Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.     

Perfusion bioreactor-based cryopreservation of 3D human mesenchymal stromal cell tissue grafts

The generation of an off-the-shelf in vitro engineered living tissue graft will likely require cryopreservation. However, the efficient addition and removal of cryoprotective agents (CPA) to cells throughout the volume of a three-dimensional (3D) tissue graft remains a significant challenge. In this work, we assessed whether a perfusion bioreactor-based method could be used to improve the viability of cryopreserved mesenchymal stromal cell- (MSC) based tissue constructs as compared to using conventional diffusion-based methods. The bioreactor was first used to saturate 3D constructs with CPA under perfused flow. Following cryopreservation, the bioreactor was then also used for the efficient removal of the CPA from the 3D tissues. We demonstrate that addition and removal of CPA under perfused flow significantly increased the viability of MSC within cryopreserved 3D tissue constructs as compared to conventional diffusion-based methods.     

Human mesenchymal stem cells tissue development in 3D PET matrices

Human mesenchymal stem cells (hMSCs) are attractive cell sources for engineered tissue constructs with broad therapeutic potential. Three-dimensional (3D) hMSC tissue development in nonwoven poly(ethylene terephthalate) (PET) fibrous matrices was investigated. HMSCs were seeded onto 3D PET scaffolds and were cultured for over 1 month. Their proliferation rates were affected by seeding density but remained much lower than those of 2D controls. Compared to 2D surfaces, hMSCs grown in 3D scaffolds secreted and embedded themselves in an extensive ECM network composed of collagen I, collagen IV, fibronectin, and laminin. HMSCs were influenced by the orientation of adjacent PET fibers to organize the ECM proteins into highly aligned fibrils. We observed the increased expressions of alpha(2)beta(1) integrin but a slight decrease in the expression of alpha(5)beta(1) integrin in 3D compared to 2D culture and found that alpha(V)beta(3) was expressed only in 2D. Paxillin expression was down-regulated in 3D culture with a concomitant change in its localization patterns. We demonstrated the multi-lineage potentials of the 3D tissue constructs by differentiating the cells grown in the scaffolds into osteoblasts and adipocytes. Taken together, these results showed that hMSCs grown in 3D scaffolds display tissue development patterns distinct from their 2D counterparts and provide important clues for designing 3D scaffolds for developing tissue engineered constructs.     

Characterization of electrospun nanocomposite scaffolds and biocompatibility with adipose-derived human mesenchymal stem cells

Electrospun nanocomposite scaffolds were fabricated by encapsulating multi-walled carbon nanotubes (MWNT) in poly (lactic acid) (PLA) nanofibers. Scanning electron microscopy (SEM) confirmed the fabrication of nanofibers, and transmission electron microscopy identified the alignment and dispersion of MWNT along the axis of the fibers. Tensile testing showed an increase in the tensile modulus for a MWNT loading of 0.25 wt% compared with electrospun nanofibrous mats without MWNT reinforcement. Conductivity measurements indicated that the confined geometry of the fibrous system requires only minute doping to obtain significant enhancements at 0.32 wt%. Adipose-derived human mesenchymal stem cells (hMSCs) were seeded on electrospun scaffolds containing 1 wt% MWNT and 0 wt% MWNT, to determine the efficacy of the scaffolds for cell growth, and the effect of MWNT on hMSC viability and proliferation over two weeks in culture. Staining for live and dead cells and DNA quantification indicated that the hMSCs were alive and proliferating through day 14. SEM images of hMSCs at 14 days showed morphological differences, with hMSCs on PLA well spread and hMSCs on PLA with 1% MWNT closely packed and longitudinally aligned.     

分离与培养人脂肪组织间充质干细胞的研究

目的：探索人脂肪组织源性间充质干细胞（ASCs）的分离、体外培养,为其广泛应用提供实验依据。方法：无菌条件下获取腹部手术病人皮下脂肪组织,酶消化法分离、培养ASCs,观察细胞形态并绘制细胞生长曲线,计算细胞群体倍增时间;对第2代细胞进行免疫组织化学染色,鉴定其表面分子CD44表达;取2—4代细胞用含体积分数为10％胎牛血清、1％青链霉素原液、1μmmol／L地塞米松、10μmmol／L胰岛素、0．5mmmol／LIBMX的高糖DMEM培养基中诱导培养一周,观察细胞形态变化,并用油红“O”染色定性。结果：人脂肪组织中含有大量间充质干细胞,呈成纤维细胞样贴壁生长,细胞群体倍增时间为55h左右;免疫化学染色鉴定CD44阳性;成脂诱导分化一周,可见细胞内有大量脂滴,油红“0”染色可见胞浆内有大量红染颗粒。结论：建立了一种自人体脂肪组织分离,培养ASCs经济简便的方法,为其能够作为组织工程理想的种子细胞及广泛应用于临床提供实验依据。     

