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.

Theory and practice of mesenchymal condensation directing tooth development and regeneration

Mesenchymal stem cells, under spatiotemporal regulation of genes and microenvironment, are capable of spontaneously aggregating into dense regions, a phenomenon known as mesenchymal condensation. Mesenchymal condensation is an evolutionarily conserved developmental event that is critical in initiating morphogenesis of teeth and systemic organs. Mesenchymal stem cells hold the intrinsic ability to self-assemble in culture, and the generation of stem cell aggregates based on this property that mimics developmental mesenchymal condensation has become a potent and promising approach in regenerative medicine. This review discusses the mesenchymal condensation principles and its role as well as mechanism in tooth morphogenesis, as well as the engineering strategies for constructing mesenchymal stem cell aggregates and their application experience in tooth regeneration. It aims to start from the perspective of "development-inspired regeneration" and provide insights into understanding stem cell developmental biology and establishing new organ regenerative strategies.

Peptide-Based Hydrogel Scaffold Facilitates Articular Cartilage Damage Repair.

Articular cartilage injury is a common disease in clinical medicine. Because of its special physiological structure and lack of blood, lymph, and nerves, its ability to regenerate once damaged is very limited. In this study, we designed and synthesized a series of self- and coassembled cartilage-inducing functional peptide molecules and constructed a coassembled functional peptide hydrogel based on phenylboronic acid-o-dihydroxy "click chemistry" cross-linking to promote aggregation and signal transduction of mesenchymal stem cells (MSCs) in the early stage and differentiation toward cartilage, thereby promoting the repair of cartilage damage. Three functional peptide molecules were produced using solid-phase peptide synthesis technology, yielding a purity higher than 95%. DOPA-FEFEFEFEGHSNGLPL (DFP) and PBA-FKFKFKFKGHAVDI (BFP) were coassembled at near-neutral pH to form hydrogels (C Gels) based on phenylboronic acid-o-dihydroxy click chemistry cross-linking and effectively loaded transforming growth factor (TGF)-β1 with a release period of up to 2 weeks. Furthermore, chondrocytes and bone marrow mesenchymal stem cells (BMSCs) were cocultured with functional peptide hydrogels, and the results displayed that the coassembled functional peptide hydrogel group C Gels significantly promoted the proliferation of chondrocytes and MSCs. The chondrocyte markers collagen type I, collagen type II, and glycosaminoglycan (GAG) in the coassembled functional peptide hydrogel group were significantly higher than those in the control group, indicating that it can induce the differentiation of MSCs into cartilage. In vivo experiments demonstrated that the size and thickness of the new cartilage in the compound gel group were the most beneficial to cartilage regeneration. These results indicated that peptide hydrogels are a promising therapeutic option for cartilage regeneration.

Harnessing mesenchymal aggregation for engineered organ‐level regeneration: Recent progress and perspective

AbstractStem cells, especially mesenchymal progenitors or mesenchymal stem cells (MSCs), possess an intrinsic property to form compact spheroid‐like assemblies, a phenomenon known as cell aggregation. In recent years, a growing body of researches have uncovered that this is a cross‐species conserved developmental event essential for initiating organogenesis in a variety of organs. Moreover, the self‐assembly property also contributes to the regenerative capacities of MSC aggregates in vivo with broad range of applications in tissue engineering. In this review, the principles of self‐assembled mesenchymal aggregation and its involvement in physiological organogenesis, as well as the construction approaches of engineering MSC aggregates and its application for organ regeneration are discussed. The authors aim to provide a speculative overview of the current understanding and the recent findings of cell aggregation, from both the developmental and the engineering perspectives, and thus offer insights into the understanding of stem cell biology and the establishment of novel organ regeneration strategies.

