vivo porcine model, the injection of MSC exosomes and hyaluronic acid has led to improved morphological, histological, and functional recovery after 4 months [43].

3. Heterogeneity of MSCs

Despite encouraging pre-clinical and clinical outcomes, inconsistencies in MSC characteristics remain a key barrier to widespread clinical application of MSC therapy in cartilage regeneration. The high degree of variability in MSC stemness and chondrogenic potential poses a challenge to the scalability, reproducibility, and standardisation of therapeutic potency [44,45]. The characterisation of MSC exosomal mechanisms of action remains limited, partly due to MSC heterogeneity. This restricts further research on MSC secretome potency, and may limit the future clinical application and commercialisation of MSC exosome therapy [46].
MSC heterogeneity can occur at multiple levels, depending on the donor, MSC tissue source, cell subset, culture conditions, and handling techniques (Table 1). There is a large degree of inter-individual variation in MSC quality, even when isolated from the same tissue source [47]. As donor age increases, MSC immunomodulation, proliferation, and differentiation potential decreases [48]. Compared to older donors, BM-MSCs from young donors have improved glycosaminoglycan deposition and increased expression of the chondrogenic markers SOX9, COL2A1, and ACAN [49]. Donor sex affects MSC properties as well. MSCs from young female donors have been shown to exhibit a shorter population doubling time and increased colony formation [50]. MSCs from donors with diseases such as osteoarthritis and osteoporosis demonstrate lower chondrogenic potential and proliferative ability, with a reduced expression of MSC surface marker CD73 [51,52]. On a genomic level, BM-MSCs from donors with a genomic deletion of glutathione S-transferase theta 1 (GSTT1) demonstrate increased scalability, with improved clonogenic and proliferative capabilities, while maintaining a robust multilineage differentiation potential [53].
MSCs are commonly derived from the bone marrow, adipose tissue, umbilical cord blood, and synovium. On an intra-individual level, cells from different tissue sources demonstrate heterogeneity in MSC characteristics. Compared to adipose-derived MSCs (AD-MSCs), BM-MSCs from the same donor exhibited a better chondrogenic potential in vitro and in vivo [54,55]. The seeding of BM-MSCs in commercially available scaffolds has led to better ECM deposition in vitro than seeding with AD-MSCs [56]; the seeding of BM-MSCs in a platelet-rich plasma bioactive scaffold has led to improved macroscopic, histological, and immunohistochemical characteristics compared to AD-MSCs in an in vivo rabbit model [57]. Although inferior in chondrogenic potential, AD-MSCs may possess an improved proliferative ability compared to BM-MSCs [58]. Umbilical cord blood-derived MSCs (UCB-MSCs) have a higher population doubling number than BM-MSCs [27]. However, compared to BM-MSCs, UCB-MSCs are reported to produce ECM of lower quantity after chondrogenesis [59]. Synovium membrane-derived MSCs (SM-MSCs) are another explored population of MSCs. Previous studies have found that SM-MSCs demonstrate an increased proliferative ability and enhanced chondrogenic potential compared to donor-matched BM-MSCs and AD-MSCs [28]. The implantation of SM-MSCs in human knee articular cartilage defects has resulted in the production of hyaline cartilaginous tissue and improved clinical outcomes [60].
Variation further exists between MSCs of a similar tissue source that are isolated from different anatomical locations. For example, a higher chondrogenic potential has been demonstrated by BM-MSCs extracted from the iliac crest and vertebral body compared to cells derived from the femoral head, possibly due to an increased involvement of the femoral head in disease processes [61]. BM-MSCs collected from the proximal femur trabeculae, through rasping, display an increased chondrogenic potential compared to BM-MSCs harvested from the main marrow compartment of the same patients through BM aspiration [62]. Among the AD-MSCs derived from various anatomical locations, the AD-MSCs from subcutaneous tissue show increased proliferative ability [63]. However, a donor-matched study has found that AD-MSCs derived from the infrapatellar fat pad demonstrate increased glycosaminoglycan production and upregulation of the chondrogenic genes ACAN and COL2A1, indicating an increased chondrogenic potential compared to AD-MSCs from subcutaneous fat [64].
During in vitro culture, MSCs form different single cell-derived clonal subpopulations within the same MSC preparation. These subpopulations vary in morphology, transcriptome, proliferative potential, and functional chondrogenic differentiation potential [65,66,67]. In fact, the MSC subpopulation phenotype may have a larger role in influencing the MSC chondrogenic potential and secretome, compared to inter-individual heterogeneity in donor age or gender [50]. Heterogeneity in MSCs is present even at an intra-colony level, despite cells being supposedly derived from a single MSC and cultured under similar conditions. Within the same colony, cell-to-cell variation exists in the morphology and gene expression [68]. Single cells differentially express immunomodulatory and chondrogenic genes, and cells with diverse properties can ultimately result from a once-homogenous colony [69,70,71].

4. Managing the Heterogeneity of MSCs for More Effective Cartilage Regeneration

In addition to selecting the appropriate donor and MSC tissue source, this section elaborates on other strategies to manage the heterogeneity of MSCs ex vivo for more effective cartilage repair, namely, through reducing the MSC culture expansion period, and selecting MSCs with a higher chondrogenic potential, through specific surface markers and biophysical attributes (Figure 1).

4.1. Reducing Expansion Period

The aforementioned diversity in MSC subpopulations emerges and becomes amplified during culture expansion, with single colonies ultimately giving way to a heterogenous mix of cells characterised by varying potencies [72,73]. Extensive passaging in vitro also results in a decreased MSC migration potential and differentiation potential, leading to the production of less cartilaginous tissue [74]. MSCs in long-term culture produce increased β-galactosidase levels compared to MSCs from aged donors, indicating that culture length may have a greater impact on MSC senescence than donor age [75]. After extended culture expansion, AD-MSCs also demonstrate an increased production of proinflammatory cytokines and a decreased production of anti-inflammatory mediators [76]. Thus, reducing the expansion period of MSCs could improve MSC homogeneity and quality for cartilage repair.
To reduce the MSC expansion period, MSC proliferation may be enhanced with the addition of bioactive factors. Growth factors, such as fibroblast growth factor 2 (FGF2), have been shown to improve MSC proliferative capabilities in vitro, reducing the population doubling time and resulting in differential expression of proliferation-related genes [77]. After four passages, FGF2 treatment increases cell yield by 256-fold without affecting the MSC chondrogenic potential [78]. Binding of the FGF receptor may activate signalling pathways, such as the Ras/Erk pathway, to increase cell proliferation downstream [79,80]. Supplementation with FGF4 also leads to a halving of the MSC population doubling time without affecting MSC pluripotency. As a ligand for the FGF2 receptor, FGF4 may exert its stimulatory effects on MSC proliferation through similar signalling pathways [81]. Heparan sulfate is another activator of FGF2 signalling through its affinity for FGF2 and the FGF2 receptor [82]. Heparan sulfate increases endogenous FGF2 production, and stabilises FGF2 to maximise the effects of FGF2 supplementation [83,84]. The addition of heparan sulfate increases MSC colony formation and growth, increasing MSC expansion by up to 13-fold while maintaining MSC multipotency [85].
Platelet-derived growth factor-BB (PDGF-BB) stimulates MSC self-renewal through the PI3K/Akt pathway [86], increasing the expression of membrane-tethered matrix metalloproteinase MT1-MMP to enhance MSC proliferation [87]. PDGF-BB exerts a dose-dependent effect on MSC numbers, increasing MSC cell count by 45% at 5 nM, and 81% at 25 nM [88]. A potent mitogen, PDGF-BB upregulates the genes associated with proliferation without promoting senescence [89].
Other bioactive additives to consider include epidermal growth factor (EGF) and insulin-like growth factor (IGF). EGF increases colony-forming units and improves MSC proliferation in vitro by 30% [90], activating β-catenin to reduce cell cycle pathway suppression [91]. Elevated autocrine IGF-1 levels also enhance MSC proliferation and the number of cell doublings while suppressing apoptosis [92]. IGF-1 supplementation stimulates proliferation in a dose-dependent manner through both the IGF-1 receptor and insulin receptor [93].
In addition to growth factors, ECM proteins have a role to play in MSC proliferation. Tropoelastin is an ECM component which interacts with the cell surface integrins αvβ3 and αvβ5. Tropoelastin supplementation stimulates MSC proliferation at a level similar to IGF-1 or FGF-2 supplementation while maintaining the MSC phenotype, and may be an attractive cost-effective alternative to the use of serum and growth factors [94]. MSCs cultured with basement membrane ECM proteins further demonstrate a 250-fold increase in proliferation capabilities, resulting in increased colony numbers and size while retaining MSC multipotency [95]. Coating culture flasks with type I collagen significantly increases MSC proliferation, boosting cell attachment and reducing cell death [96]. However, effects on the subsequent MSC chondrogenic potential have not been investigated. ECM produced from stromal cells increases cell proliferation in a three-dimensional scaffold, and enhances MSC clonogenicity while maintaining MSC stemness [97].
Lastly, biophysical stimulation through low intensity vibration, electromagnetic field exposure, and electrical stimulation may contribute to improved MSC proliferation during culture expansion. Low intensity vibration increases MSC doubling by 28%, and reduces senescence-associated β-galactosidase levels by 28% after 60 passages in vitro [98]. Loss of the MSC proliferative ability can also be reversed with the application of low intensity vibration [99]. Electromagnetic field exposure of suspended MSCs increases MSC numbers by 2.41-fold [100], possibly due to the induction of a shorter cell cycle [101]. Electrical stimulation of MSCs at 448 kHz induces cell cycle progression through ERK1/2 upregulation, enhancing MSC proliferation by 38% without affecting subsequent MSC differentiation [102].

