Vitreous cryopreservation of tissue engineered bone composed of bone marrow mesenchymal stem cells and partially demineralized bone matrix

Cryopreservation of tissue engineered products by maintaining their structure and function is a prerequisite for large-scale clinical applications. In this study, we examined the feasibility of cryopreservation of tissue engineered bone (TEB) composed of osteo-induced canine bone marrow mesenchymal stem cells (cBMSCs) and partially demineralized bone matrix (pDBM) scaffold by vitrification. A novel vitreous solution named as VS442 containing 40% dimethyl-sulfoxide (DMSO), 40% EuroCollins (EC) solution and 20% basic culture medium (BCM) was developed. After being cultured in vitro for 8 days, cell/scaffold complex in VS442 was subjected to vitreous preservation for 7 days and 3 months, respectively. Cell viability, proliferation and osteogenic differentiation of cBMSCs in TEB after vitreous cryopreservation were examined with parallel comparisons being made with those cryopreserved in VS55 vitreous solution. Compared with that cryopreserved in VS55, cell viability and subsequent proliferative ability of TEB in VS442 after being rewarmed were significantly higher as detected by live/dead staining and DNA assay. The level of alkaline phosphatase (ALP) expression and osteocalcin (OCN) deposition in VS442 preserved TEB was also higher than those in the VS55 group since 3 days post-rewarm. Both cell viability and osteogenic capability of the VS55 group were found to be declined to a negligible level within 15 days post-rewarm. Furthermore, it was observed that extending the preservation of TEB in VS442 to 3 months did not render any significant effect on its survival and osteogenic potential. Thus, the newly developed VS442 vitreous solution was demonstrated to be more efficient in maintaining cellular viability and osteogenic function for vitreous cryopreservation of TEB over VS55.     

The effect of medium composition on deposition of collagen type 1 and expression of osteogenic genes in mesenchymal stem cells derived from human adipose tissue and bone marrow

The two mesenchymal stem cell (MSC) populations that have gained most attention in relation to bone tissue engineering are adipose tissue (AT) MSCs and bone marrow (BM) MSCs. The purpose of this study was to investigate the ability of human BM-MSCs and AT-MSCs to survive, proliferate and deposit collagen type 1 when cultured on polycaprolactone nanofiber scaffolds and to ascertain the effect of medium composition on collagen type 1 formation and expression of osteogenic genes. The cells were seeded on polycaprolactone nanofiber scaffolds and cultured in three different types of media that differed by the presence of ascorbic acid, β-glycerophosphate and dexamethasone, that are typical components used for osteogenic differentiation of MSCs in vitro.In summary, AT-MSCs were proliferating significantly faster than BM-MSCs. AT-MSCs also showed better ability to deposit collagen type 1 and had a higher expression of early osteogenic markers, whereas BM-MSCs had higher expression of late osteogenic markers. This suggests that MSCs from diverse sources have different attributes and with respect to osteogenic differentiation, AT-MSCs are more immature compared to BM-MSCs. Collagen formation was depending on medium composition and the organization of collagen type 1 appeared to be influenced by the presence of dexamethasone.     

Cryopreservation of hMSCs seeded silk nanofibers based tissue engineered constructs

Long term cryopreservation of tissue engineering constructs is of paramount importance to meet off-the shelf requirements for medical applications. In the present study, the effect of cryopreservation using natural osmolytes such as trehalose and ectoin with and without conventional Me₂SO on the cryopreservation of tissue engineered constructs (TECs) was evaluated. MSCs derived from umbilical cord were seeded on electrospun nanofibrous silk fibroin scaffolds and cultured to develop TECs. TECs were subjected to controlled rate freezing using nine different freezing solutions. Among these, freezing medium consisting of natural osmolytes like trehalose (40 mM), ectoin (40 mM), catalase (100 μg) as antioxidant and Me₂SO (2.5%) was found to be the most effective. Optimality of the chosen cryoprotectants was confirmed by cell viability (PI live/dead staining), cell proliferation (MTT assay), microstructure analysis (SEM), membrane integrity (confocal microscopy) and in vitro osteogenic differentiation (ALP assay, RT-PCR and histology) study carried out with post-thaw cryopreserved TECs. The mechanical integrity of the cryopreserved scaffold was found to be unaltered.     