3D spheroids of human placenta-derived mesenchymal stem cells attenuate spinal cord injury in mice

Mesenchymal stem cell (MSC) is an absorbing candidate for cell therapy in treating spinal cord injury (SCI) due to its great potential for multiple cell differentiation, mighty paracrine secretion as well as vigorous immunomodulatory effect, of which are beneficial to the improvement of functional recovery post SCI. However, the therapeutic effects of MSC on SCI have been limited because of the gradual loss of MSC stemness in the process of expanding culture. Therefore, in this study, we aimed to maintain those beneficial properties of MSC via three-dimensional spheroid cell culture and then compared them with conventionally-cultured MSCs in the treatment of SCI both in vitro and in vivo with the aid of two-photon microscope. We found that 3D human placenta-derived MSCs (3D-HPMSCs) demonstrated a significant increase in secretion of anti-inflammatory factors and trophic factors like VEGF, PDGF, FGF via QPCR and Bio-Plex assays, and showed great potentials on angiogenesis and neurite morphogenesis when co-cultured with HUVECs or DRGs in vitro. After transplantation into the injured spinal cord, 3D-HPMSCs managed to survive for the entire experiment and retained their advantageous properties in secretion, and exhibited remarkable effects on neuroprotection by minimizing the lesion cavity, inhibiting the inflammation and astrogliosis, and promoting angiogenesis. Further investigation of axonal dieback via two-photon microscope indicated that 3D-HPMSCs could effectively alleviate axonal dieback post injury. Further, mice only treated with 3D-HPMSCs obtained substantial improvement of functional recovery on electrophysiology, BMS score, and Catwalk analysis. RNA sequencing suggested that the 3D-HPMSCs structure organization-related gene was significantly changed, which was likely to potentiate the angiogenesis and inflammation regulation after SCI. These results suggest that 3D-HPMSCs may hold great potential for the treatment of SCI.Subject terms: Spinal cord injury, Mesenchymal stem cells     

Role of nanotopography in the development of tissue engineered 3D organs and tissues using mesenchymal stem cells

Recent regenerative medicine and tissue engineering strategies(using cells, scaffolds, medical devices and gene therapy) have led to fascinating progress of translation of basic research towards clinical applications. In the past decade, great deal of research has focused on developing various three dimensional(3D) organs, such as bone, skin, liver, kidney and ear,using such strategies in order to replace or regenerate damaged organs for the purpose of maintaining or restoring organs’ functions that may have been lost due to aging, accident or disease. The surface properties of a material or a device are key aspects in determining the success of the implant in biomedicine, as the majority of biological reactions in human body occur on surfaces or interfaces. Furthermore, it has been established in the literature that cell adhesion and proliferation are, to a great extent, influenced by the micro- and nanosurface characteristics of biomaterials and devices. In addition, it has been shown that the functions of stem cells, mesenchymal stem cells in particular, could be regulated through physical interaction with specific nanotopographical cues. Therefore, guided stem cell proliferation, differentiation and function are of great importance in the regeneration of 3D tissues and organs using tissue engineering strategies. This review will provide an update on the impact of nanotopography on mesenchymal stem cells for the purpose of developing laboratory-based 3D organs and tissues, as well as the most recent research and case studies on this topic.     

Hypoxia enhances proliferation and tissue formation of human mesenchymal stem cells

Changes in oxygen concentrations affect many of the innate characteristics of stem and progenitor cells. Human mesenchymal stem cells (hMSCs) were maintained under hypoxic atmospheres (2% O(2)) for up to seven in vitro passages. This resulted in approximately 30-fold higher hMSC expansion over 6 weeks without loss of multi-lineage differentiation capabilities. Under hypoxia, hMSCs maintained their growth-rates even after reaching confluence, resulting in the formation of multiple cell layers. Hypoxic hMSCs also displayed differences in the cell and nuclear morphologies as well as enhanced ECM formation and organization. These changes in cellular characteristics were accompanied by higher mRNA levels of Oct-4 and HIF-2alpha, as well as increased expression levels of connexin-43, a protein used in gap junction formation. The results from this study demonstrated that oxygen concentrations affected many aspects of stem-cell physiology, including growth and in vitro development, and may be a critical parameter during expansion and differentiation.     