Acoustofluidic Interfaces for the Mechanobiological Secretome of MSCs

While mesenchymal stem cells (MSCs) have gained enormous attention due to their unique properties of self-renewal, colony formation, and differentiation potential, the MSC secretome has become attractive due to its roles in immunomodulation, anti-inflammatory activity, angiogenesis, and anti-apoptosis. However, the precise stimulation and efficient production of the MSC secretome for therapeutic applications are challenging problems to solve. Here, we report on Acoustofluidic Interfaces for the Mechanobiological Secretome of MSCs: AIMS. We create an acoustofluidic mechanobiological environment to form reproducible three-dimensional MSC aggregates, which produce the MSC secretome with high efficiency. We confirm the increased MSC secretome is due to improved cell-cell interactions using AIMS: the key mediator N-cadherin was up-regulated while functional blocking of N-cadherin resulted in no enhancement of the secretome. After being primed by IFN-γ, the secretome profile of the MSC aggregates contains more anti-inflammatory cytokines and can be used to inhibit the pro-inflammatory response of M1 phenotype macrophages, suppress T cell activation, and support B cell functions. As such, the MSC secretome can be modified for personalized secretome-based therapies. AIMS acts as a powerful tool for improving the MSC secretome and precisely tuning the secretory profile to develop new treatments in translational medicine.

Relationship between oxygen partial pressure and inhibition of cell aggregation of human adipose tissue-derived mesenchymal stem cells stored in cell preservation solutions

IntroductionThis study investigated the storage conditions under which cell aggregation occurs and the conditions that inhibit cell aggregation when human adipose tissue-derived mesenchymal stem cells (hADSCs) are stored in lactated Ringer’s solution (LR) supplemented with 3% trehalose and 5% dextran 40 (LR-3T-5D). MethodsWe first examined the effects of storage temperature and time on the aggregation and viability of hADSCs stored in LR and LR-3T-5D. The cells were stored at 5 °C or 25 °C for various times up to 24 h. We then evaluated the effects of storage volume (250-2,000 μL), cell density (2.5-20 × 105 cells/mL), and nitrogen gas replacement on aggregation, oxygen partial pressure (pO2), and viability of hADSCs stored for 24 h at 25 °C in LR-3T-5D. ResultsWhen stored in LR-3T-5D, viability did not change under either condition compared with pre-storage, but the cell aggregation rate increased significantly with storage at 25 °C for 24 h (p<0.001). In LR, the aggregation rate did not change under either condition, but cell viability decreased significantly after 24 h at both 5 °C and 25 °C (p < 0.05). The cell aggregation rates and pO2 tended to decrease with increasing solution volume and cell density. Nitrogen gas replacement significantly decreased the cell aggregation rate and pO2 (p < 0.05). However, there were no differences in viability among cells stored under conditions of different storage volumes, densities, and nitrogen gas replacement. ConclusionsAggregation of cells after storage at 25 °C in LR-3T-5D may be suppressed by increasing the storage volume and cell density as well as by incorporating nitrogen replacement, which lowers the pO2 in the solution.

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.

Transcriptome analysis reveals synergistic modulation of E-cadherin/N-cadherin in hMSC aggregates chondrogenesis.

N-cadherin-mediated cell adhesion is a vital inductor for mesenchymal condensation in chondrogenesis. Recent studies have revealed the involvement of E-cadherin in enhancing the multipotency of mesenchymal stem cells (MSCs) and limb development; however, the signaling crosstalk of E/N-cadherin remains unclear. This study aimed to explore the synergistic modulation of E/N-cadherin in the chondrogenic differentiation of MSC aggregates. Human E/N-cadherin-functionalized (hE/N-cad-Fc) poly (lactic-co-glycolic acid) (PLGA) microparticles (hE/N-cad-PLGA) were incorporated into the human MSC (hMSC) aggregates to upregulate the expression of the corresponding endogenous cadherin. The chondrogenic differentiation of the hMSC aggregates was initiated by hE/N-cad-PLGA, controlling the release of transforming growth factor-β (TGF-β). A transcriptome analysis was used to assess differentially expressed genes (DEGs) modulated by hE/N-cad-Fc in hMSC aggregate chondrogenesis. Gene functions and signaling pathways were assessed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The associated biological pathways were assessed by a protein-protein interaction (PPI) network analysis, and the results were further confirmed by real-time quantitative PCR (qPCR) and western blotting. A total of 1083 DEGs, comprising 111 upregulated and 972 downregulated genes, were discovered to be related to the enhanced chondrogenic differentiation modulated by hE/N-cad-Fc. The GO and KEGG functional enrichment analyses revealed that hE/N-cad-Fc synergistically regulated the p53-related survival signaling pathway. PPI analysis revealed that mitogen-activated protein kinases (MAPK) caspase regulation is a core aspect of the chondrogenic differentiation process, confirmed by western blotting. To the best of our knowledge, our study is the first to reveal that the synergistic modulation of E/N-cadherin enhances the chondrogenic differentiation of hMSCs via the ERK1/2-p53 signaling axis.

