Ebook: Stem Cells and Regenerative Medicine

Regenerative medicine is a branch of translational medicine. Through tissue engineering, cell biology, molecular biology and other techniques, it promotes the self-repair, regeneration, or constructions of new tissue/organ in vitro or in vivo, and improves or restores the functions of injured tissue/organ. Biomaterials, seed cells and local microenvironment are the three major elements of regenerative medicine.

Stem cells are one of the most important kind of seed cells for regenerative medicine. Stem cell research is also one of the most active research areas. Stem cells have self-renewal and directed differentiation characteristics. In addition to the above characteristics, mesenchymal stem cells can also support bone marrow hematopoiesis and regulate immune response. Stem cells, particularly perinatal stem cells, have broad prospects in clinical treatment. They have been successfully used in the treatment of bone and joint defects, spinal cord injury, Crohn’s disease, macular degeneration, diabetes, graft versus host disease, Alzheimer’s disease, and other diseases. Therefore, stem cell research has enormous potential market value. Currently, 14 kinds of stem cell products have been approved by authorities in the world, the majority of which are mesenchymal stem cell products. Biomaterials can not only serve as a scaffold for tissue repair, but also promote stem cell colonization, growth and microenvironment reconstruction, and support the regeneration and repair of defective tissue/organ, for example, spinal cord and myocardium.

As the global aging problem intensifies, the prevalence of degenerative diseases such as cardiovascular and cerebrovascular diseases, cancer, diabetes, and Alzheimer’s disease, is gradually increasing. At the same time, more than ten thousand of patients in the world who suffer from organ failure or severe organ damage due to illness or accident are waiting for organ transplants every day. However, organ transplantation has many problems, including insufficient source of organs, expensive cost, and immune rejection. The rapid development in stem cell and regenerative medicine technology makes it possible to repair or replace aging and damaged tissues with stem cells or in vitro regeneration of tissue/organs. The strategy involves direct infusion of stem cells or stem cell-related factors to the lesion site, in vitro culture of tissues and organs through bioreactors, construction of functional organs by 3D-bioprinting, etc. In addition, stem cell modification using gene reprogramming techniques can be used to treat certain genetic diseases or malignant tumors caused by genetic defects. All these treatments have brought new hope to human beings to overcome major diseases.

In June 19–21, 2018, the 8th International Symposium China-Europe “Stem Cells and Regenerative Medicine” was held in Wuhan, China. At this meeting, researchers from China and France in the field of stem cells and regenerative medicine broadly discussed topics of the basic research and clinical application of mesenchymal stem cells, the construction and application of new biomaterials, biomechanics of bone tissue engineering, cellular immunotherapy, etc. The selected papers from the symposium are published in this special issue.

The guest editors would like to thank all participants and authors for their cooperation and efforts.

Yun Chen

Department of Bio-medical Engineering, School of Basic Medical Sciences, Wuhan University Wuhan, China President of the organizing committee

Jean-François Stoltz

CNRS-UMR 7563, Biopôle, Faculty Medicine, VandÂIJuvre-lès-Nancy 54500, France

Yinping Li

Department of Pathophysiology, School of Basic Medical Sciences, Wuhan University Wuhan, China

Mesenchymal stromal cells (MSC) represent an interesting cell source for cell therapy, nevertheless their use in clinic involve an expansion step which could affect their quality because of replicative aging. Moreover, the proliferation rate depends on the MSC source. This work compares the proliferation kinetics at early and late passage of young and old Bone Marrow MSC (BM-MSC) and Wharton’s Jelly MSC (WJ-MSC) to know if senescence could affect their proliferation properties. Phenotype, cell cycle analysis, relative size and cell structure determination were performed by flow cytometry. Senescence was estimated by β-Galactosidase activity. Immunofluorescent staining was performed for proliferation antigen Ki67, protection enzyme against oxidative stress superoxide dismutase 2 (SOD2) and cytoskeleton. Results showed better proliferation properties for WJ-MSC compared to young and old BM-MSC. For WJ-MSC the high proliferation rate was linked to a lower senescent cells percentage and SOD2 expression. On the contrary, old BM-MSC have a low proliferation rate linked to an increase in G0/G1 cell cycle phase, an increase in cell size, a higher senescent cells percentage and SOD2 expression. The comprehension of the process of MSC proliferation and the development of strategies to prevent senescence could help to improve therapeutic efficacy.