Involvement of stretch-activated ion channels in strain-regulated glycosaminoglycan synthesis in mesenchymal stem cell-seeded 3D scaffolds

The objective of this study was to determine if cyclic tensile strain would regulate the rate of glycosaminoglycan synthesis via stretch-activated ion channels in adult mesenchymal stem cells seeded in a collagen type I-glycosaminoglycan scaffold and treated with TGF-beta1. The application of 10% cyclic tensile loading at 1Hz for 7 days significantly increased the rate of glycosaminoglycan synthesis, as assessed using [(35)S] sulphate incorporation. This increase was attenuated in the presence of a stretch-activated ion channel inhibitor (10microM gadolinium chloride) demonstrating the involvement, in part, of these ion channels in the mechanotransduction pathway that couples cyclic tensile loading to matrix synthesis.     

Isolation and identification of mesenchymal stem cells from human lipoma tissue

Lipoma is a benign neoplasm of normal fat cells that appears as a soft, movable swelling, often with a slight yellowish coloration. Human mesenchymal stem cells (MSCs) that have been isolated from bone marrow, blood, and other adult tissues including adipose tissue have the potential to be useful candidates for therapy. No literature had reported about stem cells from lipoma tissue. Here, a new cell culture method is described and utilized to greatly accelerate the growth rate and prolong the lifespan of lipoma-derived MSCs. Cells produced in early cultures display characteristics similar to those previously reported for multipotential stem cells, including a high frequency of anchorage-independent growth in soft agar and a lack of gap junctional intercellular communication in cell types with serpiginous morphology. These cells can differentiate into adipocytes, osteoblasts, and chondrocytes after induction. In conclusion, lipoma-derived stem cells possessing the characteristics of MSCs are described for the first time.     

Cell culture density affects the proliferation activity of human adipose tissue stem cells

In this study, we investigated the effect of cell density on the proliferation activity of human mesenchymal stem cells (MSCs) derived from adipose tissue (AT‐MSCs) over time in culture. Passage #4 (P4) and #12 (P12) AT‐MSCs from two donors were plated at a density of 200 (culture condition 1, CC1) or 5000 (culture condition 2, CC2) cells cm^?2. After 7 days of incubation, P4 and P12 AT‐MSCs cultured in CC1 were thin and spindle‐shaped, whereas those cultured in CC2 had extensive cell‐to‐cell contacts and an expanded cell volume. In addition, P4 and P12 AT‐MSCs in CC1 divided more than three times, while those in CC2 divided less than once on average. Flow cytometric analysis using 5(6)‐carboxyfluorescein diacetate N‐succinimidyl ester dye showed that the fluorescence intensity of AT‐MSCs was lower in CC1 than in CC2. Furthermore, expression of proliferation‐associated genes, such as CDC45L, CDC20A and KIF20A, in P4 AT‐MSCs was higher in CC1 than in CC2, and this difference was also observed in P12 AT‐MSCs. These data demonstrated that cell culture density affects the proliferation activity of MSCs, suggesting that it is feasible to design a strategy to prepare suitable MSCs using specific culture conditions. Copyright © 2016 John Wiley & Sons, Ltd.     

人脐带间充质干细胞在组织工程中的研究进展

人脐带间充质干细胞是一类具有自我更新、增殖和多向分化潜能的干细胞,具有来源广泛、易于采集、保存和运输、无异体排斥、避免伦理争议等诸多优点.流式细胞仪分析发现人脐带间充质干细胞高表达间质细胞标志(CD44、CD105)、整合素受体(CD29、CD49b、CD49c、CD51),不表达造血系标志(CD34、CD45)人白细胞抗原HLA-DR和内皮细胞标志CD31.人脐带间充质干细胞在体内外可以分化为骨细胞、软骨细胞、肝细胞、心肌细胞、骨骼肌细胞以及神经元细胞等.目前人脐带间充质干细胞在组织工程骨、人工血管以及基因治疗等临床应用研究中已逐渐深入,并已显示出广阔的应用前景.本文就人脐带间充质干细胞的生物学特性及其在组织工程中的研究作一综述.     