Co-aggregation of MSC/chondrocyte in a dynamic 3D culture elevates the therapeutic effect of secreted extracellular vesicles on osteoarthritis in a rat model

Extracellular vesicles (EVs) have therapeutic effects on osteoarthritis (OA). Some recent strategies could elevate EV's therapeutic properties including cell aggregation, co-culture, and 3D culture. It seems that a combination of these strategies could augment EV production and therapeutic potential. The current study aims to evaluate the quantity of EV yield and the therapeutic effect of EVs harvested from rabbit mesenchymal stem cells (MSCs) aggregates, chondrocyte aggregates, and their co-aggregates in a dynamic 3D culture in a rat osteoarthritis model. MSC and chondrocytes were aggregated and co-aggregated by spinner flasks, and their conditioned medium was collected. EVs were isolated by size exclusion chromatography and characterized in terms of size, morphology and surface markers. The chondrogenic potential of the MSC-ag, Cho-ag and Co-ag EVs on MSC micromass differentiation in chondrogenic media were assessed by qRT-PCR, histological and immunohistochemical analysis. 50 μg of MSC-ag-EVs, Cho-ag-EVs and Co-ag-EVs was injected intra-articularly per knee of OA models established by monoiodoacetate in rats. After 8 weeks follow up, the knee joints were harvested and analyzed by radiographic, histological and immunohistochemical features. MSC/chondrocyte co-aggregation in comparison to MSC or chondrocyte aggregation could increase EV yield during dynamic 3D culture by spinner flasks. Although MSC-ag-, Cho-ag- and Co-ag-derived EVs could induce chondrogenesis similar to transforming growth factor-beta during in vitro study, Co-ag-EV could more effectively prevent OA progression than MSC-ag- and Cho-ag-EVs. Our study demonstrated that EVs harvested from the co-aggregation of MSCs and chondrocytes could be considered as a new therapeutic potential for OA treatment.

Fabrication of scaffold-free mesenchyme tissue bands by cell self-aggregation technique for potential use in tissue regeneration

Three-dimensional cell constructs comprising only tissue-specific cells and extracellular matrix secreted by them would be ideal transplants, but their fabrication in a cell aggregation manner without cell scaffolds relies on random cell self-aggregation, making the control of their size and shape difficult. In this study, we propose a method to fabricate band-shaped tissues by inducing the self-aggregation of cell sheets using the developed cell self-aggregation technique (CAT). Acting as cell aggregation stoppers, silicone semicircular pillars were attached to two positions equidistant from both short ends of the rounded rectangular culture groove and coated with a specifically charged biomimetic polymer as a CAT-inducing surface. Mesenchymal stem cells, chondrocytes, and skeletal myoblast cells seeded on the surface of the culture grooves formed band-shaped aggregates between the two aggregation stoppers following spontaneous detachment with aggregation of the cell sheet from the outer edge of the grooves during day one of culture. The aggregated chondrocyte band matured into a cartilage-like plate with an abundant cartilage matrix while retaining its band shape after two weeks of chondrogenic cultivation. Additionally, the aggregates of mesenchymal stem cells and myoblast cell bands could patch the induced collagen membrane derived from rat subcutaneous tissue like a bandage immediately after their formation and successfully mature into fat and muscle tissues, respectively. These results indicate that, depending on the cell type, scaffold-free band-shaped cell aggregates produced by CAT have the potential to achieve tissue regeneration that follows the shape of the defect via in vitro maturation culture or in vivo organization.

