Human Mesenchymal Stem Cell Aggregates Research Articles

Synergistic effects of biological stimuli and flexion induce microcavities promote hypertrophy and inhibit chondrogenesis during in vitro culture of human mesenchymal stem cell aggregates.

Interzone/cavitation are key steps in early stage joint formation that have not been successfully developed in vitro. Further, current models of endochondral ossification, an important step in early bone formation, lack key morphology morphological structures such as microcavities found during development in vivo. This is possibly due to the lack of appropriate strategies for incorporating chemical and mechanical stimuli that are thought to be involved in joint development. We designed a bioreactor system and investigated the synergic effect of chemical stimuli (chondrogenesis-inducing [CIM] and hypertrophy-inducing medium [HIM]) and mechanical stimuli (flexion) on the growth of human mesenchymal stem cells (hMSCs) based linear aggregates under different conditions over 4 weeks of perfusion culture. Computational studies were used to evaluate tissue stress qualitatively. After harvesting, both Safranin-O and hematoxylin & eosin (H&E) staining histology demonstrated microcavity structures and void structures in the region of higher stresses for tissue aggregates cultured only in HIM under flexion. In comparison to either HIM treatment or flexion only, increased glycosaminoglycan (GAG) content in the extracellular matrix (ECM) at this region indicates the morphological change resembles the early stage of joint cavitation; while decreased type II collagen (Col II), and increased type X collagen (Col X) and vascular endothelial growth factor (VEGF) with a clear boundary in the staining section indicates it resembles the early stage of ossification. Further, cell alignment analysis indicated that cells were mostly oriented toward the direction of flexion in high-stress region only in HIM under flexion, resembling cell morphology in both joint cavitation and hypertrophic cartilage in growth plate. Collectively, our results suggest that flexion and HIM inhibit chondrogenesis and promote hypertrophy and development of microcavities that resemble the early stage of joint cavitation and endochondral ossification. We believe the tissue model described in this work can be used to develop in vitro models of joint tissue for applications such as pathophysiology and drug discovery.

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

Extended Ischemic Recovery After Implantation of Human Mesenchymal Stem Cell Aggregates Indicated by Sodium MRI at 21.1T.

Extended therapeutic application remains a significant issue in the use of stem cell therapies to treat ischemic stroke. Along these lines, neurological recovery in a rodent model of ischemic stroke was evaluated following implantation of human mesenchymal stem cell aggregates (hMSC-agg), labeled with micron-sized particles of iron oxide, directly into the lateral ventricle contralateral to the ischemic lesion hemisphere. Longitudinally, disease progression and response to hMSC-agg therapy were assessed by 1H and 23Na magnetic resonance imaging (MRI) at 21.1T to investigate cellular localization, migration, and recovery over an extended timeframe. MRI provides quantifiable metrics of tissue status through sodium distributions in addition to traditional proton imaging. Quantitative 23Na MRI revealed a significant decrease of sodium concentrations following hMSC aggregate implantation, indicating recovery of homeostasis. This result correlates positively with extended neurological recovery assessed by behavioral analysis and immunohistochemistry. These findings demonstrate the potential of implanted hMSC aggregate therapy to provide extended treatment for ischemic stroke, as well as the robustness of MRI for monitoring such approaches. This method potentially can be translated to a clinical setting for the assessment of extended cell therapy efficacy.

Injectable Hyaluronic Acid Hydrogel Loaded with Functionalized Human Mesenchymal Stem Cell Aggregates for Repairing Infarcted Myocardium.

Conventional strategies of stem cell injection in treating myocardial infarction (MI) remain a challenge because of low retention rate and insufficient secretion of exogenous cytokines for efficiently improving the microenvironment in the infarcted myocardium, thus hampering the therapeutic effect. Herein, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human VE-cad-Fc fusion protein are fabricated and integrated with human mesenchymal stem cells (hMSCs) to construct functionalized MSC aggregates (FMAs). This fusion protein can effectively promote the paracrine activity of MSCs. The FMA is encapsulated with an injectable hyaluronic acid (HA)-based hydrogel, which is prepared by Schiff base reaction between oxidized HA (OHA) and hydrazided HA (HHA). The OHA@HHA hydrogel loading FMA is injected into the infarcted myocardium of rats, thereby efficiently improving the MI microenvironment in terms of decreased expressions of inflammatory cytokines and upregulated secretion of angiogenic factors compared to the plain hydrogel only and hydrogel encapsulating MSCs. The results of both echocardiography and histological analyses demonstrate the efficient reconstruction of cardiac function and structure and revascularization in the infarct myocardium. The delivery of functionalized stem cell aggregates with an injectable hydrogel offers a promising strategy for treating myocardial infarction and may be expanded to other tissue repair and reconstruction.

Serum-Free Culture of Human Mesenchymal Stem Cell Aggregates in Suspension Bioreactors for Tissue Engineering Applications.