Cryopreservation of rat MSCs by use of a programmed freezer with magnetic field

Mesenchymal stem cells (MSCs) can be used for the regeneration of various tissues and cryopreservation of MSCs is so important for regenerative medicine. The purpose of this study was to evaluate the influences of cryopreservation on MSCs by use of a programmed freezer with a magnetic field (CAS freezer). MSCs were isolated from bone marrow of rat femora. The cells were frozen by a CAS freezer with 10% dimethyl sulfoxide (Me2SO) and cryopreserved for 7 days at a temperature of −150 °C. Immediately after thawing, the number of survived cells was counted. The cell proliferation also examined after 48 h culture. Next, MSCs were frozen by two different freezers; CAS freezer and a conventional programmed freezer without magnetic field. Then, osteogenic and adipogenic differentiations of cryopreserved cells were examined. As a result, survival and proliferation rates of MSCs were significantly higher in CAS freezer than in the non-magnetic freezer. Alizarin positive reaction, large amount of calcium quantification, and greater alkaline phosphatase activity were shown in both the non-cryopreserved and CAS groups after osteogenic differentiation. Moreover, Oil Red O staining positive reaction and high amount of PPARγ and FABP4 mRNAs were shown in both the non-cryopreserved and CAS groups after adipogenic differentiation. From these findings, it is shown that a CAS freezer can maintain high survival and proliferation rates of MSCs and maintain both adipogenic and osteogenic differentiation abilities. It is thus concluded that CAS freezer is available for cryopreservation of MSCs, which can be applied to various tissue regeneration.     

Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells

In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds (Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy.     

The effects of β-tricalcium phosphate 3D scaffold in-situ cryopreservation on the migration rate and osteogenic ability of mesenchymal stem cells

To readily supply seed cells for tissue engineering and ensure their constant availability for experiments, it is imperative to establish an in-situ cryopreservation method for cell storage. We investigated the effects of a β-tricalcium phosphate (β-TCP) 3D scaffold in-situ cryopreservation method on the migration rate and osteogenic ability of mesenchymal stem cells (MSCs). Compared to using a 2D plate culture and trypsinized cryopreservation, MSCs on β-TCP 3D scaffolds demonstrated a higher amplification rate, and the harvest and survival rates (HSR) increased from 55.9 to 81.3% when the 3D in-situ cryopreservation method was applied. The cell migration rate and alkaline phosphatase (ALP) activity were unaffected after in-situ cryopreservation, and unexpectedly, the Specific ALP activity of migrating cells was higher than that of non-cryopreserved cells, suggesting that the cell-scaffold combination could be cryopreserved using the present protocol without loss of proliferative or osteogenic potential. These findings highlight a methodology for 3D scaffold in-situ cryopreservation and passage for MSC production in bone tissue engineering, and present the possibility of designing a perfusion cells/scaffold factory for scale-up production.     

Long bone mesenchymal stem cells (Lb-MSCs): clinically reliable cells for osteo-diseases