High-efficient engineering of osteo-callus organoids for rapid bone regeneration within one month

Large bone defects that cannot form a callus tissue are often faced with long-time recovery. Developmental engineering-based strategies with mesenchymal stem cell (MSC) aggregates have shown enhanced potential for bone regeneration. However, MSC aggregates are different from the physiological callus tissues, which limited the further endogenous osteogenesis. This study aims to achieve engineering of osteo-callus organoids for rapid bone regeneration in cooperation with bone marrow-derived stem cell (BMSC)-loaded hydrogel microspheres (MSs) by digital light-processing (DLP) printing technology and stepwise-induction. The printed MSC-loaded MSs aggregated into osteo-callus organoids after chondrogenic induction and showed much higher chondrogenic efficiency than that of traditional MSC pellets. Moreover, the osteo-callus organoids exhibited stage-specific gene expression pattern that recapitulated endochondral ossification process, as well as a synchronized state of cell proliferation and differentiation, which highly resembled the diverse cell compositions and behaviors of developmentally endochondral ossification. Lastly, the osteo-callus organoids efficiently led to rapid bone regeneration within only 4 weeks in a large bone defect in rabbits which need 2–3 months in previous tissue engineering studies. The findings suggested that in vitro engineering of osteo-callus organoids with developmentally osteogenic properties is a promising strategy for rapid bone defect regeneration and recovery.

Light-controlled scaffold- and serum-free hard palatal-derived mesenchymal stem cell aggregates for bone regeneration.

Cell aggregates that mimic in vivo cell-cell interactions are promising and powerful tools for tissue engineering. This study isolated a new, easily obtained, population of mesenchymal stem cells (MSCs) from rat hard palates named hard palatal-derived mesenchymal stem cells (PMSCs). The PMSCs were positive for CD90, CD44, and CD29 and negative for CD34, CD45, and CD146. They exhibited clonogenicity, self-renewal, migration, and multipotent differentiation capacities. Furthermore, this study fabricated scaffold-free 3D aggregates using light-controlled cell sheet technology and a serum-free method. PMSC aggregates were successfully constructed with good viability. Transplantation of the PMSC aggregates and the PMSC aggregate-implant complexes significantly enhanced bone formation and implant osseointegration in vivo, respectively. This new cell resource is easy to obtain and provides an alternative strategy for tissue engineering and regenerative medicine.

Duo Cadherin-Functionalized Microparticles Synergistically Induce Chondrogenesis and Cartilage Repair of Stem Cell Aggregates.

Mesenchymal stem cell (MSC) aggregates incorporated with microparticles of functional materials have shown promising prospects in the field of cell therapy for cartilage repair. Given the importance of cadherins in modulating the stemness and chondrogenesis of MSCs, the use of transforming growth factor β1 (TGFβ1)-loaded poly (lactic-co-glycolic acid) (PLGA)-based composite microparticles inspired by duo cadherin (human E- and N-cadherin fusion proteins) to construct a bioartificial stem cell niche in engineered human MSC (hMSC) aggregates to promote chondrogenesis and cartilage regeneration is proposed. The hE/N-cadherin-functionalized PLGA/chitosan-heparin-TGFβ1 (Duo hE/N-cad@P/C-h-TGFβ1) microparticles spatiotemporally upregulates the endogenous E/N-cadherin expression of hMSC aggregates which further amplifies the chondrogenic differentiation and modulate paracrine and anti-inflammatory functions of hMSCs toward constructing a favorable microenvironment for chondrogenesis. The Duo hE/N-cad@P/C-h-TGFβ1 microparticles finely regulate the response of hMSCs to biochemical and mechanical signal stimuli in the microenvironment through the cadherin/catenin-Yes-associated protein signal transduction, which inhibits the hypertrophy of hMSC-derived chondrocytes. Furthermore, immunofluorescent and histological examinations show that the Duo hE/N-cad@P/C-h-TGFβ1 microparticles significantly improve regeneration of cartilage and subchondral bone in vivo. Together, the application of duo cadherin-functionalized microparticles is considered an innovative material-wise approach to exogenously activate hMSC aggregates for functional applications in regenerative medicine.

