Lakshmi Selvaratnam

Human peripheral blood derived mesenchymal stem cells demonstrate similar characteristics and chondrogenic differentiation potential to bone marrow derived mesenchymal stem cells

The use of mesenchymal stem cells (MSCs) for cartilage repair has generated much interest owing to their multipotentiality. However, their significant presence in peripheral blood (PB) has been a matter of much debate. The objectives of this study are to isolate and characterize MSCs derived from PB and, compare their chondrogenic potential to MSC derived from bone marrow (BM). PB and BM derived MSCs from 20 patients were isolated and characterized. From 2 ml of PB and BM, 5.4 ± 0.6 million and 10.5 ± 0.8 million adherent cells, respectively, were obtained by cell cultures at passage 2. Both PB and BM derived MSCs were able to undergo tri‐lineage differentiation and showed negative expression of CD34 and CD45, but positively expressed CD105, CD166, and CD29. Qualitative and quantitative examinations on the chondrogenic potential of PB and BM derived MSCs expressed similar cartilage specific gene (COMP) and proteoglycan levels, respectively. Furthermore, the s‐GAG levels expressed by chondrogenic MSCs in cultures were similar to that of native chondrocytes. In conclusion, this study demonstrates that MSCs from PB maintain similar characteristics and have similar chondrogenic differentiation potential to those derived from BM, while producing comparable s‐GAG expressions to chondrocytes.

Treatment Outcomes of Alginate-Embedded Allogenic Mesenchymal Stem Cells Versus Autologous Chondrocytes for the Repair of Focal Articular Cartilage Defects in a Rabbit Model:

Background: Mesenchymal stem cells (MSCs) represent a promising alternative form of cell-based therapy for cartilage injury. However, the capacity of MSCs for chondrogenesis has not been fully explored. In particular, there is presently a lack of studies comparing the effectiveness of MSCs to conventional autologous chondrocyte (autoC) treatment for regeneration of full-thickness cartilage defects in vivo. Hypothesis: Treatment with allogenic undifferentiated MSCs (alloMSCs) results in superior cartilage tissue regeneration profiles when compared with autoC for repair of focal articular cartilage defects. Study Design: Controlled laboratory study. Methods: Full-thickness articular cartilage defects were created on the weightbearing surface of the medial femoral condyles in both knees of New Zealand White rabbits (N = 30). Six weeks after the defect was induced, the right knee was treated with either alloMSCs (n = 12) or autoC (n = 18), while the left knee remained untreated (control). The rabbits were sacrificed at 6 months after treatment for assessment of cartilage tissue regeneration, which included the Brittberg morphologic score, histologic grading by O’Driscoll score, and quantitative analysis of glycosaminoglycans per total protein content. Results: Apart from significantly higher Brittberg scores in the alloMSC treatment group (8.8 ± 0.8) versus the autoC treatment group (6.6 ± 0.8) (P = .04), both treatments showed similar cartilage regenerative profiles. All outcome measures were significantly higher in the treatment groups compared with their respective controls (P < .05). Conclusion: AlloMSCs have similar effectiveness as autoC for repair of focal cartilage defects. Both treatments resulted in superior tissue regeneration compared with untreated defects. Clinical Relevance: The results have an implication of supporting the potential use of MSCs for cartilage repair after sports injuries or diseases, in view of similar efficacy but less patient morbidity and potential cost savings as compared with conventional autoC therapy.

Isolation, characterization and the multi‐lineage differentiation potential of rabbit bone marrow‐derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are recognized by their plastic adherent ability, fibroblastic‐like appearance, expression of specific surface protein markers, and are defined by their ability to undergo multi‐lineage differentiation. Although rabbit bone marrow‐derived MSCs (rbMSCs) have been used extensively in previous studies especially in translational research, these cells have neither been defined morphologically and ultrastructurally, nor been compared with their counterparts in humans in their multi‐lineage differentiation ability. A study was therefore conducted to define the morphology, surface marker proteins, ultrastructure and multi‐lineage differentiation ability of rbMSCs. Herein, the primary rbMSC cultures of three adult New Zealand white rabbits (at least 4 months old) were used for three independent experiments. rbMSCs were isolated using the gradient‐centrifugation method, an established technique for human MSCs (hMSCs) isolation. Cells were characterized by phase contrast microscopy observation, transmission electron microscopy analysis, reverse transcriptase‐polymerase chain reaction (PCR) analysis, immunocytochemistry staining, flow cytometry, alamarBlue® assay, histological staining and quantitative (q)PCR analysis. The isolated plastic adherent cells were in fibroblastic spindle‐shape and possessed eccentric, irregular‐shaped nuclei as well as rich inner cytoplasmic zones similar to that of hMSCs. The rbMSCs expressed CD29, CD44, CD73, CD81, CD90 and CD166, but were negative (or dim positive) for CD34, CD45, CD117 and HLD‐DR. Despite having similar morphology and phenotypic expression, rbMSCs possessed significantly larger cell size but had a lower proliferation rate as compared with hMSCs. Using established protocols to differentiate hMSCs, rbMSCs underwent osteogenic, adipogenic and chondrogenic differentiation. Interestingly, differentiated rbMSCs demonstrated higher levels of osteogenic (Runx2) and chondrogenic (Sox9) gene expressions than that of hMSCs (P < 0.05). There was, however, no difference in the adipogenic (Pparγ) expressions between these cell types (P > 0.05). rbMSCs possess similar morphological characteristics to hMSCs, but have a higher potential for osteogenic and chondrogenic differentiation, despite having a lower cell proliferation rate than hMSCs. The characteristics reported here may be used as a comprehensive set of criteria to define or characterize rbMSCs.

