Single cell derived spheres of umbilical cord mesenchymal stem cells enhance cell stemness properties, survival ability and therapeutic potential on liver failure
Introduction
Umbilical cord-derived mesenchymal stem cells (UCMSCs) are isolated from Wharton's jelly of umbilical cord and characterized by the abilities of self-renewal, multilineage differentiation, extensive proliferation and paracrine. They have exhibited multiple advantages such as easy isolation and harvest, lack of ethical concerns and tumorigenicity, and low immunogenicity [1,2]. As a result, UCMSCs hold significant promise for tissue engineering and regenerative medicine applications [3]. To date, UCMSCs have been widely used in multiple therapeutic studies such as acute lung injury, insulin-resistant diabetes, Alzheimer's disease (AD), acute myocardial infarction, graft-versus-host diseases (GVHD), aplastic anemia, arthrophlogosis, liver disease, spinal cord injury, systemic lupus erythematosus and stroke [[4], [5], [6], [7], [8], [9], [10], [11], [12], [13], [14]].
In spite of many advantages, mesenchymal stem cells (MSCs) are still confronting challenges in clinical applications, such as requirement of large quantity of cells for therapy, heterogeneity of cell quality, low survival ability after transplantation, the attenuation of MSC characteristics in two-dimensional (2D) culture and ambiguous mechanism of MSCs function for disease therapy. The top priority for MSC therapy is to produce sufficient number of high-quality MSCs in vitro to meet clinical demand.
Generally, MSCs isolated from human tissues are cultured in 2D dishes as monolayer for fast expansion. However, 2D culture condition in vitro is far different from the native growth niche in vivo. It was reported that long-term 2D culture evoked a series of changes in MSCs including cellular senescence, immunogenicity, and losses of their stemness properties and paracrine activity [15,16]. Furthermore, 2D culture affected the genetic expression of cells and remodeled the internal structure of cells [[15], [16], [17], [18]].
Three-dimensional (3D) culture artificially creates an environment in which cells are permitted to grow or interact with their surroundings in all three dimensions. Therefore, 3D culture was widely regarded as a more preferable and closer physiological microenvironment for cells growth [[19], [20], [21]]. So far, many 3D culture methods have been developed to form MSC spheroids, such as hanging drop, magnetically levitate, chitosan membrane culture, microgravity bioreactor and rotating culture [[22], [23], [24], [25], [26]]. The common fundamental of these spheroid-formation methods was to provide cells with a suspension culture condition where the 3D spheroids were formed mainly relying on cell-cell adhesion and interaction that led to self-assembly tendency of MSCs [27,28]. That is, this type of spheroid is initially formed and derived from the aggregation of many individual cells. Here, we name this type of spheroid as multiple cells derived spheroid (MCDS). Growing evidences demonstrated that, by comparing with 2D culture, 3D MCDS culture enhanced the characteristics of MSCs on cell survival, factor secretion, stemness maintenance, migration, anti-senescence in vitro and enhanced the capacities of anti-inflammatory, angiogenesis, tissue repair and regeneration in vivo [[29], [30], [31], [32], [33], [34], [35]].
However, in spite of many advantages reported, obvious defects restrict the direct application of MCDS-cultured MSCs in clinic. First, the MCDS is initially composed of many individual MSCs that have difference viability, which causes the heterogeneity of cell quality in whole spheroid. Second, large size of MCDS results in different distributions of nutrients, oxygen, and waste metabolism between the core and periphery of spheroid. Third, the cells in core of MCDS are subjected to hostile metabolic stresses and tend to undergo apoptosis [28]. For example, lack of extracellular matrix (ECM) support may cause anoikis in the core of MCDS [36]. Forth, large size (diameter > 100 μm) lets MCDS unable to be directly injected into the body for the risk of blood vessel blockage. Therefore, MCDSs generally have to be dissociated into single cells by enzyme before vein-injection [25,37]. However, this dissociation process causes more cell damage and impairs cell viability [38].
The formation of single cell derived sphere (SCDS) reflects the potential of a cell to behave as a stem cell in vitro. Therefore, it has been widely used to evaluate self-renewal and differentiation at the single cell level and then to identify stem cells capacity [39]. Here, we speculate that the formation of SCDS can enhance MSCs stemness and then optimize the quality of MSCs to meet the demand of clinical application. To investigate this hypothesis, we developed a novel approach to produce UCMSC SCDS by combining 2D-pattern of single cell on chip with 3D culture. This technique enables single cells to be patterned on cell-array chip preventing from multicellular aggregation and cultured to form 3D spheres. Our in vitro results demonstrated that, compared to 2D- and MCDS-cultured UCMSCs, SCDS-cultured UCMSCs showed greater properties in many aspects such as stemness maintenance, anti-apoptosis, anti-senescence, hypoxia resistance and cell migration. In vivo results showed that SCDS culture improved cell survival and angiogenesis in UCMSC derived xenografts, and improved therapeutic potential of UCMSCs for carbon tetrachloride (CCl4)-induced acute liver failure (ALF). Our research indicates that SCDS culture may serve as a simple and effective strategy for MSCs optimization to meet clinical application.
Section snippets
Isolation, culture and identification of primary UCMSCs
Human umbilical cords were obtained from full-term infants delivered by caesarean section at Nanjing Drum Tower Hospital. The volunteers obtained written informed consent. The cords were delivered in a sterilized jar filled cold phosphate buffered saline (PBS) to the laboratory. The isolation and primary culture of UCMSCs was according to the method previously reported [40]. To isolate the UCMSCs, the mesenchymal tissue from the Wharton's jelly was minced into small pieces (0.5–1 cm3), evenly
Isolation and identification of UCMSCs
After 20 days of culture, primary cells grew out from the attached tissue blocks cells and began to assemble into groups. Most cells were spindle-shaped with prominent nucleoli. Tissue blocks were removed and cells were cultured to 80% confluence. These cells were named as P0 (passage 0) cells that were ready for passaging. Flow cytometry analysis showed that the isolated UCMSCs expressed high level of MSC markers such as CD44, CD73, CD90 and CD105, but negatively expressed CD11b, CD19, CD34,
Discussion
MSCs can be used as a powerful source in stem cell-based regenerative medicine for their abilities of differentiation, homing and paracrine [30,[63], [64], [65]]. The most realistic problem met in clinic is how to produce sufficient number of high-quality MSCs in vitro. Clinically, the conventional dose of UCMSCs is about 0.5 × 106–1 × 106/kg body/time, and total 4–8 times of administration is needed for one patient in whole process of therapy. Two-D culture is widely used to obtain a large
Conclusions
In this study, we developed a novel method to produce SCDS of UCMSCs based on 2D pattern of single cell on chip and 3D culture. For the first time, we reported that SCDS culture could markedly optimize UCMSCs for potentially meeting the demand of clinical application. Moreover, cell array chip is an appropriate tool for large-scale preparation of SCDSs and different size of MCDSs. In the future, SCDS-cultured UCMSCs may be hopefully used as high-quality cell source for regenerative medicine,
Acknowledgements
We thank Zhijun Zhang, Renjun Pei, Guosheng Cheng, and Qiangbin Wang for insightful comments on the study, Xiang He for her excellent technical assistance. This research was supported by funds from the Ministry of Science and Technology (MOST) (Grant 2017YFA0104301), National Natural Science Foundation of China (Grant 31870975), Suzhou Science and Technology Development Program (Grant SYG201835), Development Program of Ministry of Science and Technology (2016YFC1000802 and 2016YFC1000801), and
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These authors are equal contribution to this work.