Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.     

New insights on the reparative cells in bone regeneration and repair

Bone possesses a remarkable repair capacity to regenerate completely without scar tissue formation. This unique characteristic, expressed during bone development, maintenance and injury (fracture) healing, is performed by the reparative cells including skeletal stem cells (SSCs) and their descendants. However, the identity and functional roles of SSCs remain controversial due to technological difficulties and the heterogeneity and plasticity of SSCs. Moreover, for many years, there has been a biased view that bone marrow is the main cell source for bone repair. Together, these limitations have greatly hampered our understanding of these important cell populations and their potential applications in the treatment of fractures and skeletal diseases. Here, we reanalyse and summarize current understanding of the reparative cells in bone regeneration and repair and outline recent progress in this area, with a particular emphasis on the temporal and spatial process of fracture healing, the sources of reparative cells, an updated definition of SSCs, and markers of skeletal stem/progenitor cells contributing to the repair of craniofacial and long bones, as well as the debate between SSCs and pericytes. Finally, we also discuss the existing problems, emerging novel technologies and future research directions in this field.     

干细胞在牙齿再生研究中的应用

干细胞是一类能够自我更新并分化形成多种组织细胞类型的原始细胞.基于其特殊的生物学性质,干细胞可作为器官再生的理想种子细胞.干细胞已成功被诱导为神经、心肌、皮肤、软骨、肝脏、胰岛、造血等不同类型组织细胞,为神经损伤、退行性变、胰岛素依赖性糖尿病、造血功能障碍等多种难活性疾病提供替代疗法.牙齿是人体中唯一的能在成体中再次发育而且结构相对简单的一个器官,因此牙齿再生已成为组织工程研究领域的热点.目前,在模式动物小鼠以及小型猪中已开展许多利用干细胞进行釉质、本质、牙髓以及牙周韧带等牙齿组织再生和整牙再生的研究.本文系统地概括了不同来源的干细胞(胚胎干细胞、成体干细胞和诱导多能干细胞)在牙齿再生研究中的应用.其中,成体干细胞具有来源广泛、便于采集培养以及不导入外源基因等优势,在牙齿再生的基础研究和临床应用中具有更大的价值.     

Transdifferentiated Mesenchymal Stem Cells as Alternative Therapy in Supporting Nerve Regeneration and Myelination

1. Aims: Demyelination plays a crucial role in neurodegenerative processes and traumatic disorders. One possibility to achieve remyelination and subsequent restoration of neuronal function is to provide an exogenous source of myelinating cells via transplantation. In this context, mesenchymal stem cells (MSCs) have attracted interest. They are multipotent stem cells that differentiate into cells of the mesodermal lineage like bone, cartilage, fat, and muscle. Although adult, their differentiation potential is remarkable, and they are able to transdifferentiate.2. Methods: We transformed cultivated rat MSCs into myelinating cells by using a cytokine cocktail. Transdifferentiated MSCs were characterized by an enhanced expression of LNGF-receptor, Krox20, and CD104, and a decreased expression of BMP receptor-1A as compared to untreated MSCs. The myelinating capacity was evaluated in vitro and in vivo. Therefore, PC12 cells, normally unmyelinated, were cocultivated with MSCs, transdifferentiated MSCs, and Schwann cells, or the respective cells were grafted into an autologous muscle conduit bridging a 2-cm gap in the rat sciatic nerve. Myelination of PC12 cells was demonstrated by electron microscopy. In vivo, after 3 and 6 weeks regeneration including myelination was monitored histologically and morphometrically. Autologous nerves and cell-free muscle grafts were used as control.3. Results: Schwann cells and transdifferentiated MSCs were able to myelinate PC12 cells after 14 days in vitro. In vivo, autologous nerve grafts demonstrated the best results in all regenerative parameters. An appropriate myelination was noted in the Schwann cell groups and, albeit with restrictions, in the transdifferentiated MSC groups, while regeneration in the MSC groups and in the cell-free groups was impaired.4. Conclusion: Our findings demonstrate that it may be possible to differentiate MSCs into therapeutically useful cells for clinical applications in myelin defects.     

非牙源性干细胞在牙齿再生研究中的应用

近年来,干细胞治疗研究获取的成果为牙齿的修复和再生研究奠定了有力的理论基础.根据牙齿的发育特征,牙齿再生需要牙源性的上皮干细胞和牙源性的间充质干细胞,目前研究表明,牙源性的间充质干细胞可应用于牙齿再生,例如牙髓干细胞和牙周韧带干细胞.但是,人牙源性上皮干细胞仅存在于胚胎期,萌发后的牙齿并不存在牙上皮干细胞,因此学者们开始探索将非牙源性干细胞替代牙源性上皮细胞应用于牙齿再生研究.以下概述了胚胎干细胞、成体干细胞和诱导性多潜能性干细胞等非牙源性干细胞在牙齿再生中的研究进展.     