Sulfated lactose-modified chitosan. A novel synthetic glycosaminoglycan-like polysaccharide inducing chondrocyte aggregation

Lactose-modified chitosan (CTL) is sulfated using SO3·py or SO3·DMF as sulfating agents. The two products are characterized by elemental analysis, FT-IR, 1H,13C-DEPT-HSQC and 1H,13C-HSQC-TOCSY experiments which allow the extent and selectivity of chemical sulfation to be determined. Dynamic Light Scattering shows a pH-dependent association of the sulfated polysaccharides which are described as flexible by the Smidsrød's B parameter and the intrinsic viscosity at infinite ionic strength. Shear viscosity and intrinsic viscosity show that sulfation protocols lead to chain scission which is more pronounced when SO3·DMF is used. The sulfated samples are able to induce aggregation of human bone marrow mesenchymal stem cells, resulting in the formation of smaller nodules compared to the unmodified CTL sample. Over time, the sample with the higher degree of sulfation allows further aggregation between cell clusters while the sample with the lower degree of sulfation shows dissolution of the aggregates.

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.

Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an invitro invasion of cancer cells.

IntroductionThe objective of this study is to design a co-culture system of cancer cells and three-dimensional (3D) mesenchymal stem cells (MSC) aggregates for the in vitro evaluation of cancer invasion.MethodsFirst, the MSC of an immunosuppressive phenotype (MSC2) were prepared by the MSC stimulation of polyriboinosinic polyribocytidylic acid. By simple mixing MSC2 and gelatin hydrogel microspheres (GM) in a U-bottomed well of 96 well plates which had been pre-coated with poly (vinyl alcohol), 3D MSC2 aggregates incorporating GM were obtained. The amount of chemokine (C–C motif) ligand 5 (CCL5) secreted from the MSC2 aggregates incorporating GM. Finally, an invasion assay was performed to evaluate the cancer invasion rate by co-cultured cancer cells and the 3D MSC2 incorporating GM.ResultsThe amount of CCL5 secreted for the 3D MSC2 aggregates incorporating GM was significantly higher than that of two-dimensional (2D) MSC, 2D MSC2, and 3D MSC aggregates incorporating GM. When MDA-MB-231 human breast cancer cells were co-cultured with the 3D MSC2 aggregates incorporating GM, the invasion rate of cancer cells was significantly high compared with that of 2D MSC or 2D MSC2 and 3D MSC aggregates incorporating GM. In addition, high secretion of matrix metalloproteinase-2 was observed for the 3D MSC2 aggregates/cancer cells system.ConclusionsIt is concluded that the co-culture system of 3D MSC2 aggregates incorporating GM and cancer cells is promising to evaluate the invasion of cancer cells in vitro.

In vivo study of the angiogenesis potential of bone marrow-derived mesenchymal stem cell aggregates in their niche like environment.

Sufficient blood vessel formation in bioengineered tissues is essential in order to keep the viability of the organs. Impaired development of blood vasculatures results in failure of the implanted tissue. The cellular source which is seeded in the scaffold is one of the crucial factors involved in tissue engineering methods. Considering the notable competence of Bone Marrow derived Mesenchymal Stem Cell aggregates for tissue engineering purposes, in this study BM-aggregates and expanded BM-MSCs were applied without any inductive agent or co-cultured cells, in order to investigate their own angiogenesis potency in vivo. BM-aggregates and BM-MSC were seeded in Poly-L Lactic acid (PLLA) scaffold and implanted in the peritoneal cavity of mice. Immunohistochemistry results indicated that there was a significant difference (p < 0.050) in CD31+ cells between PLLA scaffolds contained cultured BM-MSC; PLLA scaffolds contained BM-aggregates and empty PLLA. According to morphological evidence, obvious connections with recipient vasculature and acceptable integration with surroundings were established in MSC and aggregate-seeded scaffolds. Our findings revealed cultured BM-MSC and BM-aggregates, capacity in order to develop numerous connections between PLLA scaffold and recipient's vasculature which is crucial to the survival of tissues, and considerable tendency to develop constructs containing CD31+ endothelial cells which can contribute in vessel's tube formation.