Mesenchymal stem cells (MSCs) have been designated as the most reliable cells in clinics to treat osteo-diseases because of their versatile nature. MSCs, isolated from long bone (Lb-MSCs) are rarely reported and named as RIA-MSCs because of the reamer–irrigator–aspirator (RIA) device. The potential of these cells in the treatment of non-union bone fractures made them the ideal candidates to be studied for clinical practices. In this work, effect of cryopreservation on the proliferation and differentiation capabilities of long bone MSCs (Lb-MSCs) has been studied. For this purpose, Lb-MSCs were isolated via RIA device and characterized using flow cytometry and differentiation assays. Cells were cryopreserved for 3, 6 and 12 months and thereafter were characterized using differentiation assays and genetic markers specific for osteogenic, chondrogenic, and adipogenic potential quantitatively by qRT-PCR. Lb-MSCs were found expressing MSC characteristic markers defining their identity. The population doubling time (PDT) was about 2.5 ± 0.5 days and colonies appeared after 7–10 days. Differentiation potential and gene expression of 3, 6 and 12 months cryopreserved Lb-MSCs were unaltered. The results show that cryopreservation did not have an effect on the differentiation potential of human Lb-MSCs. Therefore, our work offers Lb-MSCs as clinically cells for treating osteo-diseases.     

The biocompatibility of calcium phosphate cements containing alendronate-loaded PLGA microparticles in?vitro

The composite of poly-lactic-co-glycolic acid (PLGA) and calcium phosphate cements (CPC) are currently widely used in bone tissue engineering. However, the properties and biocompatibility of the alendronate-loaded PLGA/CPC (APC) porous scaffolds have not been characterized. APC scaffolds were prepared by a solid/oil/water emulsion solvent evaporation method. The morphology, porosity, and mechanical strength of the scaffolds were characterized. Bone marrow mesenchymal stem cells (BMSCs) from rabbit were cultured, expanded and seeded on the scaffolds, and the cell morphology, adhesion, proliferation, cell cycle and osteogenic differentiation of BMSCs were determined. The results showed that the APC scaffolds had a porosity of 67.43 ± 4.2% and pore size of 213 ± 95 µm. The compressive strength for APC was 5.79 ± 1.21 MPa, which was close to human cancellous bone. The scanning electron microscopy, cell counting kit-8 assay, flow cytometry and ALP activity revealed that the APC scaffolds had osteogenic potential on the BMSCs in vitro and exhibited excellent biocompatibility with engineered bone tissue. APC scaffolds exhibited excellent biocompatibility and osteogenesis potential and can potentially be used for bone tissue engineering.     

Crosstalks between integrin alpha 5 and IGF2/IGFBP2 signalling trigger human bone marrow-derived mesenchymal stromal osteogenic differentiation

Background  

The potential of mesenchymal stromal cells (MSCs) to differentiate into functional bone forming cells provides an important tool for bone regeneration. The identification of factors that trigger osteoblast differentiation in MSCs is therefore critical to promote the osteogenic potential of human MSCs. In this study, we used microarray analysis to identify signalling molecules that promote osteogenic differentiation in human bone marrow stroma derived MSCs.     

Assessment of the proliferation of human mesenchymal stromal cells in the presence of human demineralised bone matrix

Tissue engineering integrates discoveries from biochemistry, cell and molecular biology, material science and biomedical engineering to produce innovative three-dimensional composites that can be used to replace or correct damaged tissues and organs. Precise classification of osteoinductive properties of human demineralised bone is often the problem, because it varies from batch to batch. An in vitro assay using bone marrow derived human mesenchymal stromal cells (hMSCs) was developed to improve the classification of the osteoinductive quality of demineralised bone matrix. In this study, three-dimensional, partially demineralised bone scaffolds were investigated for their ability to induce osteogenic differentiation of hMSCs in vitro. Proliferation of the hMSCs was measured by the CelTiter 96? AQueous One Solution Cell assay. Chemical structure was evaluated using quantitative and qualitative X-ray analysis. Scanning electron microscopy revealed primary proliferation of the cells cultivated 14 days and showed elevations in the content of Ca²⁺. These results demonstrate that partially demineralised human bone material supports osteogenic differentiation of hMSCs in vitro. This study documents that in vitro test using hMSCs can be used for classification of the osteoinductive quality of human demineralised bone matrix.     

