Correction: A comparative analysis of stem cells derived from young rabbit knee joints: the potentially superior performance of decellularized extracellular matrix pretreated infrapatellar fat pad stem cells on nanofiber scaffolds

IntroductionOsteoarthritis (OA) remains a significant clinical challenge, necessitating improved strategies for cartilage repair. Stem cells and scaffolds have crucial roles in tissue repair and regeneration. In this study, we comprehensively investigated the proliferation and differentiation potential of infrapatellar fat pad stem cells (IFPSCs), synovium-derived stem cells (SDSCs), and bone marrow stem cells (BMSCs) from unpretreated knee joints in young rabbits, and after decellularized extracellular matrix (dECM) deposition by stem cell pretreatment in vitro.MethodsWe also examined adhesion and differentiation effects of poly-L-lactic acid (PLLA) and poly-D, L-lactic acid (PDLLA) scaffolds after inoculation with the three stem cell types. We conducted osteogenic, adipogenic, and chondrogenic induction studies using three unpretreated stem cell groups, nine stem cell groups cross-preconditioned with different dECM types, and six stem cell groups cultured on nanofiber PLLA and PDLLA scaffolds. Staining and PCR analyses were then performed.ResultsIn vitro studies indicated that without pretreatment, IFPSCs exhibited the highest proliferation capacity, followed by SDSCs, while BMSCs had the lowest proliferation rate. After cross-pretreatment with dECMs from different sources, IFPSCs pretreated with IECM (decellularized extracellular matrix deposited by IFPSCs) showed the greatest proliferation. BMSCs displayed the highest osteogenic potential, while SDSCs and IFPSCs showed greater chondrogenic potential. No significant differences were observed in adipogenic potential among the three groups. BMSCs exhibited reduced osteogenic potential after pretreatment with all three dECMs, whereas IFPSCs and SDSCs showed enhanced osteogenic potential following SECM and IECM pretreatment, respectively. Additionally, all 3 cell types showed reduced lipogenic potential after pretreatment with the three dECM types. For chondrogenesis, BECM pretreatment were suitable for enhancing the chondrogenic potential of all 3 cell types. Furthermore, BMSCs and IFPSCs exhibited better adhesion and survival than SDSCs on electrospun scaffolds, which mimicked dECM structures. Besides, BMSCs and IFPSCs are more suitable for PLLA to promote osteogenic, adipogenic, and chondrogenic differentiation, whereas SDSCs are better suited for PDLLA.DiscussionOverall, it is anticipated that IFPSCs can be expanded with BECM pretreatment in vitro, and when combined with degradable nanofiber PLLA scaffolds in vivo, will facilitate better OA repair.

Bony defects are a common problem in musculoskeletal surgery. Replacement with autologous bone grafts is limited by availability of transplant material. Sterilized cancellous bone, while being osteoconductive, has limited osteoinductivity. Nanofiber scaffolds are currently used for several purposes due to their capability of imitating the extracellular matrix. Furthermore, they allow modification to provide functional properties. Previously we showed that electrospun nanofiber scaffolds can be used for bone tissue regeneration. While aiming to use the osteoinductive capacities of collagen type-I nanofibers we saw reduced scaffold pore sizes that limited cellular migration and thus colonization of the scaffolds. Aim of the present study was the incorporation of mesenchymal stem cells into the electrospinning process of a nanofiber scaffold to produce cell-seeded nanofiber scaffolds for bone replacement. After construction of a suitable spinning apparatus for simultaneous electrospinning and spraying with independently controllable spinning and spraying devices and extensive optimization of the spinning process, in vitro and in vivo evaluation of the resulting scaffolds was conducted. Stem cells isolated from rat femora were incorporated into PLLA (poly-l-lactide acid) and PLLA-collagen type-I nanofiber scaffolds (PLLA Col I Blend) via simultaneous electrospinning and –spraying. Metabolic activity, proliferation and osteoblastic differentiation were assessed in vitro. For in vivo evaluation scaffolds were implanted into critical size defects of the rat scull. After 4 weeks, animals were sacrificed and bone healing was analyzed using CT-scans, histological, immunhistochemical and fluorescence evaluation. Successful integration of mesenchymal stem cells into the scaffolds was achieved by iteration of spinning and spraying conditions regarding polymer solvent, spinning distance, the use of a liquid counter-electrode, electrode voltage and spinning duration. In vivo formation of bone tissue was achieved. Using a PLLA scaffold, comparable results for the cell-free and cell-seeded scaffolds were found, while the cell-seeded PLLA-collagen scaffolds showed significantly better bone formation when compared to the cell-free PLLA-collagen scaffolds. These results provide support for the future use of cell-seeded nanofiber scaffolds for large bony defects.