Hydrogel microspheres for spatiotemporally controlled delivery of RNA and silencing gene expression within scaffold-free tissue engineered constructs

Delivery systems for controlled release of RNA interference (RNAi) molecules, including small interfering (siRNA) and microRNA (miRNA), have the potential to direct stem cell differentiation for regenerative musculoskeletal applications. To date, localized RNA delivery platforms in this area have focused predominantly on bulk scaffold-based approaches, which can interfere with cell-cell interactions important for recapitulating some native musculoskeletal developmental and healing processes in tissue regeneration strategies. In contrast, scaffold-free, high density human mesenchymal stem cell (hMSC) aggregates may provide an avenue for creating a more biomimetic microenvironment. Here, photocrosslinkable dextran microspheres (MS) encapsulating siRNA-micelles were prepared via an aqueous emulsion method and incorporated within hMSC aggregates for localized and sustained delivery of bioactive siRNA. siRNA-micelles released from MS in a sustained fashion over the course of 28 days, and the released siRNA retained its ability to transfect cells for gene silencing. Incorporation of fluorescently labeled siRNA (siGLO)-laden MS within hMSC aggregates exhibited tunable siGLO delivery and uptake by stem cells. Incorporation of MS loaded with siRNA targeting green fluorescent protein (siGFP) within GFP-hMSC aggregates provided sustained presentation of siGFP within the constructs and prolonged GFP silencing for up to 15 days. This platform system enables sustained gene silencing within stem cell aggregates and thus shows great potential in tissue regeneration applications. Statement of SignificanceThis work presents a new strategy to deliver RNA-nanocomplexes from photocrosslinked dextran microspheres for tunable presentation of bioactive RNA. These microspheres were embedded within scaffold-free, human mesenchymal stem cell (hMSC) aggregates for sustained gene silencing within three-dimensional cell constructs while maintaining cell viability. Unlike exogenous delivery of RNA within culture medium that suffers from diffusion limitations and potential need for repeated transfections, this strategy provides local and sustained RNA presentation from the microspheres to cells in the constructs. This system has the potential to inhibit translation of hMSC differentiation antagonists and drive hMSC differentiation toward desired specific lineages, and is an important step in the engineering of high-density stem cell systems with incorporated instructive genetic cues for application in tissue regeneration.

Cyclical aggregation extends in vitro expansion potential of human mesenchymal stem cells

Mesenchymal stem cell (MSC)-based therapy has shown great promises in various animal disease models. However, this therapeutic potency has not been well claimed when applied to human clinical trials. This is due to both the availability of MSCs at the time of administration and lack of viable expansion strategies. MSCs are very susceptible to in vitro culture environment and tend to adapt the microenvironment which could lead to cellular senescence and aging. Therefore, extended in vitro expansion induces loss of MSC functionality and its clinical relevance. To combat this effect, this work assessed a novel cyclical aggregation as a means of expanding MSCs to maintain stem cell functionality. The cyclical aggregation consists of an aggregation phase and an expansion phase by replating the dissociated MSC aggregates onto planar tissue culture surfaces. The results indicate that cyclical aggregation maintains proliferative capability, stem cell proteins, and clonogenicity, and prevents the acquisition of senescence. To determine why aggregation was responsible for this phenomenon, the integrated stress response pathway was probed with salubrial and GSK-2606414. Treatment with salubrial had no significant effect, while GSK-2606414 mitigated the effects of aggregation leading to in vitro aging. This method holds the potential to increase the clinical relevance of MSC therapeutic effects from small model systems (such as rats and mice) to humans, and may open the potential of patient-derived MSCs for treatment thereby removing the need for immunosuppression.

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.