Mesenchymal stem cells (MSCs) have the capacity to differentiate towards bone, fat, and cartilage lineages. The most widely used culture and differentiation protocols for MSCs are currently limited by their use of serum-containing media and small-scale static culture vessels. Suspension bioreactors have multiple advantages over static culture vessels (e.g., scalability, control, and mechanical forces). This study sought to compare the formation and culture of 3D aggregates of human synovial fluid MSCs within suspension bioreactors and static microwell plates. It also sought to elucidate the benefits of these techniques in terms of productivity, cell number, and ability to generate aggregates containing extracellular matrix deposition. MSCs in serum-free medium were either (1) inoculated as single cells into suspension bioreactors, (2) aggregated using static microwell plates prior to being inoculated in the bioreactor environment, or (3) aggregated using microwell plates and kept in the static environment. Preformed aggregates that were size-controlled at inoculation had a greater tendency to form large, irregular super aggregates after a few days of suspension culture. The single MSCs inoculated into suspension bioreactors formed a more uniform population of smaller aggregates after a definite culture period of 8 days. Both techniques showed initial deposition of extracellular matrix within the aggregates. When the relationship between aggregate size and ECM deposition was investigated in static culture, midsized aggregates (100-300 cells/aggregate) were found to most consistently maximize sGAG and collagen productivity. Thus, this study presents a 3D tissue culture method, which avoids the clinical drawbacks of serum-containing medium that can easily be scaled for tissue culture applications.

Metabolic Reconfiguration Supports Reacquisition of Primitive Phenotype in Human Mesenchymal Stem Cell Aggregates.

Spontaneous aggregation and the associated enhancement of stemness have been observed in many anchorage dependent cells. Recently, aggregation of human mesenchymal stem cells (hMSCs) in nonadherent culture has been shown to reverse expansion-induced heterogeneity and loss of stemness and reprogram the hMSC to reacquire their primitive phenotype, a phenomenon that can significantly enhance therapeutic applications of hMSC. The objective of this study was to investigate the mechanistic basis underlying the connection between multicellular aggregation and stemness enhancement in hMSC by testing the hypothesis that cellular events induced during three-dimensional aggregation on nonadherent substratum induces changes in mitochondrial metabolism that promote the expression of stem cell genes Oct4, Sox2, and Nanog. Our results show that aggregation changes mitochondrial morphology and reduces mitochondrial membrane potential, resulting in a metabolic reconfiguration characterized by increased glycolytic and anaplerotic flux, and activation of autophagy. We further demonstrate that interrupting mitochondrial respiration in two-dimensional planar culture with small molecule inhibitors partially recapitulates the aggregation-mediated enhancement in stem cell properties, whereas enhancement of mitochondrial oxidative phosphorylation in the aggregated state reduces the aggregation-induced upregulation of Oct4, Sox2, and Nanog. Our findings demonstrate that aggregation-induced metabolic reconfiguration plays a central role in reacquisition of primitive hMSC phenotypic properties. Stem Cells 2017;35:398-410.

Cell number and chondrogenesis in human mesenchymal stem cell aggregates is affected by the sulfation level of heparin used as a cell coating.

For particular cell-based therapies, it may be required to culture mesenchymal stem cell (MSC) aggregates with growth factors to promote cell proliferation and/or differentiation. Heparin, a negatively charged glycosaminoglycan (GAG) is known to play an important role in sequestration of positively charged growth factors and, when incorporated within cellular aggregates, could be used to promote local availability of growth factors. We have developed a heparin-based cell coating and we believe that the electrostatic interaction between native heparin and the positively charged growth factors will result in (1) higher cell number in response to fibroblast growth factor-2 (FGF-2) and 2) greater chondrogenic differentiation in response to transforming growth factor-β1 (TGF-β1), compared to a desulfated heparin coating. Results revealed that in the presence of FGF-2, by day 14, heparin-coated MSC aggregates increased in DNA content 8.5 ± 1.6 fold compared to day 1, which was greater than noncoated and desulfated heparin-coated aggregates. In contrast, when cultured in the presence of TGF-β1, by day 21, desulfated heparin-coated aggregates upregulated gene expression of collagen II by 86.5 ± 7.5 fold and collagen X by 37.1 ± 4.7 fold, which was higher than that recorded in the noncoated and heparin-coated aggregates. These observations indicate that this coating technology represents a versatile platform to design MSC culture systems with pairings of GAGs and growth factors that can be tailored to overcome specific challenges in scale-up and culture for MSC-based therapeutics. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1817-1829, 2016.

Chondrogenic differentiation of human mesenchymal stem cell aggregates via controlled release of TGF‐β1 from incorporated polymer microspheres

Aggregate culture is a useful method for inducing chondrogenic differentiation of human mesenchymal stem cells (hMSC) in a three-dimensional in vitro culture environment. Conventional aggregate culture, however, typically requires repeated growth factor supplementation during media changes, which is both expensive and time-intensive. In addition, homogenous cell differentiation is limited by the diffusion of chondrogenic growth factor from the culture medium into the aggregate and peripheral cell consumption of the growth factor. We have engineered a technology to incorporate growth factor-loaded polymer microspheres within hMSC aggregates themselves. Here, we report on the system's capacity to induce chondrogenesis via sustained delivery of transforming growth factor-beta1 (TGF-beta1). Cartilage formation after 3 weeks in the absence of externally supplied growth factor approached that of aggregates cultured by conventional methods. Chondrogenesis in the central region of the aggregates is enabled at TGF-beta1 levels much lower than those required by conventional culture using exogenously supplied TGF-beta1, which is likely a result of the system's ability to overcome limitations of growth factor diffusion from cell culture media surrounding the exterior of the aggregates. Importantly, the inclusion of growth factor-releasing polymer microspheres in hMSC aggregates could enable in vivo chondrogenesis for cartilage tissue engineering applications without extensive in vitro culture.