Stem cell-based therapy has become an attractive and promising approach for the treatment of severe injuries or thus-far incurable diseases. However, the use of stem cells is often limited by a shortage of available tissue-specific stem cells; therefore, other sources of stem cells are being investigated and tested. In this respect, mesenchymal stromal/stem cells (MSCs) have proven to be a promising stem cell type. In the present study, we prepared MSCs from bone marrow (BM-MSCs) or adipose tissue (Ad-MSCs) as well as limbal epithelial stem cells (LSCs), and their growth, differentiation, and secretory properties were compared. The cells were grown on nanofiber scaffolds and transferred onto the alkali-injured eye in a rabbit model, and their therapeutic potential was characterized. We found that BM-MSCs and tissue-specific LSCs had similar therapeutic effects. Clinical characterization of the healing process, as well as the evaluation of corneal thickness, re-epithelialization, neovascularization, and the suppression of a local inflammatory reaction, were comparable in the BM-MSC- and LSC-treated eyes, but results were significantly better than in injured, untreated eyes or in eyes treated with a nanofiber scaffold alone or with a nanofiber scaffold seeded with Ad-MSCs. Taken together, the results show that BM-MSCs' therapeutic effect on healing of injured corneal surface is comparable to that of tissue-specific LSCs. We suggest that BM-MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain. Damage of ocular surface represents one of the most common causes of impaired vision or even blindness. Cell therapy, based on transplantation of stem cells, is an optimal treatment. However, if limbal stem cells (LSCs) are not available, other sources of stem cells are tested. Mesenchymal stem cells (MSCs) are a convenient type of cell for stem cell therapy. The therapeutic potential of LSCs and MSCs was compared in an experimental model of corneal injury, and healing was observed following chemical injury. MSCs and tissue-specific LSCs had similar therapeutic effects. The results suggest that bone marrow-derived MSCs can be used for ocular surface regeneration in cases when autologous LSCs are absent or difficult to obtain.

The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a hepatocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multipotency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.

Myoblasts, the contractile cells of skeletal muscle, have been invaluable for fundamental studies of muscle development and clinical applications for muscle loss. A major limitation to the myoblast-based therapeutic approach is contamination with non-contractile fibroblasts, which overgrow during cell expansion. To overcome these limitations, this study was carried out to establish a 3D culture environment using nanofiber scaffolds to enrich the myoblast population during construct formation. Poly(methyl methacrylate) (PMMA) nanofiber (PM) scaffolds were fabricated using electrospinning techniques and coated with extracellular matrix (ECM) proteins, such as collagen or laminin, in the presence or absence of genipin. A mixed population of myoblasts and fibroblasts was isolated from human skeletal muscle tissues and cultured on plain surfaces, as well as coated and non-coated PM scaffolds. PMMA can produce smooth fibers with an average diameter of 360 ± 50 nm. Adsorption of collagen and laminin on PM scaffolds is significantly enhanced in the presence of genipin, which introduces roughness to the nanofiber surface without affecting fiber diameter and mechanical properties. It was also demonstrated that laminin-coated PM scaffolds significantly enhance myoblast proliferation (0.0081 ± 0.0007 h−1) and migration (0.26 ± 0.04 μm/min), while collagen-coated PM scaffolds favors fibroblasts proliferation (0.0097 ± 0.0009 h−1) and migration (0.23 ± 0.03 μm/min). Consequently, the myoblast population was enriched on laminin-coated PM scaffolds throughout the culture process. Therefore, laminin coating of nanofiber scaffolds could be a potential scaffold for the development of a tissue-engineered muscle substitute.