4.2. Selecting MSCs Based on Specific Markers

The expression of specific cell surface markers has been linked to an increased cartilage regeneration potential of MSC subpopulations [48] (Table 2). Screening MSCs for potency allows for the selective expansion of MSC subpopulations with homogenous and desired characteristics, increasing the applicability of MSC therapy. Selection may be performed with techniques such as magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) [103], and with a combination of markers to potentially improve screening specificity [104].
The MSC surface markers specified by the International Society for Cell and Gene Therapy are CD73, CD90, and CD105 [44,105]. CD73+ MSCs possess a good chondrogenic potential, with high levels of COL2 and ACAN expression during chondrogenesis [106]. Meanwhile, CD105+ MSCs demonstrate increased proliferation, improved colony formation, and an enhanced chondrogenic potential in vitro with increased expression of SOX9, COL2, and ACAN [107,108]. Subpopulations of AD-MSCs expressing CD29 and CD105 show an increased chondrogenic potential, witha higher expression of the chondrogenic marker COL2 [109].
A member of the tumour necrosis factor receptor superfamily, CD271, is a cell surface marker that establishes MSC proliferation and differentiation capacity [110]. Compared to CD73+ MSCs, CD271+ MSCs have an increased chondrogenic potential [106]. CD271+ BM-MSCs display improved chondrogenesis in vitro and in vivo compared to control MSCs, and a reduced volume of CD271+ BM-MSCs is needed to produce a similar cartilage graft compared to unfiltered multiclonal MSC preparations [111,112]. CD271+ MSCs improve osteochondral defect healing, and maintain low angiogenesis while retaining trilineage multipotency [113]. In addition to demonstrating upregulation of the genes associated with ECM production and cell adhesion, CD271+ MSCs demonstrate downregulation of the genes associated with inflammation [114].
CD146 is a transmembrane glycoprotein expressed in MSCs but not in fibroblasts [115]. CD146 expression decreases as the MSC passage number increases, coinciding with an increase in population doubling time [116]. CD146+ MSCs produce increased glycosaminoglycans after chondrogenesis [117] and demonstrate an increased chondrogenic potential [118]. In a three-dimensional scaffold, CD146+ MSCs demonstrate spontaneous chondrogenesis [119], while intra-articular implantation of CD146+ MSCs has led to chondroprotective effects in inflammatory environments [120]. A magnetically-sorted CD146+ MSC population has also shown improved cell migration compared to a heterogenous cell population [121]. Compared to unsorted AD-MSCs, CD146+ MSCs promote long-term cartilage repair in vivo and produce less inflammation [122].
Stro-1 is another widely-employed MSC marker [123]. Stro-1 expression is associated with increased proliferation and differentiation capacity [124], along with increased immunosuppression and homing capabilities of MSCs [125]. Meanwhile, CD49f is a member of the adhesion molecular family that is also involved in the maintenance of MSC stemness, regulating MSC proliferation and differentiation through the PI3K/AKT signalling pathway [126]. CD49f+ MSCs have improved clonogenicity, adhesion, migration, and anti-apoptotic properties compared to unsorted MSCs, with MSCs demonstrating reduced CD49f expression as the passage number increases [127,128]. The glycolipid SSEA-4 may be another surface marker of interest. Compared to unsorted MSCs, SSEA-4+ MSCs exhibit improved growth and multipotency [129,130]. Increased SSEA-4 expression may also be associated with increased chondrogenic potential [131]. Although these surface markers have not been well-studied in the context of chondrogenesis and cartilage repair, the superior MSC characteristics of cells expressing Stro-1, CD49, and SSEA-4 may render these specific markers an attractive target in MSC selection for more effective clinical application.
Alternatively, genomic biomarkers, such as GSTT1, may be used for the selection of MSC donors pre-harvest. Transcriptomic analyses have revealed that homozygous negative GSTT1 MSCs demonstrate increased scalability and potency, possibly due to increased protection from DNA damage [53]. Diagnostic kits to identify prospective donors are being developed, and may reduce unnecessary MSC harvesting from donors [132]. Other hypothesised genetic biomarkers, which may be validated in future studies, include the single-nucleotide polymorphism rs144383 in GDF5, which is strongly associated with osteoarthritis [133].
With the high cell-to-cell variability present within a single MSC colony, single-cell profiling techniques may become increasingly important to identify heterogeneity within MSC subpopulations, and establish more selective biomarkers for isolating MSC subpopulations capable of more effective cartilage repair. Single-cell RNA sequencing (scRNA-seq) allows for the ongoing identification of functional MSC subpopulations through clustering [134]. Surface marker expression and the subsequent heterogeneity during chondrogenesis can be quantified to identify cell subpopulations that are less heterogenous and more chondrogenic [135]. At a genomic level, techniques such as multiple displacement amplification (MDA) and multiple annealing and looping-based amplification cycles (MALBAC) may be employed, while single-cell mass spectrometry may be used to explore cellular heterogeneity at a proteomic level [136,137].
Table
Table 2. A summary of studies that highlight specific MSC markers and their associated phenotype in cartilage regenerative therapy.
Table 2. A summary of studies that highlight specific MSC markers and their associated phenotype in cartilage regenerative therapy.
Specific MSC MarkersResultsReference
CD73+High levels of COL2 and ACAN expression during chondrogenic differentiation, with stable levels of COL1, COLX, and MMP13.[106]
CD105+Increased proliferation and improved colony formation.
Enhanced chondrogenic potential in vitro with increased expression of SOX9, COL2, and ACAN.		[107,108]
CD271+Improved chondrogenic differentiation with a higher expression of Runx2 and COL2.
Upregulation of genes associated with ECM production and cell adhesion; downregulation of genes associated with inflammation.
Improved osteochondral defect healing while maintaining low angiogenesis in an athymic rat model. 		[106,113,114]
CD146+Increased glycosaminoglycan production after chondrogenesis.
Improved chondrogenic potential and cell migration.
Chondroprotective effects during intra-articular implantation.
Promoted long-term cartilage repair in a rat osteochondral defect model and demonstrated immunomodulation.		[117,118,119,120,121,122]
Stro-1+Increased proliferation and differentiation capacity.
Increased immunosuppression and homing capabilities.		[124,125]
CD49f+Improved clonogenicity, adhesion, migration, and anti-apoptotic properties.[127]
SSEA-4Improved growth and multipotency.
Increased chondrogenicity. 		[129,130,131]
GSTT1Homozygous negative GSTT1 MSCs demonstrate increased scalability and potency.[53]

4.3. Selecting MSCs Based on Specific Biophysical Attributes

MSCs develop variation in morphology and physical properties with prolonged in vitro expansion, with multivariate biophysical analysis of culture-expanded MSCs further demonstrating a correlation between cell size, stiffness, nuclear membrane fluctuations, MSC biomolecular markers, and functionality [138,139]. High-throughput cell separation techniques are being developed for the selection of MSCs based on these properties. Multiparameter deformability cytometry classifies cells according to multiple biophysical characteristics, such as size, deformability, and morphology, through microfluidic inertial focusing, hydrodynamic stretching of cells, and high-speed video recording [140]. Since MSC differentiation results in a reorganisation of lamin A/C and an increase in heterochromatin levels [141], MSC stiffness is also directly correlated with the extent of chondrogenic differentiation [142]. The combination of small cell size, low cell stiffness, and high nuclear membrane fluctuations is indicative of high MSC clonogenicity and multipotency, and future cell isolation techniques may select for this subpopulation of MSCs [138]. The corresponding surface biomarkers of the cell mechanophenotype may also be employed for MSC sorting. For example, CD44 expression is strongly inversely related with cell elasticity, and MSC isolation based on cell stiffness may employ CD44 expression levels as a surrogate marker [143]. Alternatively, cell separation based on cell deformability using a deterministic lateral displacement device, such as those employed in isolating immune cells from whole blood cells, may be used to segregate MSC based on stiffness [144].
Functional differences in the capacity for cartilage repair are present based purely on MSC size. Using a high-throughput microfluidic label-free technology, medium-sized cells, 17 to 21 μm in size, have been demonstrated to possess the highest proliferation rate and chondrogenic potential. Selected MSCs of this size continue to highly express COL2 and ACAN after 10 passages, prolonging the MSC chondrogenic potential in vitro [145]. In addition, the secretome of MSCs within this size range promotes chondrogenesis, while the secretome of larger MSCs, more than 21 μm in size, promotes osteogenesis and adipogenesis [139,146]. Further, the secretome of larger MSCs has been found to further induce cellular senescence, indicating the negative effect of MSC heterogeneity during in vitro expansion. A selective MSC culture approach, that excludes MSCs larger than 21 μm and smaller than 17 μm at every passage, may select for MSCs with an improved proliferation and chondrogenic potential compared to a conventional MSC expansion approach [145,146].
Another potentially relevant biophysical attribute is cell and nuclei shape. MSCs take on a rounded phenotype at the start of chondrogenesis, and rounded MSCs in early lineage commitment have been found to upregulate chondrogenic genes, committing to chondrogenesis [147]. This may be associated with the MT1-MMP control of MSC commitment through ECM remodelling, in which MT1-MMP activates β1-integrin for downstream signalling through regulating MSC shape [148]. MSCs with a rounded nuclei have also been found to increase the expression of RUNX2, SOX9, and ACAN [149].

5. Conclusions

Articular cartilage injury severely impacts patients’ quality of life, driving a market for cartilage regeneration products that was worth USD 852 million in 2021 [150]. While MSC therapy has the potential to improve cartilage repair, MSCs are highly heterogenous, and inconsistencies in MSC quality prevent scalability, reproducibility, and standardisation for clinical application. Hence, large-scale clinical trials employing MSC therapy for cartilage regeneration have not been completed [151]. To hasten the advancement of MSC therapy, numerous companies have developed proprietary MSC-specific optimised expansion media that are serum- and xeno-free for the reliable expansion of MSCs. The identification of suitable genetic markers may further improve the selection of donors for high-performance MSCs in allogenic therapy. In addition, the employment of optimal biophysical attributes for the selection of desirable chondrogenic MSCs may facilitate the delivery of a more functionally consistent MSC phenotype, thus enabling the translation of MSC therapy to the clinic for improved cartilage regeneration.