Gene editing is to insert, delete, modify or replace target gene into the genome of a living organism. The most common technique is to import a recombinant vector (such as a plasmid or lentivirus containing a target-gene) in order to get one new hereditary mark to change the target cells’ function and characteristics or differentiate target cells into a particular direction. Mesenchymal stem cell (MSC) is a kind of pluripotent stem cells with high ability of self-proliferation and can be differentiated into osteoblasts, chondrocytes, adipocytes, etc. Because of its potential of multidirectional differentiation, with the development of regenerative medicine, MSC has become one of the ideal seeding cells for tissue engineering and cell therapy. Researches have shown that gene editing technique regulates the differentiation of MSCs. In this review, we mainly discuss the latest research progress in gene editing techniques applied in mesenchymal stem cells’ differentiation.

Diosmetin, an O-methylated flavone from Caucasian vetch, displays the promising effects on the treatment and prevention of the various diseases, largely through its antioxidant and anti-inflammatory properties. However, its effect on diabetes remains largely unknown. The main purpose of this study is to investigate the potential impacts of diosmetin on insulin resistance, a hallmark of obesity and diabetes, and to evaluate its underlying molecular mechanism. We found that diosmetin markedly attenuated palmitate (PA)-induced insulin resistance in the mature 3T3-L1 adipocytes, evidenced by the elevated levels of phosphorylation of Akt and its downstream AS160, and the increased 2-deoxy-D-glucose uptake when compared with PA treatment alone. Diosmetin was also found to suppress PA-induced endoplasmic reticulum (ER) stress/c-Jun N-terminal kinases (JNKs) signaling and NF-κB signaling pathways. In addition, diosmetin administration significantly decreased the mRNA levels of IL-1β and TNF-α. Furthermore, diosmetin supplementation reversed PA-induced inhibition on adiponectin (ADPN) abundance in both the cells and the culture medium. Given that ER stress is responsible for the activation of NF-κB inflammation signaling pathway and the suppression of ADPN production and secretion, these results revealed that diosmetin mitigates insulin resistance through suppressing ER stress in adipocytes.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns. It is of critical importance for clearing invading pathogens and controlling bacterial infections through modulating inflammation. However, excessive host inflammatory responses may trigger organ failure, disrupt tissue homeostasis and induce immune disorders. Emerging evidence has shown that Notch signaling interacts with components of TLRs and participates in regulating immune responses. In this review, we discussed how Notch pathways interact with TLRs pathway and how these two pathways coordinate to regulate immune response, particularly polarization of macrophages, differentiation of dendritic cells, activation of T cell subsets.

Alzheimer’s disease (AD) is the most common form of dementia in the elderly, and most AD patients show defective insulin signaling. Tau protein levels are elevated in the AD brains, and the insulin signaling is impaired in many AD patients, but the direct link between tau and insulin signaling is still missing. Here, we investigated whether increasing tau affects insulin signaling. We found that overexpression of full-length human tau in neuroblastoma Neuro2a cells decreased the constitutive activity and insulin-induced activation of insulin receptor, PDK and Akt. Overexpression of tau reduced GSK-3β phosphorylation and decreased insulin-stimulated translocation of GLUT4 from an intracellular compartment to the plasma membrane. We also demonstrated that tau overexpression increased JNK phosphorylation with upregulation of the inhibitory phosphorylation of IRS-1 at serine residues and downregulation of the tyrosine phosphorylation. Furthermore, simultaneous inhibition of JNK reversed tau overexpression-induced insulin signaling impairment. These findings suggest that intracellular accumulation of tau protein inhibits insulin signaling through activation of JNK, which provides new insights in the toxicities of tau protein in AD and tauopathies.

Purpose:

The aim of this study is to investigate the osteogenic induction of the cells in a transcriptional level.

Methods:

The dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) were isolated from human dental pulp and periodontal ligaments, respectively. The cells were identified by assessing the stem cell markers, and characterized by differentiation multipotency. During the osteogenic induction, we detected the gene expression of DSPP, RUNX2, HOSTRIX, BMP-2 and OPN on Day 3, 5, 7, 10 and 14 by real time PCR. Furthermore, the in vitro mineralization of those cells was examined by semi-quantitative determination.