Historical Perspectives and Advances in Mesenchymal Stem Cell Research for the Treatment of Liver Diseases

The osteogenic differentiation of human bone marrow stromal cells induced by nanofiber scaffolds using bioinformatics

Meeting summary: International Symposium and Workshop on Hematopoietic Stem Cells V, University of Tübingen, Germany, September 16–18, 2004

The role of chemokine activation of Rac GTPases in hematopoietic stem cell marrow homing, retention, and peripheral mobilization

Nanofibers show promise as bone tissue engineering scaffolds (BTESs). In this study, electrospun poly (lactic-co-glycolic acid) (PLGA)/silicon nitride (Si3N4) composite nanofiber membranes were formed and the osteogenesis capability of mesenchymal stem cells (MSC) from the scaffold marrow was investigated. By modifying the different properties of Si3N4 in the PLGA, two hybrid scaffolds were successfully prepared, including the PLGA/Si3N4 (1 wt.%) nanofiber scaffold and PLGA/Si3N4 (2 wt.%) nanofiber scaffold. The diameter of the fiber nanofiber scaffold PLGA/Si3N4 was decreased and the mechanical strength was increased compared to PLGA. In vitro studies showed better cell adhesion and proliferation on the PLGA/Si3N4 nanofiber scaffold compared to the PLGA nanofiber scaffold. The integration of Si3N4 promoted osteogenesis capacity by increasing the gene expression of bone-related proteins (BMP2, ALP, OPN, COL1a1, Runx2, and OCN), calcium deposits, and support of ALP activity compared to those for the PLGA nanofiber scaffold. Similarly, the PLGA/Si3N4 (2 wt.%) nanofiber scaffold showed better mechanics and biological activity compared to the PLGA/Si3N4 (1 wt.%) nanofiber scaffold. Overall, the PLGA/Si3N4 nanofiber scaffold showed potential as a promising hybrid scaffold for bone regeneration.

The induced pluripotent stem (iPS) cells of murine and human are capable to differentiate into any cell type of the body through recapitulating normal development, similarly as the embryonic stem (ES) cells. Lines of evidence support that both ES cells and iPS cells are induced to differentiate in vitro by sequential treatment of humoral cues such as growth factors and chemicals, combined with the use of certain microenvironments including extracellular matrices and scaffolds.Here, we describe the procedure to potentiate hepatic lineage cells differentiation from murine and human iPS cells, using growth factor cocktails and nanofiber scaffolds. Nanofiber scaffolds have a three-dimensional surface mimicking the fine structures of the basement membrane in vivo, allow the iPS cells to differentiate into the definitive endoderm and mature hepatocyte-like cells more efficiently than the two-dimensional conventional culture plates.

Electrospun nanofiber matrices sufficiently mimic the structural morphology of natural extracellular matrix. In this study, we aimed to examine the effects of agar/polyvinyl alcohol nanofiber (PVA) scaffold on the proliferation efficiency and differentiation potential of neonate mouse spermatogonial stem cells (SCCs). Testicular cells were isolated from testes of 40 mouse pups and were seeded in: 1) 2D cell culture plates in the absence (2D/−GF) or presence (2D/+GF) of growth factors and 2) onto agar/PVA scaffold in the absence (3D/−GF) or presence (3D/+GF) of growth factors. The cells were subsequently cultured for 4 weeks. First 2 weeks were dedicated to proliferative phase, whereas the next 2 weeks emphasized the differentiation phase. The identity of the SCCs was investigated at different time-points by flow cytometry and quantitative reverse transcription PCR (qRT-PCR) analyses against the germ cell markers, including PLZF, Id-4, Gfrα-1, Tekt-1, and Sycp-3. After 2 weeks of culture, the 3D/+GF group showed the highest percentage of PLZF-positive cells among culture systems (P < 0.05). The expression levels of pre-meiotic markers (Id-4 and Gfrα-1) decreased significantly in all groups, particularly in 3D/+GF group after 28 days of culture. Additionally, the cells in the 3D/+GF group displayed the highest expression of meiotic (Sycp-3) and post-meiotic markers (Tekt-1) 14 days after differentiation induction. Seemingly, the combination of the agar/PVA scaffold and growth factor-supplemented medium synergistically increased the differentiation rate of mouse SSCs into meiotic and post-meiotic cells. Thus, agar/PVA nanofiber scaffolds may have the potential for applications in the restoration of infertility, especially in azoospermic males. Abbreviations 2D: two dimentional; 3D: three dimentional; bFGF: basic fibroblast growth factor; BMP-4: bone morphogenetic protein 4; DMEM: Dulbecco’s modified Eagle’s medium; ECM: extracellular matrix; FCS: fetal calf serum; FTIR: Fourier-transform infrared spectroscopy; GDNF: glial cell line-derived neurotrophic factor; GF: growth factors; Gfrα-1, GDNF family co-receptor α1; Id-4, Inhibitor of DNA Binding 4; MTT: methylthiazoltetrazolium; PLZF: promyelocytic leukemia zinc finger; PVA: polyvinyl alcohol; qRT-PCR: quantitative reverse transcription PCR; RA: retinoic acid; SACS: soft agar culture system; SD: standard deviation; SEM: scanning electron microscope; SSCs: spermatogonial stem cells; Sycp-3, Synaptonemal complex protein 3; Tekt-1, Tektin 1.