骨髓间充质干细胞在脱细胞真皮基质再生过程中的初步研究

较大的腹壁缺损需要应用补片修复来缓解腹横筋膜的张力,人工合成补片的应用一定程度上实现了无张力修补的目的,但它在腹壁外科应用中有诸多的并发症,诸如复发率高,腹腔黏连,肠穿孔导致腹膜炎,侵袭性肠瘘等影响患者术后的正常生活,而脱细胞真皮基质(Acellular dermal matrix,ADM)作为一种新型的生物材料应用在腹壁外科中能解决上述人工合成补片所带来的并发症发挥强大作用且能与周围组织较好的融合,最后改建成宿主自身组织已并在多学科领域中广泛应用;骨髓间充质干细胞(Bone marrow mesenchymal stem cells,BMSCs)能参与组织自我修复,并能分化成为多种功能细胞,分泌各种生长因子,在ADM内源性转归过程中可发挥作用。本文就对骨髓间充质干细胞在ADM生物补片应用于临床疝修补术中转归机制的研究做一综述。     

PAF-Myc-Controlled Cell Stemness Is Required for Intestinal Regeneration and Tumorigenesis

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Local signals in stem cell-based bone marrow regeneration

The cellular basis of bone marrow （BM） tissue development and regeneration is mediated through hematopoietic stem cells （HSCs） and mesenchymal stem cells （MSCs）. Local interplays between hematopoietic cells and BM stromal cells （BMSCs） determine the reconstitution of hematopoiesis after myelosuppression. Here we review the BM local signals in control of BM regeneration after insults. Hematopoietic growth factors （HGFs） and cytokines produced by BMSCs are primary factors in regulation ofBM hematopoiesis. Morphogens which are critical to early embryo development in multiple species have been added to the family of HSCs regulators, including families of Wnt proteins, Notch ligands, BMPs, and Hedgehogs. Global gene expression analysis of HSCs and BMSCs has begun to reveal signature groups of genes for both cell types. More importantly, analysis of global gene expression coupled with biochemical and biological studies of local signals during BM regeneration have strongly suggested that HGFs and cytokines may not be the primary local regulators for BM recovery, rather chemokines （SDF- 1, FGF-4） and angiogenic growth factors （VEGF-A, Ang- 1） play instructive roles in BM reconstitution after myelosuppression. A new direction of management of BM toxicity is emerging from the identification of BM regenerative regulators.     

Research progress on the hedgehog signalling pathway in regulating bone formation and homeostasis

Bone formation is a complex regeneration process that was regulated by many signalling pathways, such as Wnt, Notch, BMP and Hedgehog (Hh). All of these signalling have been demonstrated to participate in the bone repair process. In particular, one promising signalling pathway involved in bone formation and homeostasis is the Hh pathway. According to present knowledge, Hh signalling plays a vital role in the development of various tissues and organs in the embryo. In adults, the dysregulation of Hh signalling has been verified to be involved in bone‐related diseases in terms of osteoarthritis, osteoporosis and bone fracture; and during the repair processes, Hh signalling could be reactivated and further modulate bone formation. In this chapter, we summarize our current understanding on the function of Hh signalling in bone formation and homeostasis. Additionally, the current therapeutic strategies targeting this cascade to coordinate and mediate the osteogenesis process have been reviewed.     

Transplants of Human Mesenchymal Stem Cells Improve Functional Recovery After Spinal Cord Injury in the Rat

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2×10⁶ in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso–Beattie–Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.     

Mesenchymal stem cells show radioresistance in vivo

Irradiation impacts on the viability and differentiation capacity of tissue‐borne mesenchymal stem cells (MSC), which play a pivotal role in bone regeneration. As a consequence of radiotherapy, bones may develop osteoradionecrosis. When irradiating human bone‐derived MSC in vitro with increasing doses, the cells’ self‐renewal capabilities were greatly reduced. Mitotically stalled cells were still capable of differentiating into osteoblasts and pre‐adipocytes. As a large animal model comparable to the clinical situation, pig mandibles were subjected to fractionized radiation of 2 χ 9 Gy within 1 week. This treatment mimics that of a standardized clinical treatment regimen of head and neck cancer patients irradiated 30 χ 2 Gy. In the pig model, fractures which had been irradiated, showed delayed osseous healing. When isolating MSC at different time points post‐irradiation, no significant changes regarding proliferation capacity and osteogenic differentiation potential became apparent. Therefore, pig mandibles were irradiated with a single dose of either 9 or 18 Gy in vivo, and MSC were isolated immediately afterwards. No significant differences between the untreated and 9 Gy irradiated bone with respect to proliferation and osteogenic differentiation were unveiled. Yet, cells isolated from 18 Gy irradiated specimens exhibited a reduced osteogenic differentiation capacity, and during the first 2 weeks proliferation rates were greatly diminished. Thereafter, cells recovered and showed normal proliferation behaviour. These findings imply that MSC can effectively cope with irradiation up to high doses in vivo. This finding should thus be implemented in future therapeutic concepts to protect regenerating tissue from radiation consequences.     