Results:

DPSCs and PDLSCs expressed stem cell markers and were able to differentiate into multiple cell types. The gene expression of ALP, BMP-2, DSPP, OSTRIX OPN, and RUNX2 revealed a distinguished trend during osteogenesis. For the qual- itative study, semi-quantitative determination of mineralization showed that DPSCs maintain more powerful differ- entiation ability than PDLSCs under the same conditions.

Conclusions:

DPSCs are probably a more suitable candidate for tissue engineering than PDLSCs. The 7th day of osteogenesis differentiation is an appropriate time point to investigate the microenvironment involved differences between DPSCs and PDLSCs.

Almost all cells in the human body are subjected to mechanical stress. These forces can vary from a few Pascals (shear stress) to some Mega Pascals (on hip cartilage). It is now well known that mechanical forces have a critical effect on cellular physiology. However, although the main biological effects of mechanical forces are well documented, the relation between mechanical stress and physiological phenomena is mainly unknown (mechanotransduction phenomenon). In this chapter, some effects of mechanical stress on bone cells (mesenchymal stem cells, osteoblasts…) are given.

In this work, we have developed a mechanobiological model of bone remodeling involving a mineralization of bone in a moving diffuse interface separating the marrow containing all specialized cells from newly formed bone. The phase field describes the degree of mineralization within the diffuse interface at the level of individual trabeculae; it varies continuously between the lower value (no mineral) and unity (fully mineralized phase corresponding to new bone). The field equations for the mechanical, chemical, and interfacial phenomena have been written, based on the thermodynamics of irreversible processes. The kinetic equations for the internal variables are obtained from a pseudo-potential of dissipation. The combination of the balance equations for the microforce associated to the phase field and the kinetic equations lead to the Ginzburg–Landau equation satisfied by the phase field with a source term accounting for the dissipative microforce. The bone remodeling phenomena have been further coupled to the cell activity responsible for bone production/resorption. Simulations illustrating the proposed framework show the major role of mechanical energy on bone mass production and cell activity.

The renewal of living bone is continuous and influenced by many mechanical and biological factors. A reliable prediction of such phenomena should include many external sources such as mechanical, biological, electrical, neurological, and others, triggered by genetic and/or epigenetic factors. In the current work, we consider as external sources, (i) the mechanical energy developed by the applied mechanical load and sustained by the bone cells, (ii) the concentration of nutriments (oxygen and glucose) available for cell survival, and (iii) cell activity triggered by the nutriments available and the mechanical force applied. We study the bone cell interactions, and more specifically the competition between osteoblasts and osteoclasts leading the bone density kinetics over time, in order to try to predict the evolution of osteoporosis with age. Three-dimensional finite element models are developed to calculate the model variable evolutions for different bone microstructures as a function of applied mechanical load corresponding to standard body weight. The bone density evolution is then calculated and compared with literature data and show good correlation for the studied samples. This could help in the understanding and prediction of osteoporotic degradation mechanisms for better medical prevention.

Background:

Mechanical chest compression devices have been developed to improve the effectiveness of cardiopulmonary resuscitation (CPR). But the neurological benefit of mechanical chest compression is uncertain.

Objectives:

To assess the effectiveness of mechanical chest compression versus manual chest compression on the neurological outcomes in patients who suffered cardiac arrest using a meta-analysis.

Methods:

By searching the Cochrane library, Pubmed, Embase and Web of Science datebase, 10 clinical controlled trials from 1990 to July 2018 were included our study, and 6 trials among them were to compare the neurological outcomes of mechanical chest compression and manual chest compression. We used the Cochrane Collaboration Network Risk Assessment Tool and the Newcastle-Orrawa Scale (NOS) for quality assessment and RevMan 5.3 for data analysis. Use relative risk (RR) and 95% confidence interval (CI) to pool the effect.

Results:

Compared with manual chest compression, mechanical chest compression did not significantly improve survival with good neurological outcome to hospital discharge (RR 0.78, 95% CI 0.60–1.00, P = 0.05), ROSC (RR = 0.97,95% CI 0.92–1.02, p = 0.20, I² = 38%) and short-term (survival to hospital admission or survival to 4 hours) survival (RR = 1.00,95% CI 0.92–1.09, p = 0.97, I² = 0%;). In addition, compared with manual chest compression, mechanical chest compression were associated with higher survival to hospital discharge (RR = 0.86,95% CI 0.76–0.97, p = 0.01,I² = 36%).