Mutual, reciprocal SDF-1/CXCR4 interactions between hematopoietic and bone marrow stromal cells regulate human stem cell migration and development in NOD/SCID chimeric mice

Glioblastoma Multiforme (GBM) invasiveness renders complete surgical resection impossible and highly invasive Glioblastoma Initiating Cells (GICs) are responsible for tumour recurrence. Their dissemination occurs along pre-existing fibrillary brain structures comprising the aligned myelinated fibres of the corpus callosum (CC) and the laminin (LN)-rich basal lamina of blood vessels. The extracellular matrix (ECM) of these environments regulates GIC migration, but the underlying mechanisms remain largely unknown. In order to recapitulate the composition and the topographic properties of the cerebral ECM in the migration of GICs, we have set up a new aligned polyacrylonitrile (PAN)-derived nanofiber (NF) scaffold. This system is suitable for drug screening as well as discrimination of the migration potential of different glioblastoma stem cells. Functionalisation with LN increases the spatial anisotropy of migration and modulates its mode from collective to single cell migration. Mechanistically, equally similar to what has been observed for mesenchymal migration of GBM in vivo, is the upregulation of galectin-3 and integrin-β1 in Gli4 cells migrating on our NF scaffold. Downregulation of Calpain-2 in GICs migrating in vivo along the CC and in vitro on LN-coated NF underlines a difference in the turnover of focal adhesion (FA) molecules between single-cell and collective types of migration.

IntroductionMesenchymal stem cells (MSCs) have immunosuppressive activity and can differentiate into bone and cartilage; and thus seem ideal for treatment of rheumatoid arthritis (RA). Here, we investigated the osteogenesis and chondrogenesis potentials of MSCs seeded onto nano-fiber scaffolds (NFs) in vitro and possible use for the repair of RA-affected joints.MethodsMSCs derived from healthy donors and patients with RA or osteoarthritis (OA) were seeded on poly-lactic-glycolic acid (PLGA) electrospun NFs and cultured in vitro.ResultsHealthy donor-derived MSCs seeded onto NFs stained positive with von Kossa at Day 14 post-stimulation for osteoblast differentiation. Similarly, MSCs stained positive with Safranin O at Day 14 post-stimulation for chondrocyte differentiation. Surprisingly, even cultured without any stimulation, MSCs expressed RUNX2 and SOX9 (master regulators of bone and cartilage differentiation) at Day 7. Moreover, MSCs stained positive for osteocalcin, a bone marker, and simultaneously also with Safranin O at Day 14. On Day 28, the cell morphology changed from a spindle-like to an osteocyte-like appearance with processes, along with the expression of dentin matrix protein-1 (DMP-1) and matrix extracellular phosphoglycoprotein (MEPE), suggesting possible differentiation of MSCs into osteocytes. Calcification was observed on Day 56. Expression of osteoblast and chondrocyte differentiation markers was also noted in MSCs derived from RA or OA patients seeded on NFs. Lactic acid present in NFs potentially induced MSC differentiation into osteoblasts.ConclusionsOur PLGA scaffold NFs induced MSC differentiation into bone and cartilage. NFs induction process resembled the procedure of endochondral ossification. This finding indicates that the combination of MSCs and NFs is a promising therapeutic technique for the repair of RA or OA joints affected by bone and cartilage destruction.