The effect of forced growth of cells into 3D spheres using low attachment surfaces on the acquisition of stemness properties

Su, Guannan; Zhao, Yannan; Wei, Jianshu; Han, Jin Soo; Chen, Lei; Xiao, Zhifeng; Chen, Bing; Dai, Jianwu

doi:10.1016/j.biomaterials.2013.01.044

Cited by 77 publications

(72 citation statements)

References 48 publications

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“…To further show the stemness of cells on soft substrates, we examined the up-regulation of two other key marker proteins of stem cells, transcription factor OCT4 and Nanog 29,30 (Fig. 6A).…”

Section: Resultsmentioning

confidence: 99%

Actin stress in cell reprogramming

Guo

Wang

Sachs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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Cell mechanics plays a role in stem cell reprogramming and differentiation. To understand this process better, we created a genetically encoded optical probe, named actin-cpstFRET-actin (AcpA), to report forces in actin in living cells in real time. We showed that stemness was associated with increased force in actin. We reprogrammed HEK-293 cells into stem-like cells using no transcription factors but simply by softening the substrate. However, Madin-Darby canine kidney (MDCK) cell reprogramming required, in addition to a soft substrate, Harvey rat sarcoma viral oncogene homolog expression. Replating the stem-like cells on glass led to redifferentiation and reduced force in actin. The actin force probe was a FRET sensor, called cpstFRET (circularly permuted stretch sensitive FRET), flanked by g-actin subunits. The labeled actin expressed efficiently in HEK, MDCK, 3T3, and bovine aortic endothelial cells and in multiple stable cell lines created from those cells. The viability of the cell lines demonstrated that labeled actin did not significantly affect cell physiology. The labeled actin distribution was similar to that observed with GFPtagged actin. We also examined the stress in the actin cross-linker actinin. Actinin force was not always correlated with actin force, emphasizing the need for addressing protein specificity when discussing forces. Because actin is a primary structural protein in animal cells, understanding its force distribution is central to understanding animal cell physiology and the many linked reactions such as stress-induced gene expression. This new probe permits measuring actin forces in a wide range of experiments on preparations ranging from isolated proteins to transgenic animals.actin | force probe | stem cell | reprogramming | cell mechanics C ells have only three sources of free energy-chemical, electrical, and mechanical potential (1)-and life is driven by the flow of energy between them. The flow of mechanical energy has not been well studied in living cells outside of muscle, yet all cells are subject to endogenous and exogenous mechanical forces. The lack of progress in measuring these forces (stress) has primarily been due to a lack of suitable probes, but that has now been remedied with the development of genetically coded FRETbased force sensors (2). The literature shows many macroscopic effects of mechanical stress on cellular processes including cell motility, embryogenesis, stem cell replication and differentiation (3, 4), bone and muscle homeostasis, gene expression, protein folding (5), and membrane potential (6). However, these studies lacked the ability to measure stress in specific structural proteins, and the analyses have tended to treat cytoskeletal stresses as uniform, which we now know is not true (2, 7). We, and other groups (8), have shown that the distribution of forces is nonuniform in both time and space and protein specificity (9).The force sensors consist of a FRET pair (typically CFP/ YFP) connected by a protein linker, a biological analog of a mechanic...

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Section: Resultsmentioning

confidence: 99%

Actin stress in cell reprogramming

Guo

Wang

Sachs

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

103

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“…The spheroid method has also been used to generate neural stem cells [18] and cardiac stem cells [19]. It has also been reported that the spheroid microenvironment mimics the in vivo stem cell niche and is able to reprogram somatic cells into neural progenitor-like cells [20,21]. We hypothesized that lung spheroids could be generated from healthy lung tissues and might contain lung progenitor cells suitable for therapeutic applications.…”

Section: Introductionmentioning

confidence: 99%

Adult Lung Spheroid Cells Contain Progenitor Cells and Mediate Regeneration in Rodents With Bleomycin-Induced Pulmonary Fibrosis