Broad Distribution of Hepatocyte Proliferation in Liver Homeostasis and Regeneration

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Mesenchymal stromal cells from adipose tissue attached to suture material enhance the closure of enterocutaneous fistulas in a rat model

Background aimsSurgical treatment for enterocutaneous fistulas (EF) frequently fails. Cell therapy may represent a new approach to treatment. Mesenchymal stromal cells (MSCs) have high proliferative and differentiation capacity. This study aimed to investigate whether MSCs could adhere to suture filament (SF), promoting better EF healing.MethodsMSCs, 1 × 10⁶, from adipose tissue (ATMSCs) were adhered to a Polyvicryl SF by adding a specific fibrin glue formulation. Adhesion was confirmed by confocal and scanning electron microscopy (SEM). A cecal fistula was created in 22 Wistar rats by incising the cecum and suturing the opening to the surgical wound subcutaneously with four separate stitches. The animals were randomly allocated to three groups: control (CG)—five animals, EF performed; injection (IG)—eight animals 1 × 10⁶ ATMSCs injected around EF borders; and suture filament (SG): nine animals, sutured with 1 × 10⁶ ATMSCs attached to the filaments with fibrin glue. Fistulas were photographed on the operation day and every 3 days until the 21st day and analyzed by two observers using ImageJ Software.ResultsConfocal and SEM results demonstrated ATMSCs adhered to SF (ATMSCs-SF). The average reduction size of the fistula area at 21st day was greater for the SG group (90.34%, P < 0.05) than the IG (71.80%) and CG (46.54%) groups.ConclusionsATMSCs adhered to SF maintain viability and proliferative capacity. EF submitted to ATMSCs-SF procedure showed greater recovery and healing. This approach might be a new and effective tool for EF treatment.     

Mesenchymal Precursor Cells in Adult Nerves Contribute to Mammalian Tissue Repair and Regeneration

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Reviewing the role of cardiac exosomes in myocardial repair at a glance

Exosome-based therapy is an emerging novel approach for myocardial infarction (MI) treatment. Exosomes are identified as extracellular vesicles that are produced within multivesicular bodies in the cells' cytosols and then are secreted from the cells. Exosomes are 30–100 nm in diameter that are released from viable cells and are different from other secreted vesicles such as apoptotic bodies and microvesicles in their origin and contents such as RNAs, proteins, and nucleic acid. The recent advances in exosome research have demonstrated the role of these bionanovesicles in the physiological, pathological, and molecular aspects of the heart. The results of in vitro and preclinical models have shown that exosomes from different cardiac cells can improve cardiac function following MI. For example, mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) containing exosomes can affect the proliferation, survival, and differentiation of cardiac fibroblasts and cardiomyocytes. Moreover, MSCs- and CPCs-derived exosomes can enhance the migration of endothelial cells. Exosome-based therapy approaches augment the cardiac function by multiple means, such as reducing fibrosis, stimulation of vascular angiogenesis, and proliferation of cardiomyocytes that result in replacing damaged heart tissue with newly generated functional myocytes. This review article aims to briefly discuss the recent advancements in the role of secreted exosomes in myocardial repair by focusing on cardiac cells-derived exosomes.     

Combination of G-CSF Administration and Human Amniotic Fluid Mesenchymal Stem Cell Transplantation Promotes Peripheral Nerve Regeneration

Amniotic fluid mesenchymal stem cells (AFS) harbor the potential to improve peripheral nerve injury by inherited neurotrophic factor secretion, but present the drawback of the short-term survival after transplantation. Granulocyte-colony stimulating factor (G-CSF) has a diversity of functions, including anti-inflammatory and anti-apoptotic effects. This study was conducted to evaluate whether G-CSF could augment the neuroprotective effect of transplanted AFS against peripheral nerve injury. The potential involvement of anti-inflammation/anti-apoptosis effect was also investigated. Peripheral nerve injury was produced in Sprauge-Dawley rats by crushing left sciatic nerve using a vessel clamp. The AFS were embedded in fibrin glue and delivered to the injured site. G-CSF (50 μg/kg) was administrated by intra-peritoneal injection for 7 consecutive days. Cell apoptosis, inflammatory cytokines, motor function, and nerve regeneration were evaluated 7 or 28 days after injury. Crush injury induced inflammatory response, disrupted nerve integrity, and impaired nerve function in sciatic nerve. Crush injury-provoked inflammation was attenuated in groups receiving G-CSF but not in AFS only group. In transplanted AFS, marked apoptosis was detected and this event was reduced by G-CSF treatment. Increased nerve myelination and improved motor function were observed in AFS transplanted, G-CSF administrated, and AFS/G-CSF combined treatment groups. Significantly, the combined treatment showed the most beneficial effect. In conclusion, the concomitant treatment of AFS with G-CSF augments peripheral nerve regeneration which may involve the suppression of apoptotic death in implanted AFS and the attenuation of inflammatory response.