Osteogenic differentiation of GFP-labeled human umbilical cord blood derived mesenchymal stem cells after cryopreservation

The osteogenic capacity of human umbilical cord blood derived mesenchymal stem cells (UCB-MSCs) has been demonstrated both in vitro and in vivo. Therefore, cell labeling and storage are becoming necessary for researching the potential therapeutic use of UCB-MSCs for bone tissue engineering. The aim of this study was to determine the effect of cryopreservation on the osteogenic differentiation of green fluorescent protein (GFP)-marked UCB-MSCs in vitro. MSCs were isolated from full-term human UCB, expanded, transfected with the GFP gene, and then cryopreserved in liquid nitrogen for 4 weeks. After thawing, cell surface antigen markers and osteogenic potential were analyzed, and the luminescence of these cells was observed by fluorescence microscopy. The results demonstrate that cryopreservation has no effect on the cell phenotype, GFP expression or osteogenic differentiation of UCB-MSCs, showing that cryopreserved GFP-labeled UCB-MSCs might be applied for bone tissue engineering.     

Proliferation of canine bone marrow derived mesenchymal stem cells on different nanomaterial based thin film scaffolds

Stem cell niche research uses nanotechnologies to mimic the extra-cellular microenvironment to promote proliferation and differentiation. The aim of designing different scaffolds is to simulate the best structural and environmental pattern for extracellular matrix. This experiment was designed to study the proliferative behaviour of canine bone marrow deriver mesenchymal stem cells (MSCs) on different nanomaterial based thin film scaffolds of carbon nanotubes (CNT), chitosan and poly ε-caprolactone. Similar number of cells was seeded on the scaffolds and standard cell culture flask, taken as control. Cells were maintained on DMEM media and relative number of metabolically active cells was determined by MTT assay up to day six of culture. Cells proliferated on control and all the scaffolds as the days progressed. Although proliferation rate was slow but no decline of cell number was noticed on the scaffolds during the study period. Initially, the cell proliferation was lower on CNT but as time progressed no significant difference was observed compared to control. The result indicated that nanomaterial based scaffolds reduce the proliferation rate of canine MSCs. However, canine MSCs adapted and proliferated better on CNT substrate in vitro and may be used as a scaffold component in canine tissue engineering in future.     

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells(MSCs).Although MSCs are most commonly grown in media containing fetal calf serum,human platelet lysate(PL) offers an effective alternative.Bone marrow- derived MSCs grown in PLcontaining media display faster proliferation whilst maintaining good osteogenic differentiation capacity.Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo.In an alternative approach,nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices.Even though methods to coat scaffolds with PL vary,in vitro studies suggest that PL allows for MSC adhesion,migration and differentiation inside these scaffolds.Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo.This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration.To minimise inconsistency between the studies,further work is required towards standardisation of PL preparation in terms of the starting material,platelet concentration,leukocyte depletion,and the method of platelet lysis.PL quality control procedures and its "potency" assessment are urgently needed,which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation.Furthermore,different PL formulations could be tailor-made for specific bone repair indications.Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.     

Synergistic Actions of Hematopoietic and Mesenchymal Stem/Progenitor Cells in Vascularizing Bioengineered Tissues