Author Contributions

Conceptualization, D.G., Y.Y., E.H.L., J.H.P.H. and Z.Y.; resources, D.G., Y.Y., E.H.L., J.H.P.H. and Z.Y.; writing—original draft preparation, D.G., Y.Y. and Z.Y.; writing—review and editing, D.G., Y.Y., E.H.L., J.H.P.H. and Z.Y.; visualization, Y.Y.; supervision, Y.Y., E.H.L., J.H.P.H. and Z.Y.; funding acquisition, E.H.L., J.H.P.H. and Z.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Medical Research Council of Singapore (MOH-000371-00).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

3Dthree-dimensional
ACIautologous chondrocyte implantation
AD-MSCadipose-derived mesenchymal stem cell
BM-MSCbone marrow mesenchymal stem cell
ECMextracellular matrix
EGFepidermal growth factor
FACSfluorescence-activated cell sorting
FGF2fibroblast growth factor 2
GSTT1glutathione S-transferase theta 1
IGFinsulin-like growth factor
M-ACImatrix-induced autologous cartilage implantation
MACSmagnetic-activated cell sorting
MALBACmultiple annealing and looping-based amplification cycles
MDAmultiple displacement amplification
MSCmesenchymal stem cell
OATSosteochondral autograft transfer system
PDGF-BBplatelet-derived growth factor-BB
scRNA-seqsingle-cell RNA sequencing
SM-MSCsynovium membrane-derived mesenchymal stem cell
UCB-MSCumbilical cord blood-derived mesenchymal stem cell