Henry

Cores

Hensley

et al. 2015

Stem Cells Translational Medicine

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Lung diseases are devastating conditions and ranked as one of the top five causes of mortality worldwide according to the World Health Organization. Stem cell therapy is a promising strategy for lung regeneration. Previous animal and clinical studies have focused on the use of mesenchymal stem cells (from other parts of the body) for lung regenerative therapies. We report a rapid and robust method to generate therapeutic resident lung progenitors from adult lung tissues. Outgrowth cells from healthy lung tissue explants are self-aggregated into three-dimensional lung spheroids in a suspension culture. Without antigenic sorting, the lung spheroids recapitulate the stem cell niche and contain a natural mixture of lung stem cells and supporting cells. In vitro, lung spheroid cells can be expanded to a large quantity and can form alveoli-like structures and acquire mature lung epithelial phenotypes. In severe combined immunodeficiency mice with bleomycin-induced pulmonary fibrosis, intravenous injection of human lung spheroid cells inhibited apoptosis, fibrosis, and infiltration but promoted angiogenesis. In a syngeneic rat model of pulmonary fibrosis, lung spheroid cells outperformed adipose-derived mesenchymal stem cells in reducing fibrotic thickening and infiltration. Previously, lung spheroid cells (the spheroid model) had only been used to study lung cancer cells. Our data suggest that lung spheroids and lung spheroid cells from healthy lung tissues are excellent sources of regenerative lung cells for therapeutic lung regeneration. STEM CELLS TRANSLATIONAL MEDICINE 2015;4:1265-1274 SIGNIFICANCEThe results from the present study will lead to future human clinical trials using lung stem cell therapies to treat various incurable lung diseases, including pulmonary fibrosis. The data presented here also provide fundamental knowledge regarding how injected stem cells mediate lung repair in pulmonary fibrosis.

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“…Our another previous work also found that rabbit corneal stromal cells (CSCs) spheroids were positively expressed the mesenchymal and stem cell phenotypes, which contained immunopositive for vimentin and CD34, as well as mRNA expression of Nestin and Nanog [35]. There were reports that the aggregation not only helped to maintain the stemness of stem cells but also could induce some nonstem cells to acquire stem cell properties [69,70]. Bayoussef et al reported that comparison with single cells, the aggregation promoted differentiation of injection cells therapy in vitro model [65].…”

Section: Discussionmentioning

confidence: 91%

Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture

Guo

et al. 2015

Human Cell

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Human adipose-derived stem cells (hADSCs) are potential adult stem cells source for cell therapy. But hADSCs with multi-passage or cryopreservation often revealed poor growth performance. The aim of our work was to improve the activity of poor post-thaw hADSCs by simple and effective means. We describe here a simple method based on commercially available silicone micro-wells for creating hADSCs spheroids to improve viability and neural differentiation potential on poor post-thaw hADSCs. The isolated hADSCs positively expresse d CD29, CD44, CD105, and negatively expressed CD34, CD45, HLA-DR by flow cytometry. Meanwhile, they had adipogenic and osteogenic differentiation capacity. The post-thaw and post-spheroid hADSCs from poor growth status hADSCs showed a marked increase in cell proliferation by CKK-8 analysis, cell cycle analysis and Ki67/P27 quantitative polymerase chain reaction (qPCR) analysis. They also displayed an increase viability of anti-apoptosis by annexin v and propidium iodide assays and mitochondrial membrane potential assays. After 3 days of neural induction, the neural differentiation potential of post-thaw and post-spheroid hADSCs could be enhanced by qPCR analysis and western blotting analysis. These results suggested that the spheroid formation could improve the viability and neural differentiation potential of bad growth status hADSCs, which is conducive to ADSCs research and cell therapy.

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The effect of forced growth of cells into 3D spheres using low attachment surfaces on the acquisition of stemness properties

Cited by 77 publications

References 48 publications

Actin stress in cell reprogramming

Actin stress in cell reprogramming

Adult Lung Spheroid Cells Contain Progenitor Cells and Mediate Regeneration in Rodents With Bleomycin-Induced Pulmonary Fibrosis

Enhanced viability and neural differential potential in poor post-thaw hADSCs by agarose multi-well dishes and spheroid culture

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