Poor angiogenesis is a major road block for tissue repair. The regeneration of virtually all tissues is limited by angiogenesis, given the diffusion of nutrients, oxygen, and waste products is limited to a few hundred micrometers. We postulated that co-transplantation of hematopoietic and mesenchymal stem/progenitor cells improves angiogenesis of tissue repair and hence the outcome of regeneration. In this study, we tested this hypothesis by using bone as a model whose regeneration is impaired unless it is vascularized. Hematopoietic stem/progenitor cells (HSCs) and mesenchymal stem/progenitor cells (MSCs) were isolated from each of three healthy human bone marrow samples and reconstituted in a porous scaffold. MSCs were seeded in micropores of 3D calcium phosphate (CP) scaffolds, followed by infusion of gel-suspended CD34⁺ hematopoietic cells. Co-transplantation of CD34⁺ HSCs and CD34⁻ MSCs in microporous CP scaffolds subcutaneously in the dorsum of immunocompromized mice yielded vascularized tissue. The average vascular number of co-transplanted CD34⁺ and MSC scaffolds was substantially greater than MSC transplantation alone. Human osteocalcin was expressed in the micropores of CP scaffolds and was significantly increased upon co-transplantation of MSCs and CD34⁺ cells. Human nuclear staining revealed the engraftment of transplanted human cells in vascular endothelium upon co-transplantation of MSCs and CD34⁺ cells. Based on additional in vitro results of endothelial differentiation of CD34⁺ cells by vascular endothelial growth factor (VEGF), we adsorbed VEGF with co-transplanted CD34⁺ and MSCs in the microporous CP scaffolds in vivo, and discovered that vascular number and diameter further increased, likely owing to the promotion of endothelial differentiation of CD34⁺ cells by VEGF. Together, co-transplantation of hematopoietic and mesenchymal stem/progenitor cells may improve the regeneration of vascular dependent tissues such as bone, adipose, muscle and dermal grafts, and may have implications in the regeneration of internal organs.     

The Osteogenic Potential of Mesoporous Bioglasses/Silk and Non-Mesoporous Bioglasses/Silk Scaffolds in Ovariectomized Rats: In vitro and In vivo Evaluation

Silk-based scaffolds have been introduced to bone tissue regeneration for years, however, their local therapeutic efficency in bone metabolic disease condition has been seldom reported. According to our previous report, mesoporous bioactive glass (MBG)/silk scaffolds exhibits superior in vitro bioactivity and in vivo osteogenic properties compared to non-mesoporous bioactive glass (BG)/silk scaffolds, but no information could be found about their efficiency in osteoporotic (OVX) environment. This study investigated a biomaterial-based approach for improving MSCs behavior in vitro, and accelerating OVX defect healing by using 3D BG/silk and MBG/silk scaffolds, and pure silk scaffolds as control. The results of SEM, CCK-8 assay and quantitative ALP activity showed that MBG/silk scaffolds can improve attachment, proliferation and osteogenic differentiation of both O-MSCs and sham control. In vivo therapeutic efficiency was evaluated by μCT analysis, hematoxylin and eosin staining, safranin O staining and tartrate-resistant acid phosphatase, indicating accelerated bone formation with compatible scaffold degradation and reduced osteoclastic response of defect healing in OVX rats after 2 and 4 weeks treatment, with a rank order of MBG/silk > BG/silk > silk group. Immunohistochemical markers of COL I, OPN, BSP and OCN also revealed that MBG/silk scaffolds can better induce accelerated collagen and non-collagen matrix production. The findings of this study suggest that MBG/silk scaffolds provide a better environment for cell attachment, proliferation and differentiation, and act as potential substitute for treating local osteoporotic defects.     

TLR4 Activation Promotes Bone Marrow MSC Proliferation and Osteogenic Differentiation via Wnt3a and Wnt5a Signaling

Mesenchymal stem cells (MSCs) from adult bone marrow maintain their self-renewal ability and the ability to differentiate into osteoblast. Thus, adult bone marrow MSCs play a key role in the regeneration of bone tissue. Previous studies indicated that TLR4 is expressed in MSCs and is critical in regulating the fate decision of MSCs. However, the exact functional role and underlying mechanisms of how TLR4 regulate bone marrow MSC proliferation and differentiation are unclear. Here, we found that activated TLR4 by its ligand LPS promoted the proliferation and osteogenic differentiation of MSCs in vitro. TLR4 activation by LPS also increased cytokine IL-6 and IL-1β production in MSCs. In addition, LPS treatment has no effect on inducing cell death of MSCs. Deletion of TLR4 expression in MSCs completely eliminated the effects of LPS on MSC proliferation, osteogenic differentiation and cytokine production. We also found that the mRNA and protein expression of Wnt3a and Wnt5a, two important factors in regulating MSC fate decision, was upregulated in a TLR4-dependent manner. Silencing Wnt3a with specific siRNA remarkably inhibited TLR4-induced MSC proliferation, while Wnt5a specific siRNA treatment significantly antagonized TLR4-induced MSC osteogenic differentiation. These results together suggested that TLR4 regulates bone marrow MSC proliferation and osteogenic differentiation through Wnt3a and Wnt5a signaling. These finding provide new data to understand the role and the molecular mechanisms of TLR4 in regulating bone marrow MSC functions. These data also provide new insight in developing new therapy in bone regeneration using MSCs by modulating TLR4 and Wnt signaling activity.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