References

  1. Sophia Fox, A.J.; Bedi, A.; Rodeo, S.A. The Basic Science of Articular Cartilage: Structure, Composition, and Function. Sports Health 2009, 1, 461–468. [Google Scholar] [CrossRef] [Green Version]
  2. Krishnan, Y.; Grodzinsky, A.J. Cartilage diseases. Matrix Biol. 2018, 71–72, 51–69. [Google Scholar] [CrossRef] [PubMed]
  3. Greene, G.W.; Banquy, X.; Lee, D.W.; Lowrey, D.D.; Yu, J.; Israelachvili, J.N. Adaptive mechanically controlled lubrication mechanism found in articular joints. Proc. Natl. Acad. Sci. USA 2011, 108, 5255–5259. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Buckwalter, J.; Mankin, H. Articular cartilage: Part I. J. Bone Jt. Surg. 1997, 79, 600. [Google Scholar] [CrossRef]
  5. Gomoll, A.H.; Minas, T. The quality of healing: Articular cartilage. Wound Repair Regen. 2014, 22, 30–38. [Google Scholar] [CrossRef]
  6. Morales, T.I. Chondrocyte moves: Clever strategies? Osteoarthr. Cartil. 2007, 15, 861–871. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Buckwalter, J.A.; Felson, D.T. Post-Traumatic Arthritis: Definitions and Burden of Disease. In Post-Traumatic Arthritis: Pathogenesis, Diagnosis and Management; Olson, M.D.S.A., Guilak, P.F., Eds.; Springer US: Boston, MA, USA, 2015; pp. 7–15. [Google Scholar]
  8. Buckwalter, J.A.; Mankin, H.J. Instructional Course Lectures, The American Academy of Orthopaedic Surgeons—Articular Cartilage. Part II: Degeneration and Osteoarthrosis, Repair, Regeneration, and Transplantation*†. JBJS 1997, 79, 612–632. [Google Scholar] [CrossRef]
  9. Gomoll, A.H.; Farr, J.; Gillogly, S.D.; Kercher, J.; Minas, T. Surgical Management of Articular Cartilage Defects of the Knee. JBJS 2010, 92, 2470–2490. [Google Scholar]
  10. Richter, W. Mesenchymal stem cells and cartilage in situ regeneration. J. Intern. Med. 2009, 266, 390–405. [Google Scholar] [CrossRef] [PubMed]
  11. Knutsen, G.; Engebretsen, L.; Ludvigsen, T.C.; Drogset, J.O.; Grøntvedt, T.; Solheim, E.; Torbjørn, S.; Sally, R.; Vidar, I.; Oddmund, J. Autologous chondrocyte implantation compared with microfracture in the knee: A randomized trial. JBJS 2004, 86, 455–464. [Google Scholar] [CrossRef] [PubMed]
  12. Olivos-Meza, A.; Madrazo-Ibarra, A.; de León, C.I.P. Arthroscopic Technique to Treat Articular Cartilage Lesions in the Patellofemoral Joint; IntechOpen: London, UK, 2018. [Google Scholar]
  13. Gudas, R.; Gudaitė, A.; Pocius, A.; Gudienė, A.; Čekanauskas, E.; Monastyreckienė, E.; Basevičius, A. Ten-year follow-up of a prospective, randomized clinical study of mosaic osteochondral autologous transplantation versus microfracture for the treatment of osteochondral defects in the knee joint of athletes. Am. J. Sports Med. 2012, 40, 2499–2508. [Google Scholar] [CrossRef] [PubMed]
  14. Gobbi, A.; Karnatzikos, G.; Kumar, A. Long-term results after microfracture treatment for full-thickness knee chondral lesions in athletes. Knee Surg. Sports Traumatol. Arthrosc. 2014, 22, 1986–1996. [Google Scholar] [CrossRef]
  15. Orth, P.; Goebel, L.; Wolfram, U.; Ong, M.F.; Gräber, S.; Kohn, D.; Ignatius, A.; Pape, D.; Madry, H. Effect of subchondral drilling on the microarchitecture of subchondral bone: Analysis in a large animal model at 6 months. Am. J. Sports Med. 2012, 40, 828–836. [Google Scholar] [CrossRef]
  16. Richter, D.L.; Tanksley, J.A.; Miller, M.D. Osteochondral autograft transplantation: A review of the surgical technique and outcomes. Sports Med. Arthrosc. Rev. 2016, 24, 74–78. [Google Scholar] [CrossRef]
  17. Bedi, A.; Feeley, B.T.; Williams, R.J., III. Management of Articular Cartilage Defects of the Knee. JBJS 2010, 92, 994–1009. [Google Scholar] [CrossRef] [PubMed]
  18. Cerrato, R. Particulated juvenile articular cartilage allograft transplantation for osteochondral lesions of the talus. Foot Ankle Clin. 2013, 18, 79–87. [Google Scholar] [CrossRef]
  19. Niemeyer, P.; Albrecht, D.; Andereya, S.; Angele, P.; Ateschrang, A.; Aurich, M.; Baumann, M.; Bosch, U.; Erggelet, C.; Fickert, S.; et al. Autologous chondrocyte implantation (ACI) for cartilage defects of the knee: A guideline by the working group “Clinical Tissue Regeneration” of the German Society of Orthopaedics and Trauma (DGOU). Knee 2016, 23, 426–435. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Bhosale, A.M.; Richardson, J.B. Articular cartilage: Structure, injuries and review of management. Br. Med. Bull. 2008, 87, 77–95. [Google Scholar] [CrossRef]
  21. Zheng, M.-H.; Willers, C.; Kirilak, L.; Yates, P.; Xu, J.; Wood, D.; Shimmin, A. Matrix-induced autologous chondrocyte implantation (MACI®): Biological and histological assessment. Tissue Eng. 2007, 13, 737–746. [Google Scholar] [CrossRef]
  22. Caron, M.M.J.; Emans, P.J.; Coolsen, M.M.E.; Voss, L.; Surtel, D.A.M.; Cremers, A.; van Rhijn, L.W.; Welting, T.J.M. Redifferentiation of dedifferentiated human articular chondrocytes: Comparison of 2D and 3D cultures. Osteoarthr. Cartil. 2012, 20, 1170–1178. [Google Scholar] [CrossRef] [Green Version]
  23. Saris, D.; Price, A.; Widuchowski, W.; Bertrand-Marchand, M.; Caron, J.; Drogset, J.O.; Emans, P.; Podskubka, A.; Tsuchida, A.; Kili, S.; et al. Matrix-applied characterized autologous cultured chondrocytes versus microfracture: Two-year follow-up of a prospective randomized trial. Am. J. Sports Med. 2014, 42, 1384–1394. [Google Scholar] [CrossRef] [PubMed]
  24. Nazempour, A.; Van Wie, B.J. Chondrocytes, Mesenchymal Stem Cells, and Their Combination in Articular Cartilage Regenerative Medicine. Ann. Biomed. Eng. 2016, 44, 1325–1354. [Google Scholar] [CrossRef]
  25. Duan, L.; Ma, B.; Liang, Y.; Chen, J.; Zhu, W.; Li, M.; Wang, D. Cytokine networking of chondrocyte dedifferentiation in vitro and its implications for cell-based cartilage therapy. Am. J. Transl. Res. 2015, 7, 194–208. [Google Scholar]
  26. Reissis, D.; Tang, Q.O.; Cooper, N.C.; Carasco, C.F.; Gamie, Z.; Mantalaris, A.; Tsiridis, E. Current clinical evidence for the use of mesenchymal stem cells in articular cartilage repair. Expert Opin. Biol. Ther. 2016, 16, 535–557. [Google Scholar] [CrossRef] [PubMed]
  27. Kern, S.; Eichler, H.; Stoeve, J.; Klüter, H.; Bieback, K. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells 2006, 24, 1294–1301. [Google Scholar] [CrossRef] [PubMed]
  28. Yoshimura, H.; Muneta, T.; Nimura, A.; Yokoyama, A.; Koga, H.; Sekiya, I. Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle. Cell Tissue Res. 2007, 327, 449–462. [Google Scholar] [CrossRef] [PubMed]
  29. Hubka, K.M.; Dahlin, R.L.; Meretoja, V.V.; Kasper, F.K.; Mikos, A.G. Enhancing chondrogenic phenotype for cartilage tissue engineering: Monoculture and coculture of articular chondrocytes and mesenchymal stem cells. Tissue Eng. Part B Rev. 2014, 20, 641–654. [Google Scholar] [CrossRef] [Green Version]
  30. Ankrum, J.A.; Ong, J.F.; Karp, J.M. Mesenchymal stem cells: Immune evasive, not immune privileged. Nat. Biotechnol. 2014, 32, 252–260. [Google Scholar] [CrossRef] [Green Version]
  31. Zhang, R.; Ma, J.; Han, J.; Zhang, W.; Ma, J. Mesenchymal stem cell related therapies for cartilage lesions and osteoarthritis. Am. J. Transl. Res. 2019, 11, 6275–6289. [Google Scholar]
  32. Anderson, J.A.; Little, D.; Toth, A.P.; Moorman, C.T.; Tucker, B.S.; Ciccotti, M.G.; Guilak, T. Stem Cell Therapies for Knee Cartilage Repair: The Current Status of Preclinical and Clinical Studies. Am. J. Sports Med. 2013, 42, 2253–2261. [Google Scholar] [CrossRef] [Green Version]
  33. Park, Y.-B.; Ha, C.-W.; Lee, C.-H.; Yoon, Y.C.; Park, Y.-G. Cartilage Regeneration in Osteoarthritic Patients by a Composite of Allogeneic Umbilical Cord Blood-Derived Mesenchymal Stem Cells and Hyaluronate Hydrogel: Results from a Clinical Trial for Safety and Proof-of-Concept with 7 Years of Extended Follow-Up. Stem Cells Transl. Med. 2016, 6, 613–621. [Google Scholar] [CrossRef] [PubMed]
  34. Hashimoto, Y.; Nishida, Y.; Takahashi, S.; Nakamura, H.; Mera, H.; Kashiwa, K.; Yoshiya, S.; Inagaki, Y.; Uematsu, K.; Tanaka, Y.; et al. Transplantation of autologous bone marrow-derived mesenchymal stem cells under arthroscopic surgery with microfracture versus microfracture alone for articular cartilage lesions in the knee: A multicenter prospective randomized control clinical trial. Regen. Ther. 2019, 11, 106–113. [Google Scholar] [CrossRef]
  35. Saris, T.F.F.; de Windt, T.S.; Kester, E.C.; Vonk, L.A.; Custers, R.J.H.; Saris, D.B.F. Five-Year Outcome of 1-Stage Cell-Based Cartilage Repair Using Recycled Autologous Chondrons and Allogenic Mesenchymal Stromal Cells: A First-in-Human Clinical Trial. Am. J. Sports Med. 2021, 49, 941–947. [Google Scholar] [CrossRef]
  36. Wu, L.; Prins, H.-J.; Helder, M.N.; van Blitterswijk, C.A.; Karperien, M. Trophic effects of mesenchymal stem cells in chondrocyte co-cultures are independent of culture conditions and cell sources. Tissue Eng. Part A 2012, 18, 1542–1551. [Google Scholar] [CrossRef] [PubMed]
  37. Tan, S.S.H.; Tjio, C.K.E.; Wong, J.R.Y.; Wong, K.L.; Chew, J.R.J.; Hui, J.H.P.; Toh, W.S. Mesenchymal stem cell exosomes for cartilage regeneration: A systematic review of preclinical in vivo studies. Tissue Eng. Part B Rev. 2021, 27, 1–13. [Google Scholar] [CrossRef]