The effect of TGF-β1 and β-estradiol on glycosaminoglycan and type II collagen distribution in articular chondrocyte cultures

Articular cartilage extracellular matrix (ECM) plays a crucial role in regulating chondrocyte functions via cell—matrix interaction, cytoskeletal organization and integrin‐mediated signaling. Factors such as interleukins, basic fibroblast growth factor (bFGF), bone morphogenic proteins (BMPs) and insulin‐like growth factor (IGF) have been shown to modulate the synthesis of extracellular matrix in vitro. However, the effects of TGF‐β1 and β‐estradiol in ECM regulation require further investigation, although there have been suggestions that these factors do play a positive role. To establish the role of these factors on chondrocytes derived from articular joints, a study was conducted to investigate the effects of TGF‐β1 and β‐estradiol on glycosaminoglycan secretion and type II collagen distribution (two major component of cartilage ECM in vivo). Thus, chondrocyte cultures initiated from rabbit articular cartilage were treated with 10 ng/ml of TGF‐β1, 10 nM of β‐estradiol or with a combination of both factors. Sulphated glycosaminoglycan (GAG) and type II collagen levels were then measured in both these culture systems. The results revealed that the synthesis of GAG and type II collagen was shown to be enhanced in the TGF‐β1 treated cultures. This increase was also noted when TGF‐β1 and β‐estradiol were both used as culture supplements. However, β‐estradiol alone did not appear to affect GAG or type II collagen deposition. There was also no difference between the amount of collagen type II and GAG being expressed when chondrocyte cultures were treated with TGF‐β1 when compared with cultures treated with combined factors. From this, we conclude that although TGF‐β1 appears to stimulate chondrocyte ECM synthesis, β‐estradiol fails to produce similar effects. The findings of this study confirm that contrary to previous claims, β‐estradiol has little or no effect on chondrocyte ECM synthesis. Furthermore, the use of TGF‐β1 may be useful in future studies looking into biological mechanisms by which ECM synthesis in chondrocyte cultures can be augmented, particularly for clinical application.

Autologous chondrocyte transplantation in the repair of full-thickness focal cartilage damage in rabbits

Purpose. To compare the efficacy of autologous chondrocyte transplantation (ACT) versus non-operative measures for cartilage repair in rabbits. Methods. Nine New Zealand white rabbits were used. Identical focal defects were created in the articular cartilage of both knees. One month later, the right knee was repaired via ACT, while the left knee was left untreated (control group). The quality of cartilage tissues in both knees was compared 3 months later, according to the quantitative analysis of glycosaminoglycan (GAG) in the cartilage and macroscopic examination of histology using the Brittberg/International Cartilage Research Society (ICRS) score. Results. Microscopic examination showed enhanced regeneration following ACT repair. Quantification analysis revealed significantly higher cellular expression of GAG in the ACT-treated knees (1.12 vs 0.81 μg GAGs/mg protein, p=0.008). The mean Brittberg/ICRS score was significantly higher in the treated knees (6.00 vs 1.89, p=0.007). Conclusion. ACT is superior to non-operative measures for repairing focal cartilage defects, as determined by favourable histological and immunohistological outcomes at the cellular level.

Identification of Pathways Mediating Growth Differentiation Factor5-Induced Tenogenic Differentiation in Human Bone Marrow Stromal Cells

To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.