Donor age of human platelet lysate affects proliferation and differentiation of mesenchymal stem cells

The regenerative potential declines upon aging. This might be due to cell-intrinsic changes in stem and progenitor cells or to influences by the microenvironment. Mesenchymal stem cells (MSC) raise high hopes in regenerative medicine. They are usually culture expanded in media with fetal calf serum (FCS) or other serum supplements such as human platelet lysate (HPL). In this study, we have analyzed the impact of HPL-donor age on culture expansion. 31 single donor derived HPLs (25 to 57 years old) were simultaneously compared for culture of MSC. Proliferation of MSC did not reveal a clear association with platelet counts of HPL donors or growth factors concentrations (PDGF-AB, TGF-β1, bFGF, or IGF-1), but it was significantly higher with HPLs from younger donors (<35 years) as compared to older donors (>45 years). Furthermore, HPLs from older donors increased activity of senescence-associated beta-galactosidase (SA-βgal). HPL-donor age did not affect the fibroblastoid colony-forming unit (CFU-f) frequency, immunophenotype or induction of adipogenic differentiation, whereas osteogenic differentiation was significantly lower with HPLs from older donors. Concentrations of various growth factors (PDGF-AB, TGF-β1, bFGF, IGF-1) or hormones (estradiol, parathormone, leptin, 1,25 vitamin D3) were not associated with HPL-donor age or MSC growth. Taken together, our data support the notion that aging is associated with systemic feedback mechanisms acting on stem and progenitor cells, and this is also relevant for serum supplements in cell culture: HPLs derived from younger donors facilitate enhanced expansion and more pronounced osteogenic differentiation.     

Functional studies of mesenchymal stem cells derived from adult human adipose tissue

Recent evidence suggests that cells with the properties of human mesenchymal stem cells (hMSCs) can be derived from adult peripheral tissues, including adipose tissue, muscle and dermis. We isolated hMSCs from the stromal-vascular portion of subcutaneous adipose tissue from seven adult subjects. These cells could be readily differentiated into cells of the chondrocyte, osteocyte and adipocyte lineage demonstrating their multipotency. We studied the functional properties of hMSCs-derived adipocytes and compared them with adipocytes differentiated from hMSCs obtained from bone marrow (BM-hMSC). The two cell types displayed similar lipolytic capacity upon stimulation with catecholamines, including a pronounced antilipolytic effect mediated through alpha2A-adrenoceptors, a typical trait in human but not rodent fat cells. Furthermore, both cell types secreted the fat cell-specific factors leptin and adiponectin in comparable amounts per time unit. The fat tissue-derived hMSCs retained their differentiation capacity up to at least fifteen passages. We conclude that hMSCs derived from adult human adipose tissue can be differentiated into fully functional adipocytes with a similar, if not identical, phenotype as that observed in cells derived from BM-hMSCs. Human adipose-tissue-derived MSCs could therefore constitute an efficient and easily obtainable renewable cellular source for studies of adipocyte biology.     

Various seeding methods for tissue development of human umbilical-cord-derived mesenchymal stem cells in 3-dimensional PET matrix

Mesenchymal stem cells derived from human umbilical cords (hUCMSCs) are attractive as a new cell source for tissue engineering. It is essential to investigate and optimize the seeding process of these cells for the success of cell culture and tissue regeneration in vitro. In this study, a static seeding method (SSM), a centrifugal seeding method (CSM), and a novel method-cycling filtration seeding method (CFSM) are evaluated in terms of seeding efficiency, cell damage, and distribution inside the scaffolds, cell proliferation, and osteogenic differentiation. Cells were seeded on three-dimensional (3-D) nonwoven PET discs at a density of 1×10⁴ cells/disc, followed by 21 days of cell culture and 20 days of osteogenic differentiation. Cells grown in 3-D conditions exhibited higher metabolic activity than those grown on a 2-D control surface. The CSM and CFSM groups showed higher seeding efficiency, proliferation capacity, and differentiation potential. H&E staining indicated a more uniform spatial distribution of cells in CFSM groups. LDH level measurements suggested that more cell damage was caused by the CFSM process. Above all, the results showed that the cells maintained their proliferation ability and differentiation potential ex vivo during approximately 7 weeks of culture. The CSM and CFSM are recommended for hUCMSC tissue engineering, although the seeding parameters still require further investigation and optimization.