  38. Zhang, S.; Chuah, S.J.; Lai, R.C.; Hui, J.H.P.; Lim, S.K.; Toh, W.S. MSC exosomes mediate cartilage repair by enhancing proliferation, attenuating apoptosis and modulating immune reactivity. Biomaterials 2018, 156, 16–27. [Google Scholar] [CrossRef]
  39. Toh, W.S.; Lai, R.C.; Hui, J.H.P.; Lim, S.K. MSC exosome as a cell-free MSC therapy for cartilage regeneration: Implications for osteoarthritis treatment. Semin. Cell Dev. Biol. 2017, 67, 56–64. [Google Scholar] [CrossRef]
  40. Caplan, A.I. Adult mesenchymal stem cells for tissue engineering versus regenerative medicine. J. Cell. Physiol. 2007, 213, 341–347. [Google Scholar] [CrossRef]
  41. Melief, S.M.; Schrama, E.; Brugman, M.H.; Tiemessen, M.M.; Hoogduijn, M.J.; Fibbe, W.E.; Roelofs, H. Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells 2013, 31, 1980–1991. [Google Scholar] [CrossRef]
  42. Pers, Y.-M.; Maumus, M.; Bony, C.; Jorgensen, C.; Noël, D. Contribution of microRNAs to the immunosuppressive function of mesenchymal stem cells. Biochimie 2018, 155, 109–118. [Google Scholar] [CrossRef] [PubMed]
  43. Zhang, S.; Wong, K.L.; Ren, X.; Teo, K.Y.W.; Afizah, H.; Choo, A.B.H.; Lai, R.C.; Lim, S.K.; Hui, J.H.P.; Toh, W.S. Mesenchymal Stem Cell Exosomes Promote Functional Osteochondral Repair in a Clinically Relevant Porcine Model. Am. J. Sports Med. 2022, 50, 788–800. [Google Scholar] [CrossRef] [PubMed]
  44. Zhou, T.; Yuan, Z.; Weng, J.; Pei, D.; Du, X.; He, C.; Lai, P. Challenges and advances in clinical applications of mesenchymal stromal cells. J. Hematol. Oncol. 2021, 14, 24. [Google Scholar] [CrossRef]
  45. Levy, O.; Kuai, R.; Siren, E.M.J.; Bhere, D.; Milton, Y.; Nissar, N.; De Biasio, M.; Heinelt, M.; Reeve, B.; Abdi, R.; et al. Shattering barriers toward clinically meaningful MSC therapies. Sci. Adv. 2020, 6, eaba6884. [Google Scholar] [CrossRef] [PubMed]
  46. Elahi, F.M.; Farwell, D.G.; Nolta, J.A.; Anderson, J.D. Preclinical translation of exosomes derived from mesenchymal stem/stromal cells. Stem Cells 2019, 38, 15–21. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Kim, M.; Erickson, I.E.; Huang, A.H.; Garrity, S.T.; Mauck, R.L.; Steinberg, D.R. Donor Variation and Optimization of Human Mesenchymal Stem Cell Chondrogenesis in Hyaluronic Acid. Tissue Eng. Part A 2018, 24, 1693–1703. [Google Scholar] [CrossRef] [PubMed]
  48. Zha, K.; Li, X.; Yang, Z.; Tian, G.; Sun, Z.; Sui, X.; Dai, Y.; Liu, S.; Guo, Q. Heterogeneity of mesenchymal stem cells in cartilage regeneration: From characterization to application. NPJ Regen. Med. 2021, 6, 14. [Google Scholar] [CrossRef]
  49. Kanawa, M.; Igarashi, A.; Ronald, V.S.; Higashi, Y.; Kurihara, H.; Sugiyama, M.; Saskianti, T.; Pan, H.; Kato, Y. Age-dependent decrease in the chondrogenic potential of human bone marrow mesenchymal stromal cells expanded with fibroblast growth factor-2. Cytotherapy 2013, 15, 1062–1072. [Google Scholar] [CrossRef]
  50. Siegel, G.; Kluba, T.; Hermanutz-Klein, U.; Bieback, K.; Northoff, H.; Schäfer, R. Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells. BMC Med. 2013, 11, 146. [Google Scholar] [CrossRef] [Green Version]
  51. Camernik, K.; Mihelič, A.; Mihalič, R.; Haring, G.; Herman, S.; Presen, D.M.; Janež, A.; Trebše, R.; Marc, J.; Zupan, J. Comprehensive analysis of skeletal muscle- and bone-derived mesenchymal stem/stromal cells in patients with osteoarthritis and femoral neck fracture. Stem Cell Res. Ther. 2020, 11, 146. [Google Scholar] [CrossRef]
  52. Murphy, J.M.; Dixon, K.; Beck, S.; Fabian, D.; Feldman, A.; Barry, F. Reduced chondrogenic and adipogenic activity of mesenchymal stem cells from patients with advanced osteoarthritis. Arthritis Rheum. 2002, 46, 704–713. [Google Scholar] [CrossRef]
  53. Sathiyanathan, P.; Samsonraj, R.M.; Tan, C.L.L.; Ling, L.; Lezhava, A.; Nurcombe, V.; Stanton, L.W.; Cool, S.M. A genomic biomarker that identifies human bone marrow-derived mesenchymal stem cells with high scalability. Stem Cells 2020, 38, 1124–1136. [Google Scholar] [CrossRef]
  54. Afizah, H.; Yang, Z.; Hui, J.H.; Ouyang, H.-W.; Lee, E.-H. A comparison between the chondrogenic potential of human bone marrow stem cells (BMSCs) and adipose-derived stem cells (ADSCs) taken from the same donors. Tissue Eng. 2007, 13, 659–666. [Google Scholar] [CrossRef]
  55. Hui, J.H.; Li, L.; Teo, Y.-H.; Ouyang, H.-W.; Lee, E.-H. Comparative study of the ability of mesenchymal stem cells derived from bone marrow, periosteum, and adipose tissue in treatment of partial growth arrest in rabbit. Tissue Eng. 2005, 11, 904–912. [Google Scholar] [CrossRef]
  56. Kohli, N.; Wright, K.T.; Sammons, R.L.; Jeys, L.; Snow, M.; Johnson, W.E.B. An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair. Cartilage 2015, 6, 252–263. [Google Scholar] [CrossRef] [Green Version]
  57. Xie, X.; Wang, Y.; Zhao, C.; Guo, S.; Liu, S.; Jia, W.; Tuan, R.S.; Zhang, C. Comparative evaluation of MSCs from bone marrow and adipose tissue seeded in PRP-derived scaffold for cartilage regeneration. Biomaterials 2012, 33, 7008–7018. [Google Scholar] [CrossRef]
  58. Peng, L.; Jia, Z.; Yin, X.; Zhang, X.; Liu, Y.; Chen, P.; Ma, K.; Zhou, C. Comparative analysis of mesenchymal stem cells from bone marrow, cartilage, and adipose tissue. Stem Cells Dev. 2008, 17, 761–774. [Google Scholar] [CrossRef] [PubMed]
  59. Contentin, R.; Demoor, M.; Concari, M.; Desancé, M.; Audigié, F.; Branly, T.; Galéra, P. Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering. Stem Cell Rev. Rep. 2020, 16, 126–143. [Google Scholar] [CrossRef]
  60. Fellows, C.R.; Matta, C.; Zakany, R.; Khan, I.M.; Mobasheri, A. Adipose, Bone Marrow and Synovial Joint-Derived Mesenchymal Stem Cells for Cartilage Repair. Front. Genet. 2016, 7, 213. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  61. Herrmann, M.; Hildebrand, M.; Menzel, U.; Fahy, N.; Alini, M.; Lang, S.; Benneker, L.; Verrier, S.; Stoddart, M.J.; Bara, J.J. Phenotypic characterization of bone marrow mononuclear cells and derived stromal cell populations from human iliac crest, vertebral body and femoral head. Int. J. Mol. Sci. 2019, 20, 3454. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  62. Sivasubramaniyan, K.; Ilas, D.; Harichandan, A.; Bos, P.K.; Santos, D.L.; De Zwart, P.; Koevoet, W.J.; Owston, H.; Bühring, H.-J.; Jones, E.; et al. Bone Marrow–Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration. Am. J. Sports Med. 2018, 46, 3521–3531. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  63. Lee, N.E.; Kim, S.J.; Yang, S.-J.; Joo, S.-Y.; Park, H.; Lee, K.W.; Yang, H.-M.; Park, J.B. Comparative characterization of mesenchymal stromal cells from multiple abdominal adipose tissues and enrichment of angiogenic ability via CD146 molecule. Cytotherapy 2017, 19, 170–180. [Google Scholar] [CrossRef] [PubMed]
  64. Lopa, S.; Colombini, A.; Stanco, D.; De Girolamo, L.; Sansone, V.; Moretti, M. Donor-matched mesenchymal stem cells from knee infrapatellar and subcutaneous adipose tissue of osteoarthritic donors display differential chondrogenic and osteogenic commitment. Eur. Cell Mater. 2014, 27, 298–311. [Google Scholar] [PubMed]
  65. Wang, Z.; Chai, C.; Wang, R.; Feng, Y.; Huang, L.; Zhang, Y.; Xiao, X.; Yang, S.; Zhang, Y.; Zhang, X. Single-cell transcriptome atlas of human mesenchymal stem cells exploring cellular heterogeneity. Clin. Transl. Med. 2021, 11, e650. [Google Scholar] [CrossRef]
  66. Sun, C.; Wang, L.; Wang, H.; Huang, T.; Yao, W.; Li, J.; Zhang, X. Single-cell RNA-seq highlights heterogeneity in human primary Wharton’s jelly mesenchymal stem/stromal cells cultured in vitro. Stem Cell Res. Ther. 2020, 11, 149. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  67. Ando, W.; Kutcher, J.J.; Krawetz, R.; Sen, A.; Nakamura, N.; Frank, C.B.; Hart, D.A. Clonal analysis of synovial fluid stem cells to characterize and identify stable mesenchymal stromal cell/mesenchymal progenitor cell phenotypes in a porcine model: A cell source with enhanced commitment to the chondrogenic lineage. Cytotherapy 2014, 16, 776–788. [Google Scholar] [CrossRef]
  68. Rennerfeldt, D.A.; Van Vliet, K.J. Concise Review: When Colonies Are Not Clones: Evidence and Implications of Intracolony Heterogeneity in Mesenchymal Stem Cells. Stem Cells 2016, 34, 1135–1141. [Google Scholar] [CrossRef] [Green Version]
  69. Cote, A.J.; McLeod, C.M.; Farrell, M.J.; McClanahan, P.D.; Dunagin, M.C.; Raj, A.; Mauck, R.L. Single-cell differences in matrix gene expression do not predict matrix deposition. Nat. Commun. 2016, 7, 10865. [Google Scholar] [CrossRef] [Green Version]
  70. Freeman, B.T.; Jung, J.P.; Ogle, B.M. Single-cell RNA-Seq of bone marrow-derived mesenchymal stem cells reveals unique profiles of lineage priming. PLoS ONE 2015, 10, e0136199. [Google Scholar] [CrossRef]
  71. Rennerfeldt, D.A.; Raminhos, J.S.; Leff, S.M.; Manning, P.; Van Vliet, K.J. Emergent heterogeneity in putative mesenchymal stem cell colonies: Single-cell time lapsed analysis. PLoS ONE 2019, 14, e0213452. [Google Scholar] [CrossRef] [Green Version]
  72. McLeod, C.; Mauck, R. On the origin and impact of mesenchymal stem cell heterogeneity: New insights and emerging tools for single cell analysis. Eur. Cells Mater. 2017, 34, 217. [Google Scholar] [CrossRef]