Steroid regulation of proliferation and osteogenic differentiation of bone marrow stromal cells: A gender difference

Bone marrow mesenchymal stem cells (MSCs) are considered a potential cell source for stem cell-based bone tissue engineering. However, noticeable limitations of insufficient supply and reduction of differentiation potential impact the feasibility of their clinical application. This study investigated the in vitro function of steroids and gender differences on the proliferation and differentiation of rat MSCs. Bone marrow MSCs of age-matched rats were exposed to proliferation and osteogenic differentiation media supplements with various concentrations of 17β-estradiol (E2) and dexamethasone. Cell proliferation was measured by MTS assay; osteogenic markers and steroid-associated growth factors and receptors were evaluated by ELISA and real-time PCR. The results revealed that supplements of E2 and dexamethasone increase MSC proliferation in a biphasic manner. The optimal dose and interaction of steroids required to improve MSC proliferation effectively varied depending on the gender of donors. Supplementation of E2 effectively improves osteogenic differentiation markers including ALP, osteocalcin and calcium levels for MSCs isolated from both male and female donors. The mRNA of TGF-β1 and BMP-7 are also up-regulated. However, effective doses to maximally improve osteogenic potentials and growth factors for MSCs are different between male and female donors. The relationship between steroid receptors, osteogenic markers and cytokines are also varied by genders. The outcomes of the present study strongly indicate that steroids potentially function as an effective modulator to improve the capacity of MSCs in bone regeneration. It provides crucial information for improving and optimizing MSCs for future clinical application of bone regeneration.     

Colonization of collagen scaffolds by adipocytes derived from mesenchymal stem cells of the common marmoset monkey

In regenerative medicine, human cell replacement therapy offers great potential, especially by cell types differentiated from immunologically and ethically unproblematic mesenchymal stem cells (MSCs). In terms of an appropriate carrier material, collagen scaffolds with homogeneous pore size of 65 μm were optimal for cell seeding and cultivating. However, before clinical application and transplantation of MSC-derived cells in scaffolds, the safety and efficiency, but also possible interference in differentiation due to the material must be preclinically tested. The common marmoset monkey (Callithrix jacchus) is a preferable non-human primate animal model for this aim due to its genetic and physiological similarities to the human.Marmoset bone marrow-derived MSCs were successfully isolated, cultured and differentiated in suspension into adipogenic, osteogenic and chondrogenic lineages by defined factors. The differentiation capability could be determined by FACS. Specific marker genes for all three cell types could be detected by RT-PCR. Furthermore, MSCs seeded on collagen I scaffolds differentiated in adipogenic lineage showed after 28 days of differentiation high cell viability and homogenous distribution on the material which was validated by calcein AM and EthD staining. As proof of adipogenic cells, the intracellular lipid vesicles in the cells were stained with Oil Red O. The generation of fat vacuoles was visibly extensive distinguishable and furthermore determined on the molecular level by expression of specific marker genes. The results of the study proved both the differential potential of marmoset MSCs in adipogenic, osteogenic and chondrogenic lineages and the suitability of collagen scaffolds as carrier material undisturbing differentiation of primate mesenchymal stem cells.