Conclusion:

Mechanical chest compression devices should not be recommended to be used conventionally during CPR in adult patients with cardiac arrest.

Cellulose/poly (γ-glutamic acid) (cellulose/γ-PGA, CPGA) sponges were prepared by freeze-drying method. Scanning electron microscopy (SEM), Fourier transformed infrared spectroscopy (FTIR), and X-ray diffraction patterns (XRD) were used to characterize physicochemical properties of the sponges. The mechanical testing results showed that the sponges with the addition of γ-PGA possessed good compression performance, the corresponding stresses were 381, 506, 363, and 296 kPa at 60% deformation, respectively. The swelling ratios of the sponges were all more than 1200 times, showing good water swelling property. The MTT assay indicated that the sponges was no cytotoxicity. The experimental results of this article suggest the novel cellulose/γ-PGA sponges have very good cytocompatibility, it could be as a potential carrier material applied in skin tissue engineering.

Background:

A kind of biopolymer microsphere based on halloysite nanotubes (HNTs) were loaded insulin for oral administration.

Objective:

In order to increase the bioavailability of insulin and overcome the gastrointestinal (GI) enzymatic degradation for delivery of insulin by oral administrationfor the treatment of diabetes.

Methods:

Fourier Transform Infrared (FTIR), X-ray diffraction (XRD), transmission electron microscope (TEM), scanning electron microscopy (SEM), swelling property, release profiles, biocompatibility, acute oral toxicity and HE staining were tested.

Results:

INS could be successfully loaded on the HNTs, and the biopolymer microspheres exhibited an egg-box structure which contributed INS loading. Besides, composite microspheres with smart a pH responsive act as an insulin delivery system.

Conclusion:

The intelligent pH response system for oral delivery of insulin was a relatively safe and effective drug delivery system, and it has great potential to achieve insulin oral administration for the treatment of diabetes.

Emerging evidence highlights the important role of nervous microenvironment in cancer development. However, few in vitro models are suitable for investigating neuron-cancer interaction. In recent years, a number of new materials and techniques at micro/nano-scale have been reported, in particular in studies of neuronal development, neuron-cancer interaction, and cancer therapy. They offer new capabilities to fabricate materials and devices in controlling cell behavior and cellular microenvironment. Here, we summarize the basic principles of these micro/nano-scale materials and techniques, and highlight the important advances in this research field of neuron-cancer interface.

In order to enable the advent of personalized medicine, we need solid in vitro models, taking into account the 3D microenvironment of the tumor, the human component, and the vasculature, to test both the toxicity and efficacy of new molecules. Today, the drug screenings are performed on 3D cell lines models and Patient-Derived Xenografts models. These models are not able to recapitulate the whole tumor microenvironment and vasculature, with hopeless return to the patient. Here, we propose our in vitro model, based on Patient-Derived Organoids, in a vascularized 3D environment, including mesenchymal stem cells. We believe that this will greatly contribute to the development of personalized medicine, since we are working on both healthy tissue (toxicity) and tumor tissue (efficacy). The second step of this model will be the integration of immune cells based on recent discoveries.

Bone marrow mesenchymal stem cells (BMSCs) are promising tool for cell therapy. However, their biological behaviors and functions could be altered by the surrounding microenvironment. In this study, we intend to investigate the effects of septic microenvironment on the biological behaviors of BMSCs. Sepsis was induced in healthy Sprague-Dawley rats through cecal ligation puncture procedure. BMSCs were isolated and expanded from these septic animals in vitro. BMSCs from sham control rats were cultured as control. Morphology of cells was detected by light microscope. Proliferation and self-renewal of BMSCs were detected through cell counting kit-8 and colony forming unit-fibroblasts test. Cell cycle and apoptosis were detected by flow cytometry. Cell migration was assessed through transwell assay. Senescence-associated molecules were analyzed through Western-blot. Capacity of differentiation and activity of senescence-associated β-galactosidase (β-gal) were detected as well. Our results showed that the ability of proliferation and migration was significantly inhibited in BMSCs from septic animals, and cell cycle was blocked in G1 phase with enhanced expression of P21 and P16. Furthermore, expression of sirtuin1 was decreased and activity of β-gal in these cells was significantly enhanced. These results indicated that septic microenvironment induced a senescence-associated phenotype in BMSCs.