  73. Wilson, A.; Webster, A.; Genever, P. Nomenclature and heterogeneity: Consequences for the use of mesenchymal stem cells in regenerative medicine. Regen. Med. 2019, 14, 595–611. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  74. Tan, A.; Alegre-Aguarón, E.; O’Connell, G.; VandenBerg, C.; Aaron, R.; Vunjak-Novakovic, G.; Bulinski, J.C.; Ateshian, G.; Hung, C. Passage-dependent relationship between mesenchymal stem cell mobilization and chondrogenic potential. Osteoarthr. Cartil. 2015, 23, 319–327. [Google Scholar] [CrossRef] [Green Version]
  75. Siennicka, K.; Zołocińska, A.; Dębski, T.; Pojda, Z. Comparison of the Donor Age-Dependent and In Vitro Culture-Dependent Mesenchymal Stem Cell Aging in Rat Model. Stem Cells Int. 2021, 2021, 6665358. [Google Scholar] [CrossRef] [PubMed]
  76. Jeske, R.; Yuan, X.; Fu, Q.; Bunnell, B.A.; Logan, T.M.; Li, Y. In vitro culture expansion shifts the immune phenotype of human adipose-derived mesenchymal stem cells. Front. Immunol. 2021, 12, 621744. [Google Scholar] [CrossRef]
  77. Solchaga, L.A.; Penick, K.; Porter, J.D.; Goldberg, V.M.; Caplan, A.I.; Welter, J.F. FGF-2 enhances the mitotic and chondrogenic potentials of human adult bone marrow-derived mesenchymal stem cells. J. Cell. Physiol. 2005, 203, 398–409. [Google Scholar] [CrossRef] [PubMed]
  78. Solchaga, L.A.; Penick, K.; Goldberg, V.M.; Caplan, A.I.; Welter, J.F. Fibroblast growth factor-2 enhances proliferation and delays loss of chondrogenic potential in human adult bone-marrow-derived mesenchymal stem cells. Tissue Eng. Part A 2010, 16, 1009–1019. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  79. Cleary, M.A.; van Osch, G.J.; Brama, P.A.; Hellingman, C.A.; Narcisi, R. FGF, TGFβ and Wnt crosstalk: Embryonic to in vitro cartilage development from mesenchymal stem cells. J. Tissue Eng. Regen. Med. 2015, 9, 332–342. [Google Scholar] [CrossRef]
  80. Choi, S.-C.; Kim, S.-J.; Choi, J.-H.; Park, C.-Y.; Shim, W.-J.; Lim, D.-S. Fibroblast growth factor-2 and-4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the PI3K-Akt and ERK1/2 signaling pathways. Stem Cells Dev. 2008, 17, 725–736. [Google Scholar] [CrossRef] [Green Version]
  81. Farré, J.; Farré, J.; Roura, S.; Farré, J.; Roura, S.; Prat-Vidal, C.; Hove-Madsen, L.; Cairo, J.J.; Godia, F.; Bragos, R.; et al. FGF-4 increases in vitro expansion rate of human adult bone marrow-derived mesenchymal stem cells. Growth Factors 2007, 25, 71–76. [Google Scholar] [CrossRef]
  82. Wijesinghe, S.J.; Ling, L.; Murali, S.; Qing, Y.H.; Hinkley, S.F.; Carnachan, S.M.; Bell, T.J.; Swaminathan, K.; Hui, J.H.; van Wijnen, A.J.; et al. Affinity selection of FGF2-binding heparan sulfates for ex vivo expansion of human mesenchymal stem cells. J. Cell. Physiol. 2017, 232, 566–575. [Google Scholar] [CrossRef]
  83. Titmarsh, D.M.; Tan, C.L.; Glass, N.R.; Nurcombe, V.; Cooper-White, J.J.; Cool, S.M. Microfluidic Screening Reveals Heparan Sulfate Enhances Human Mesenchymal Stem Cell Growth by Modulating Fibroblast Growth Factor-2 Transport. Stem Cells Transl Med. 2017, 6, 1178–1190. [Google Scholar] [CrossRef]
  84. Ling, L.; Ren, X.; Cao, X.; Hassan, A.B.M.; Mah, S.; Sathiyanathan, P.; Smith, R.A.A.; Tan, C.L.; Eio, M.; Samsonraj, R.M.; et al. Enhancing the Efficacy of Stem Cell Therapy with Glycosaminoglycans. Stem Cell Rep. 2020, 14, 105–121. [Google Scholar] [CrossRef] [Green Version]
  85. Helledie, T.; Dombrowski, C.; Rai, B.; Lim, Z.X.; Hin, I.L.H.; Rider, D.A.; Stein, G.S.; Hong, W.; Van Wijnen, A.J.; Hui, J.H.; et al. Heparan sulfate enhances the self-renewal and therapeutic potential of mesenchymal stem cells from human adult bone marrow. Stem Cells Dev. 2012, 21, 1897–1910. [Google Scholar] [CrossRef] [PubMed]
  86. Gharibi, B.; Ghuman, M.S.; Hughes, F.J. Akt- and Erk-mediated regulation of proliferation and differentiation during PDGFRβ-induced MSC self-renewal. J. Cell Mol. Med. 2012, 16, 2789–2801. [Google Scholar] [CrossRef]
  87. Sun, X.; Gao, X.; Zhou, L.; Sun, L.; Lu, C. PDGF-BB-induced MT1-MMP expression regulates proliferation and invasion of mesenchymal stem cells in 3-dimensional collagen via MEK/ERK1/2 and PI3K/AKT signaling. Cell. Signal. 2013, 25, 1279–1287. [Google Scholar] [CrossRef]
  88. Lienemann, P.S.; Devaud, Y.R.; Reuten, R.; Simona, B.R.; Karlsson, M.; Weber, W.; Koch, M.; Lutolf, M.P.; Milleret, V.; Ehrbar, M. Locally controlling mesenchymal stem cell morphogenesis by 3D PDGF-BB gradients towards the establishment of an in vitro perivascular niche. Integr. Biol. 2014, 7, 101–111. [Google Scholar] [CrossRef]
  89. Grigorieva, O.; Arbatskiy, M.; Novoseletskaya, E.; Dyachkova, U.; Ishkin, A.; Kalinina, N.; Efimenko, A. Platelet-Derived Growth Factor Induces SASP-Associated Gene Expression in Human Multipotent Mesenchymal Stromal Cells but Does Not Promote Cell Senescence. Biomedicines 2021, 9, 1290. [Google Scholar] [CrossRef] [PubMed]
  90. Tamama, K.; Kawasaki, H.; Wells, A. Epidermal Growth Factor (EGF) Treatment on Multipotential Stromal Cells (MSCs). Possible Enhancement of Therapeutic Potential of MSC. J. Biomed. Biotechnol. 2010, 2010, 795385. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  91. Knight, C.; James, S.; Kuntin, D.; Fox, J.; Newling, K.; Hollings, S.; Pennock, R.; Genever, P. Epidermal growth factor can signal via β-catenin to control proliferation of mesenchymal stem cells independently of canonical Wnt signalling. Cell. Signal. 2019, 53, 256–268. [Google Scholar] [CrossRef]
  92. Hu, C.; Wu, Y.; Wan, Y.; Wang, Q.; Song, J. Introduction of hIGF-1 Gene into Bone Marrow Stromal Cells and Its Effects on the Cell’s Biological Behaviors. Cell Transplant. 2008, 17, 1067–1081. [Google Scholar] [CrossRef] [Green Version]
  93. Youssef, A.; Han, V.K.M. Low Oxygen Tension Modulates the Insulin-Like Growth Factor-1 or -2 Signaling via Both Insulin-Like Growth Factor-1 Receptor and Insulin Receptor to Maintain Stem Cell Identity in Placental Mesenchymal Stem Cells. Endocrinology 2016, 157, 1163–1174. [Google Scholar] [CrossRef] [Green Version]
  94. Yeo, G.C.; Weiss, A.S. Soluble matrix protein is a potent modulator of mesenchymal stem cell performance. Proc. Natl. Acad. Sci. USA 2019, 116, 2042–2051. [Google Scholar] [CrossRef] [Green Version]
  95. Lindner, U.; Kramer, J.; Behrends, J.; Driller, B.; Wendler, N.-O.; Boehrnsen, F.; Rohwedel, J.; Schlenke, P. Improved proliferation and differentiation capacity of human mesenchymal stromal cells cultured with basement-membrane extracellular matrix proteins. Cytotherapy 2010, 12, 992–1005. [Google Scholar] [CrossRef]
  96. Somaiah, C.; Kumar, A.; Mawrie, D.; Sharma, A.; Patil, S.D.; Bhattacharyya, J.; Swaminathan, R.; Jaganathan, B.G. Collagen Promotes Higher Adhesion, Survival and Proliferation of Mesenchymal Stem Cells. PLoS ONE 2015, 10, e0145068. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  97. Antebi, B.; Zhang, Z.; Wang, Y.; Lu, Z.; Chen, X.-D.; Ling, J. Stromal-cell-derived extracellular matrix promotes the proliferation and retains the osteogenic differentiation capacity of mesenchymal stem cells on three-dimensional scaffolds. Tissue Eng. Part C Methods 2015, 21, 171–181. [Google Scholar] [CrossRef] [Green Version]
  98. Bas, G.; Loisate, S.; Hudon, S.F.; Woods, K.; Hayden, E.J.; Pu, X.; Beard, R.; Oxford, J.T.; Uzer, G. Low Intensity Vibrations Augment Mesenchymal Stem Cell Proliferation and Differentiation Capacity during in vitro Expansion. Sci. Rep. 2020, 10, 9369. [Google Scholar] [CrossRef] [PubMed]
  99. Touchstone, H.; Bryd, R.; Loisate, S.; Thompson, M.; Kim, S.; Puranam, K.; Senthilnathan, A.N.; Pu, X.; Beard, R.; Rubin, J.; et al. Recovery of stem cell proliferation by low intensity vibration under simulated microgravity requires LINC complex. NPJ Microgravity 2019, 5, 11. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  100. Miskon, A.; Abdul Hamid, H.; Ramasamy, R.; Mohd Tawil, S. (Eds.) Enhanced proliferation potential of human umbilical cord mesenchymal stem cells through suspension induction and electromagnetic field exposure. In International Conference on the Development of Biomedical Engineering in Vietnam; Springer: Berlin/Heidelberg, Germany, 2017. [Google Scholar]
  101. Hamid, H.A.; Sarmadi, V.H.; Prasad, V.; Ramasamy, R.; Miskon, A. Electromagnetic field exposure as a plausible approach to enhance the proliferation and differentiation of mesenchymal stem cells in clinically relevant scenarios. J. Zhejiang Univ. Sci. B. 2022, 23, 42–57. [Google Scholar] [CrossRef] [PubMed]
  102. Hernández-Bule, M.L.; Paíno, C.L.; Trillo, M.Á.; Úbeda, A. Electric Stimulation at 448 kHz Promotes Proliferation of Human Mesenchymal Stem Cells. Cell. Physiol. Biochem. 2014, 34, 1741–1755. [Google Scholar] [CrossRef]
  103. Sutermaster, B.A.; Darling, E.M. Considerations for high-yield, high-throughput cell enrichment: Fluorescence versus magnetic sorting. Sci. Rep. 2019, 9, 227. [Google Scholar] [CrossRef] [Green Version]
  104. Gothard, D.; Greenhough, J.; Ralph, E.; Oreffo, R.O. Prospective isolation of human bone marrow stromal cell subsets: A comparative study between Stro-1-, CD146- and CD105-enriched populations. J. Tissue Eng. 2014, 5, 2041731414551763. [Google Scholar] [CrossRef] [Green Version]