A Preliminary Study of Human Amniotic Membrane as a Potential Chondrocyte Carrier

Purpose: To investigate the feasibility of using processed human amniotic membrane (HAM) to support the attachment and proliferation of chondrocytes in vitro which in turn can be utilised as a cell delivery vehicle in tissue engineering applications. Methods: Fresh HAM obtained from patients undergoing routine elective caesarean sections was harvested, processed and dried using either freeze drying (FD) or air drying (AD) methods prior to sterilisation by gamma irradiation. Isolated, processed and characterised rabbit autologous chondrocytes were seeded on processed HAM and cultured for up to three weeks. Cell attachment and proliferation were examined qualitatively using inverted brightfield microscopy. Results: Processed HAM appeared to allow cell attachment when implanted with chondrocytes. Although cells seeded on AD and FD HAM did not appear to attach as strongly as those seeded on glycerol preserved intact human amniotic membrane, these cells to be proliferated in cell culture conditions. Conclusion: Preliminary results show that processed HAM promotes chondrocyte attachment and proliferation.

Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

Chondrogenic differentiation of mesenchymal stromal cells (MSCs) in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM) was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG) analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05) chondrogenic but lower hypertrophic (p < 0.05) gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

Characterization of undifferentiated human bone marrow and blood derived mesenchymal stem cells and their potential for chondrogenic differentiation

In recent years, the potential of cartilage tissue engineering techniques employing mesenchymal stem cells (MSCs) to repair damaged human cartilage and defects has generated much interest. Traditionally, sources of MSCs included patient’s own bone marrow. However, little have been reported on adult peripheral blood (PB) as a source of MSCs, which has been a subject of much debate amongst scientists owing to its extremely low density in PB and the difficulties associated with extracting MSCs from PB. The objectives of this study were to isolate MSCs derived from bone marrow and peripheral blood as a source and to assess their potential to undergo in vitro chondrogenesis using a biocompatible three-dimensional scaffold. In defining MSCs, the cells isolated from its source must meet these 3 criteria: (i) adherence to plastic when maintained in culture; (ii) positive expression of several antigens such as CD29, CD105, CD166; (iii) ability to differentiate into osteoblasts, adipocytes & chondrocytes under in vitro inductive conditions. PB samples (2 ml) were collected and mononuclear cells extracted and separated using Ficoll–Paque PLUS via centrifugation. Subsequently, suspended cells were removed after 5 days of culture, and adherent cells left to grow. Cells were detached upon reaching 80-90% confluence and sub-cultured up to 4 passages prior to further experiments. MSC antigens were recognized by monoclonal antibodies CD29, CD105 and CD166. To distinguish MSCs from hematopoietic stem cells, CD34 surface markers were used as negative controls. The characterized human blood-derived progenitor cells were cultured in three-dimensional alginate scaffolds using chondrogenic induction medium to promote chondrogenesis. Chondrogenesis was quantitated by sulphated glycosaminoglycan (S-GAG) production measured by 1,9-dimethylmethylene blue (DMMB) assay. Furthermore, chondrogenic-MSCs were examined and histologically compared using Safranin O staining to that of human chondrocytes as a means to determine chondrogenic transformation. Gene expression analysis was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR) of differentiated human blood-derived progenitor cells using chondrocyte (cartilage cell)-specific phenotypic markers. The results showed that the cell derived in our processing technique share similar characteristics with adult MSCs and chondrocytes. Induction of chondrogenesis has been demonstrated in human blood-derived progenitor cells, which could provide a ready source of chondrocytes for engineering biological therapies. In the practical sense, PB isolation would prove to be less invasive, less expensive and less traumatic for patients to undergo therapy as compared to the 2-stage procedure of current available tissue engineering technique.

Factors Influencing the Successful Isolation and Expansion of Aging Human Mesenchymal Stem Cells

Abstract Most studies highlight mesenchymal stem cells (MSCs) extracted primarily from bone marrow (BM), very few report the use of peripheral blood (PB), often due to the associated low seeding density and difficulties with extraction techniques. As ageing populations are becoming more predominant globally, together with escalating demands for MSC transplantation and tissue regeneration, obtaining quality MSCs suitable for induced differentiation and biological therapies becomes increasingly important. In this study, BM and PB were obtained from elderly patients and extracted MSCs grown in vitro to determine their successful isolation and expansion. Patients’ socio-demographic background and other medical information were obtained from medical records. Successful and failed cultures were correlated with key demographic and medical parameters. A total of 112 samples (BM or PB) were used for this study. Of these, 50 samples (44.6%) were successfully cultured according to standardised criteria with no signs of contamination. Our comparative analyses demonstrated no statistical correlation between successful MSC cultures and any of the six demographic or medical parameters examined, including sample quantity, age, sex, race, habits and underlying comorbidities of sample donors. In conclusion, the present study demonstrates that typical demographics and comorbidities do not influence successful MSC isolation and expansion in culture.