Objective:

Exploring the clinical efficacy of autologous platelet-rich plasma (PRP), which has become a new approach to treat surgical wounds, especially in the treatment of osteoarthritis and refractory wounds.

Method:

PRP is derived from the centrifugation of autologous blood and plays an essential role in tissue repairing process. It contains a variety of growth factors that can stimulate cell proliferation and differentiation and facilitate healing of soft tissue, joints and wounds. Applying PRP in surgical practice can greatly reduce bleeding and enhance soft tissue healing and bone regeneration. We applied PRP extraction in the treatment of osteoarthritis and refractory wound patients.

Results:

(1) Osteoarthritis: 110 knee osteoarthritis patients with 220 affected knees, 10 cases of 20 joints were relieved after first PRP injection, 50 cases of 100 joints were relieved after the second injection, 44 cases of 88 joints were relieved after 3 times of injection, only 6 cases of 12 joints receives no significant effect. The effective rate of this group is 94.5%.

Conclusion: Intracavitary injection of PRP in the knee joint can effectively treat knee osteoarthritis and obtain favorable results in the short term, particularly in the treatment of patients with lower age or mild knee joint degeneration.

(2) Refractory wound: 18 cases, 12 cases of a traumatic wound, 2 cases of pressure ulcers, 1 cases of venous ulcer, 2 cases of diabetic ulcer foot, 1 case of chronic osteomyelitis, all healed.

Conclusion: PRP has obvious therapeutic effects on soft tissue wound reparation, without side effects, and painless. It can inhibit the growth of bacteria in the wound area, and effectively facilitate the repairing of soft tissue defect and the healing of bone and tendon wound. It is a safe, minimally invasive and efficacious treatment.

Cancers represent one of the most major causes of mortality worldwide. Solid tumors which are the most prevalent cancers have a complex microenvironment where neovascularization reduces the efficiency of anti-tumor treatments. Thus, advanced selective anti-cancer therapies targeting the neovascularization are required. Strategies targeting neovascularization are different by the nature of their target: molecular (pro-angiogenic growth factors or their receptors) or cellular (endothelial cells, endothelial progenitor or stem cells). The targeting nano-formulations are the most promising strategy. The size of nanoparticles promotes an adequate diffusion within the tumor despite of its abnormal vasculature and their accumulation. This selective therapies supported by nanotechnologies would help to take up the challenge of reducing tumor aggressiveness by targeting and normalizing its vascularization. Monotherapy targeting neo-vascularization is not suitable with the complexity of microenvironment. Their association with different anti-tumor selective therapies is required. New three-dimensional microfluidic models appear to be particularly adequate to evaluate the efficiency of anti-angiogenic therapies and the synergistic effect of their associations.

Febrile seizures (FS) is the most common type of seizures in children. Evidence indicating cognitive effects that FS exert that persist into adulthood is inconsistent. To investigate the mechanisms underlying long-term cognitive effects induced by FS, we examined specific measures in adult rats previously subjected to experimental FS. Rats were separated into hyperthermia-seizure (HS) group which experienced serious seizures, and hyperthermia-non-seizure (HN) group which did not experience seizures after each hyperthermia inductions. Based on Morris water maze and novel object recognition tests, we found that adult rats exhibited better spatial and non-spatial learning and memory performance in HS group. To further illustrate the mechanism of this phenomenon, we found greater long-term potentiation (LTP) in hippocampal area CA3, higher expression of synaptic plasticity-related factors (CREB, p-CREB and synapsin), and raised synaptic curvatures in HS group compare with HN group. Therefore, we conclude that low-intensity seizures may promote hippocampus-associated learning and memory functions by enhancing synaptic plasticity. Our findings may be useful for understanding the pathogenesis of long-term changes in cognitive function induced by FS.

In orthodontics, mechanical forces are applied onto brackets bonded on dental crowns to correct dental malocclusions, inducing biological reactions within the periodontium. These phenomena lead to orthodontic tooth movement through bone remodeling. The aim of our work is to develop a numerical model with experimental and histological data, trying to simulate at best the initial conditions of the in-vivo conditions. For this, we initially generated an experimental set-up to quantify friction and applied forces into three brackets of interest. We then defined by histology the cellular and vascular density within the periodontal ligament and surrounding bone on human samples. With the help of these informations, a preliminary mechanobiological coupling is put forward, to be integrated within a finite element numerical model in order to predict tooth displacement.