  105. Huynh, N.P.; Zhang, B.; Guilak, F. High-depth transcriptomic profiling reveals the temporal gene signature of human mesenchymal stem cells during chondrogenesis. FASEB J. 2019, 33, 358–372. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  106. Arufe, M.C.; De la Fuente, A.; Fuentes, I.; de Toro, F.J.; Blanco, F.J. Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes. J. Cell Biochem. 2010, 111, 834–845. [Google Scholar] [CrossRef] [Green Version]
  107. Fan, W.; Li, J.; Wang, Y.; Pan, J.; Li, S.; Zhu, L.; Guo, C.; Yan, Z. CD105 promotes chondrogenesis of synovium-derived mesenchymal stem cells through Smad2 signaling. Biochem. Biophys. Res. Commun. 2016, 474, 338–344. [Google Scholar] [CrossRef]
  108. Lv, X.-J.; Zhou, G.-D.; Liu, Y.; Liu, X.; Chen, J.-N.; Luo, X.-S.; Cao, Y.-L. In vitro proliferation and differentiation of adipose-derived stem cells isolated using anti-CD105 magnetic beads. Int. J. Mol. Med. 2012, 30, 826–834. [Google Scholar] [CrossRef] [Green Version]
  109. Rada, T.; Reis, R.L.; Gomes, M.E. Distinct Stem Cells Subpopulations Isolated from Human Adipose Tissue Exhibit Different Chondrogenic and Osteogenic Differentiation Potential. Stem Cell Rev. Rep. 2011, 7, 64–76. [Google Scholar] [CrossRef] [Green Version]
  110. Calabrese, G.; Giuffrida, R.; Furno, D.L.; Parrinello, N.L.; Forte, S.; Gulino, R.; Colarossi, C.; Schinocca, L.R.; Giuffrida, R.; Cardile, V.; et al. Potential Effect of CD271 on Human Mesenchymal Stromal Cell Proliferation and Differentiation. Int. J. Mol. Sci. 2015, 16, 15609–15624. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  111. Petters, O.; Schmidt, C.; Thuemmler, C.; Peinemann, F.; Zscharnack, M.; Somerson, J.S.; Schulz, R.M. Point-of-care treatment of focal cartilage defects with selected chondrogenic mesenchymal stromal cells—An in vitro proof-of-concept study. J. Tissue Eng. Regen. Med. 2018, 12, 1717–1727. [Google Scholar] [CrossRef] [PubMed]
  112. Mifune, Y.; Matsumoto, T.; Murasawa, S.; Kawamoto, A.; Kuroda, R.; Shoji, T.; Kuroda, T.; Fukui, T.; Kawakami, Y.; Kurosaka, M.; et al. Therapeutic superiority for cartilage repair by CD271-positive marrow stromal cell transplantation. Cell Transpl. 2013, 22, 1201–1211. [Google Scholar] [CrossRef]
  113. Kohli, N.; Al-Delfi, I.R.T.; Snow, M.; Sakamoto, T.; Miyazaki, T.; Nakajima, H.; Uchida, K.; Johnson, W.E.B. CD271-selected mesenchymal stem cells from adipose tissue enhance cartilage repair and are less angiogenic than plastic adherent mesenchymal stem cells. Sci. Rep. 2019, 9, 3194. [Google Scholar] [CrossRef] [Green Version]
  114. Kuçi, S.; Kuçi, Z.; Schäfer, R.; Spohn, G.; Winter, S.; Schwab, M.; Salzmann-Manrique, E.; Klingebiel, T.; Bader, P. Molecular signature of human bone marrow-derived mesenchymal stromal cell subsets. Sci. Rep. 2019, 9, 1774. [Google Scholar] [CrossRef] [Green Version]
  115. Halfon, S.; Abramov, N.; Grinblat, B.; Ginis, I. Markers distinguishing mesenchymal stem cells from fibroblasts are downregulated with passaging. Stem Cells Dev. 2011, 20, 53–66. [Google Scholar] [CrossRef] [PubMed]
  116. Yu, Y.; Park, Y.S.; Kim, H.S.; Kim, H.Y.; Jin, Y.M.; Jung, S.-C.; Ryu, K.-H.; Jo, I. Characterization of long-term in vitro culture-related alterations of human tonsil-derived mesenchymal stem cells: Role for CCN1 in replicative senescence-associated increase in osteogenic differentiation. J. Anat. 2014, 225, 510–518. [Google Scholar] [CrossRef] [PubMed]
  117. Hagmann, S.; Frank, S.; Gotterbarm, T.; Dreher, T.; Eckstein, V.; Moradi, B. Fluorescence activated enrichment of CD146+ cells during expansion of human bone-marrow derived mesenchymal stromal cells augments proliferation and GAG/DNA content in chondrogenic media. BMC Musculoskelet. Disord. 2014, 15, 322. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  118. Su, X.; Zuo, W.; Wu, Z.; Chen, J.; Wu, N.; Ma, P.; Xia, Z.; Jiang, C.; Ye, Z.; Liu, S.; et al. CD146 as a new marker for an increased chondroprogenitor cell sub-population in the later stages of osteoarthritis. J. Orthop. Res. 2015, 33, 84–91. [Google Scholar] [CrossRef]
  119. Scioli, M.G.; Storti, G.; Bielli, A.; Sanchez, M.; Scimeca, M.; Gimble, J.M.; Cervelli, V.; Orlandi, A. CD146 expression regulates osteochondrogenic differentiation of human adipose-derived stem cells. J. Cell. Physiol. 2022, 237, 589–602. [Google Scholar] [CrossRef]
  120. Wu, C.-C.; Liu, F.-L.; Sytwu, H.-K.; Tsai, C.-Y.; Chang, D.-M. CD146+ mesenchymal stem cells display greater therapeutic potential than CD146– cells for treating collagen-induced arthritis in mice. Stem Cell Res. Ther. 2016, 7, 23. [Google Scholar] [CrossRef] [Green Version]
  121. Al Bahrawy, M. Comparison of the Migration Potential through Microperforated Membranes of CD146+ GMSC Population versus Heterogeneous GMSC Population. Stem Cells Int. 2021, 2021, 5583421. [Google Scholar] [CrossRef] [PubMed]
  122. Li, X.; Guo, W.; Zha, K.; Jing, X.; Wang, M.; Zhang, Y.; Hao, C.; Gao, S.; Chen, M.; Yuan, Z.; et al. Enrichment of CD146(+) Adipose-Derived Stem Cells in Combination with Articular Cartilage Extracellular Matrix Scaffold Promotes Cartilage Regeneration. Theranostics 2019, 9, 5105–5121. [Google Scholar] [CrossRef] [PubMed]
  123. Lin, G.; Liu, G.; Banie, L.; Wang, G.; Ning, H.; Lue, T.F.; Lin, C.-S. Tissue distribution of mesenchymal stem cell marker Stro-1. Stem Cells Dev. 2011, 20, 1747–1752. [Google Scholar] [CrossRef]
  124. Fitter, S.; Gronthos, S.; Ooi, S.S.; Zannettino, A.C.W. The Mesenchymal Precursor Cell Marker Antibody STRO-1 Binds to Cell Surface Heat Shock Cognate 70. Stem Cells 2017, 35, 940–951. [Google Scholar] [CrossRef] [Green Version]
  125. Bensidhoum, M.; Chapel, A.; Francois, S.; Demarquay, C.; Mazurier, C.; Fouillard, L.; Bouchet, S.; Bertho, J.M.; Gourmelon, P.; Aigueperse, J.; et al. Homing of in vitro expanded Stro-1- or Stro-1+ human mesenchymal stem cells into the NOD/SCID mouse and their role in supporting human CD34 cell engraftment. Blood 2004, 103, 3313–3319. [Google Scholar] [CrossRef]
  126. Yu, K.; Yang, S.; Jung, J.; Kim, H.; Ko, K.; Han, D.W.; Park, S.; Choi, S.W.; Kang, S.; Schöler, H.; et al. CD49f Enhances Multipotency and Maintains Stemness Through the Direct Regulation of OCT4 and SOX2. Stem Cells 2012, 30, 876–887. [Google Scholar] [CrossRef]
  127. Zha, K.; Li, X.; Tian, G.; Yang, Z.; Sun, Z.; Yang, Y.; Wei, F.; Huang, B.; Jiang, S.; Li, H.; et al. Evaluation of CD49f as a novel surface marker to identify functional adipose-derived mesenchymal stem cell subset. Cell Prolif. 2021, 54, e13017. [Google Scholar] [CrossRef] [PubMed]
  128. Yang, Z.; Dong, P.; Fu, X.; Li, Q.; Ma, S.; Wu, D.; Kang, N.; Liu, X.; Yan, L.; Xiao, R. CD49f Acts as an Inflammation Sensor to Regulate Differentiation, Adhesion, and Migration of Human Mesenchymal Stem Cells. Stem Cells 2015, 33, 2798–2810. [Google Scholar] [CrossRef]
  129. Campbell, D.D.; Pei, M. Surface Markers for Chondrogenic Determination: A Highlight of Synovium-Derived Stem Cells. Cells 2012, 1, 1107–1120. [Google Scholar] [CrossRef] [Green Version]
  130. Rosu-Myles, M.; McCully, J.; Fair, J.; Mehic, J.; Menendez, P.; Rodriguez, R.; Westwood, C. The globoseries glycosphingolipid SSEA-4 is a marker of bone marrow-derived clonal multipotent stromal cells in vitro and in vivo. Stem Cells Dev. 2013, 22, 1387–1397. [Google Scholar] [CrossRef] [PubMed]
  131. Pei, M.; He, F.; Kish, V.L. Expansion on extracellular matrix deposited by human bone marrow stromal cells facilitates stem cell proliferation and tissue-specific lineage potential. Tissue Eng. Part. A. 2011, 17, 3067–3076. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  132. Srinivasan, A.; Sathiyanathan, P.; Yin, L.; Liu, T.M.; Lam, A.; Ravikumar, M.; Smith, R.A.A.; Loh, H.P.; Zhang, Y.; Ling, L.; et al. Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion. Cytotherapy 2022, 24, 456–472. [Google Scholar] [CrossRef]
  133. Wang, T.; Zhang, J.; Liao, J.; Zhang, F.; Zhou, G. Donor genetic backgrounds contribute to the functional heterogeneity of stem cells and clinical outcomes. Stem Cells Transl. Med. 2020, 9, 1495–1499. [Google Scholar] [CrossRef]
  134. Zheng, G.; Xie, Z.-Y.; Wang, P.; Wu, Y.-F.; Shen, H.-Y. Recent advances of single-cell RNA sequencing technology in mesenchymal stem cell research. World J. Stem Cells 2020, 12, 438. [Google Scholar] [CrossRef]
  135. Dicks, A.; Wu, C.-L.; Steward, N.; Adkar, S.S.; Gersbach, C.A.; Guilak, F. Prospective isolation of chondroprogenitors from human iPSCs based on cell surface markers identified using a CRISPR-Cas9-generated reporter. Stem Cell Res. Ther. 2020, 11, 66. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  136. Zhang, L.; Vertes, A. Single-cell mass spectrometry approaches to explore cellular heterogeneity. Angew. Chem. Int. Ed. 2018, 57, 4466–4477. [Google Scholar] [CrossRef]
  137. Wu, J.; Tzanakakis, E.S. Deconstructing stem cell population heterogeneity: Single-cell analysis and modeling approaches. Biotechnol. Adv. 2013, 31, 1047–1062. [Google Scholar] [CrossRef] [Green Version]
  138. Lee, W.C.; Shi, H.; Poon, Z.; Nyan, L.M.; Kaushik, T.; Shivashankar, G.V.; Chan, J.K.Y.; Lim, C.T.; Han, J.; Van Vliet, K.J. Multivariate biophysical markers predictive of mesenchymal stromal cell multipotency. Proc. Natl. Acad. Sci. USA 2014, 111, E4409–E4418. [Google Scholar] [CrossRef] [Green Version]
  139. Poon, Z.; Lee, W.C.; Guan, G.; Nyan, L.M.; Lim, C.T.; Han, J.; Van Vliet, K.J. Bone marrow regeneration promoted by biophysically sorted osteoprogenitors from mesenchymal stromal cells. Stem Cells Transl. Med. 2015, 4, 56–65. [Google Scholar] [CrossRef] [PubMed]
  140. Lin, J.; Kim, D.; Tse, H.T.; Tseng, P.; Peng, L.; Dhar, M.; Karumbayaram, S.; Di Carlo, D. High-throughput physical phenotyping of cell differentiation. Microsyst. Nanoeng. 2017, 3, 17013. [Google Scholar] [CrossRef] [Green Version]
  141. Heo, S.J.; Driscoll, T.P.; Thorpe, S.D.; Nerurkar, N.L.; Baker, B.M.; Yang, M.T.; Chen, C.S.; Lee, D.A.; Mauck, R.L. Differentiation alters stem cell nuclear architecture, mechanics, and mechano-sensitivity. eLife 2016, 5, e18207. [Google Scholar] [CrossRef]
  142. Sarem, M.; Otto, O.; Tanaka, S.; Shastri, V.P. Cell number in mesenchymal stem cell aggregates dictates cell stiffness and chondrogenesis. Stem Cell Res. Ther. 2019, 10, 10. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  143. Dempsey, M.E.; Chickering, G.R.; González-Cruz, R.D.; Fonseca, V.C.; Darling, E.M. Discovery of surface biomarkers for cell mechanophenotype via an intracellular protein-based enrichment strategy. Cell. Mol. Life Sci. 2022, 79, 320. [Google Scholar] [CrossRef]
  144. Zeming, K.K.; Vernekar, R.; Chua, M.T.; Quek, K.Y.; Sutton, G.; Krüger, T.; Kuan, W.S.; Han, J. Label-Free Biophysical Markers from Whole Blood Microfluidic Immune Profiling Reveal Severe Immune Response Signatures. Small 2021, 17, e2006123. [Google Scholar] [CrossRef]
  145. Yin, L.; Wu, Y.; Yang, Z.; Tee, C.A.; Denslin, V.; Lai, Z.; Lim, C.T.; Lee, E.H.; Han, J. Microfluidic label-free selection of mesenchymal stem cell subpopulation during culture expansion extends the chondrogenic potential in vitro. Lab. Chip. 2018, 18, 878–889. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  146. Yin, L.; Yang, Z.; Wu, Y.; Denslin, V.; Yu, C.C.; Tee, C.A.; Lim, C.T.; Han, J.; Lee, E.H. Label-free separation of mesenchymal stem cell subpopulations with distinct differentiation potencies and paracrine effects. Biomaterials 2020, 240, 119881. [Google Scholar] [CrossRef] [PubMed]
  147. Gao, L.; McBeath, R.; Chen, C.S. Stem Cell Shape Regulates a Chondrogenic Versus Myogenic Fate Through Rac1 and N-Cadherin. Stem Cells 2010, 28, 564–572. [Google Scholar] [CrossRef] [Green Version]
  148. Tang, Y.; Rowe, R.G.; Botvinick, E.L.; Kurup, A.; Putnam, A.J.; Seiki, M.; Weaver, V.M.; Keller, E.T.; Goldstein, S.; Dai, J.; et al. MT1-MMP-Dependent Control of Skeletal Stem Cell Commitment via a β1-Integrin/YAP/TAZ Signaling Axis. Dev. Cell 2013, 25, 402–416. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  149. McBride, S.H.; Knothe Tate, M.L. Modulation of stem cell shape and fate A: The role of density and seeding protocol on nucleus shape and gene expression. Tissue Eng. Part A 2008, 14, 1561–1572. [Google Scholar] [CrossRef]
  150. Zion Market Research. Insights on Global Cartilage Repair/Regeneration Market Size & Share To Hit USD 1962.53 Million by 2028, Exhibit a CAGR of 14.9%; Zion Market Research: Pune, India, 2022. [Google Scholar]
  151. Negoro, T.; Takagaki, Y.; Okura, H.; Matsuyama, A. Trends in clinical trials for articular cartilage repair by cell therapy. NPJ Regen. Med. 2018, 3, 17. [Google Scholar] [CrossRef] [Green Version]
Bioengineering 10 00355 g001 550
Figure 1. Managing the heterogeneity of MSCs. The heterogeneity of MSCs can be managed ex vivo through (a) reducing MSC culture expansion with the addition of bioactive factors, extracellular matrix proteins, and biophysical stimulation, (b) selecting MSCs with a higher chondrogenic potential based on specific surface markers, and (c) selecting MSCs with a higher chondrogenic potential based on specific biophysical attributes.
Figure 1. Managing the heterogeneity of MSCs. The heterogeneity of MSCs can be managed ex vivo through (a) reducing MSC culture expansion with the addition of bioactive factors, extracellular matrix proteins, and biophysical stimulation, (b) selecting MSCs with a higher chondrogenic potential based on specific surface markers, and (c) selecting MSCs with a higher chondrogenic potential based on specific biophysical attributes.
Bioengineering 10 00355 g001
Table
Table 1. A summary of studies that highlight sources of MSC heterogeneity and their corresponding outcomes in cartilage regenerative therapy.
Table 1. A summary of studies that highlight sources of MSC heterogeneity and their corresponding outcomes in cartilage regenerative therapy.
Source of HeterogeneityStudy DetailsResultsReference
DonorBM-MSCs isolated from 53 donors (25 female, 28 male; 13 to 80 years old).Highly clonogenic BM-MSCs were more frequent in preparations from younger female donors.[50]
BM-MSCs isolated from 17 donors (25 to 81 years old).BM-MSCs from young donors showed improved glycosaminoglycan deposition and increased expression of the chondrogenic markers SOX9, COL2A1, and ACAN.[49]
BM-MSCs isolated from donors with primary osteoarthritis, osteoporosis, and healthy donors.BM-MSCs from patients produced chondrogenic pellets of reduced diameter.[51]
BM-MSCs isolated from donors with advanced osteoarthritis and healthy donors.BM-MSCs from patients had a reduced proliferative capacity and a significant reduction in in vitro chondrogenic activity.[52]
TissueAD-MSCs and BM-MSCs isolated from the same donor.Collagen II and proteoglycans were synthesized only in the BM-MSCs in vitro.[54]
AD-MSCs, BM-MSCs, and MSCs from periosteum isolated from the same donor.Bone marrow and periosteum yielded more homogenous MSCs than fat, improving the correction of physeal arrest in a rabbit model.[55]
BM-MSCs and AD-MSCs seeded onto two different scaffolds: Chondro-Gide or Alpha Chondro Shield.Chondro-Gide seeded with BM-MSCs had the highest MSC proliferation and deposition of ECM tissue.[56]
BM-MSCs and AD-MSCs in a platelet-rich plasma scaffold in an osteochondral defect rabbit model.BM-MSCs demonstrated improved morphological, histological, and immunohistochemical characteristics, higher cartilage-specific gene and protein expression, as well as subchondral bone regeneration.[57]
BM-MSCs, AD-MSCs, and cartilage-derived MSCs from adult Sprague Dawley rats.AD-MSCs have the highest proliferation potential according to growth curve, cell cycle, and telomerase activity analyses.[58]
BM-MSCs, AD-MSCs, and UCB-MSCs.UCB-MSCs could be cultured the longest and showed the highest proliferation capacity.[27]
Equine-derived BM-MSCs and UCB-MSCs.BM-MSCs synthesized ECM of higher quality with a more homogenous distribution of type IIB collagen.[59]
SM-MSCs, AD-MSCs, and BM-MSCs isolated from the same donor.SM-MSCs had the greatest potential for both proliferation and chondrogenesis.[28]
Tissue locationBM-MSCs isolated from the iliac crest, vertebral body, and femoral head.BM-MSCs from the iliac crest and vertebral body demonstrated higher chondrogenic potential.[61]
BM-MSCs isolated from femur trabeculae through rasping and from the main marrow compartment.BM-MSCs from femur trabeculae displayed increased chondrogenic potential.[62]
AD-MSCs from superficial subcutaneous, deep subcutaneous, omentum, mesentery, and retroperitoneum. AD-MSCs from subcutaneous tissue show increased proliferative ability and a higher level of CD146 expression.[63]
Donor-matched AD-MSCs from knee infrapatellar and subcutaneous adipose tissue of osteoarthritic donors.AD-MSCs from the infrapatellar fat pad demonstrated increased glycosaminoglycan production and upregulation of the chondrogenic genes ACAN and COL2A1.[64]
SubpopulationSingle-cell RNA sequencing of human primary Wharton’s jelly-derived MSCs from three donors.Differentially expressed gene analysis found several distinct subpopulations of MSCs that differ in proliferation, development, and inflammation response.[66]
Single-cell RNA sequencing of BM-MSCs (three donors), AD-MSCs (three donors), UCB-MSCs (two donors), and dermis-derived MSCs (three donors).MSC subpopulations were substantially heterogeneous in immune regulation, antigen processing/presentation, and senescence. [65]
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Goh, D.; Yang, Y.; Lee, E.H.; Hui, J.H.P.; Yang, Z. Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review. Bioengineering 2023, 10, 355. https://doi.org/10.3390/bioengineering10030355

AMA Style

Goh D, Yang Y, Lee EH, Hui JHP, Yang Z. Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review. Bioengineering. 2023; 10(3):355. https://doi.org/10.3390/bioengineering10030355

Chicago/Turabian Style

Goh, Doreen, Yanmeng Yang, Eng Hin Lee, James Hoi Po Hui, and Zheng Yang. 2023. "Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review" Bioengineering 10, no. 3: 355. https://doi.org/10.3390/bioengineering10030355

APA Style

Goh, D., Yang, Y., Lee, E. H., Hui, J. H. P., & Yang, Z. (2023). Managing the Heterogeneity of Mesenchymal Stem Cells for Cartilage Regenerative Therapy: A Review. Bioengineering, 10(3), 355. https://doi.org/10.3390/bioengineering10030355

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