Identification of seven novel variants in the ß-globin gene in transfusion-dependent and normal patients.

INTRODUCTION: Abnormality in HBB results in an inherited recessive blood disorder, which can be caused by variants at the transcriptional or translational level affecting the stability and the production of the HBB chain. The severity of the disease relies on the variant's characteristics. This study aimed to identify the common ß-globin HBB variants in the population of the Eastern Province, which has the highest prevalence of blood diseases in Saudi Arabia. MATERIAL AND METHODS: Direct sequence of ß-globin HBB gene, and alpha-globin HBA1 and HBA2 genes was performed on a total of 545 blood samples (transfusion-dependent: 215, 106 men and 109 women; normal healthy subjects: 330, 197 men and 133 women) collected from Saudi Arabian participants in the Eastern region. RESULTS: A total of 36 variants in HBB gene were revealed with 11 variants that have been reported for the first time in Saudi Arabia, including 7 novel variants that have been identified for the first time in HBB gene. The novel variants consisted of two exonic (HBB:c.252C>T; HBB:c.281G>T) and five intronic variants (c.316-183_316-168del; c.315+241T>A; c.315+376T>C; c.316-114C>G; c.315+208T>G) at HBB gene. The novel exonic variants and three (c.316-183_316-168del; c.315+241T>A; c.315+376T>C) intronic variants were co-inherited with α deletion. CONCLUSIONS: This current study updated the HBB gene variations with newly identified variants of HBB gene and co-inheritance with α-globin deletions. The identified ß-globin mutations will strengthen the genetic reference that could aid in characterizing mutations that are associated with phenotype of thalassemia in a specific region.

Hemoglobin A2 (HbA2) has a measure of unreliability in diagnosing ß-thalassemia trait (ß-TT).

INTRODUCTION: Detection of ß-thalassemia trait or carriers (ß-TT) depends significantly on an increase in Hemoglobin A2 (HbA2) levels, which is found at low levels (<3%) in normal healthy individuals and elevated levels (≥3.5%) in ß-TT individuals. The study was designed to evaluate the reliability of the diagnostic parameter HbA2 in the differentiation of ß-TT and non-ß-TT in Saudis. METHODS: The widely used high performance liquid chromatography (Variant II Bio-Rad) was used to measure HbA2 levels in blood. Sanger sequencing was used to screen the variation in globin genes (HBB, HBD, HBA1, and HBA2). All the study subjects were divided into ßTT and non-ßTT (wild) categories based on the presence or absence of HBB variations and further sub-divided into false positive, true positive, false negative, and true negative, based on HbA2 values. RESULTS: Out of 288 samples, 96 had HBB gene mutations. Of the 96 ß-TT samples, sickle cell trait (SCT) samples (n = 58) were excluded, while the remaining (38 ß-TT) were included in the detailed analysis: seven subjects with the HBB mutation had normal HbA2 (<3%), and three were borderline (3.1-3.9%). The remainder (n = 28) had an elevated HbA2 level (>4%). Based on HbA2 analysis alone, both these groups would be incorrectly diagnosed as normal. Similarly, of the 189 non-ß-TT samples, 179 had normal HbA2, eight had borderline HbA2, and two had a HbA2 level above 4%. Based on HbA2 analysis alone, borderline and >4% HbA2 individuals, negative for ß-TT, can be incorrectly diagnosed as carriers. CONCLUSION: Given the percentage of samples falling in the HbA2 "borderline" and "normal" categories, it can be concluded that HbA2 has a measure of unreliability in the diagnosis of ß-thalassemia carriers.

Collagen type XII and versican are present in the early stages of cartilage tissue formation by both redifferentating passaged and primary chondrocytes.

Current approaches to cartilage tissue engineering require a large number of chondrocytes. Although chondrocyte numbers can be expanded in monolayer culture, the cells dedifferentiate and unless they can be redifferentiated are not optimal to use for cartilage repair. We took advantage of the differential effect of culture conditions on the ability of passaged and primary chondrocytes to form cartilage tissue to dissect out the extracellular matrix (ECM) molecules produced and accumulated in the early stages of passaged cell cartilage tissue formation as we hypothesized that passaged bovine cells that form cartilage accumulate a pericellular matrix that differs from cells that do not form cartilage. Twice passaged bovine chondrocytes (P2) (cartilage forming), or as a control primary chondrocytes (P0) (which do not generate cartilage), were cultured on three-dimensional membrane inserts in serum-free media. P2 redifferentiation was occurring during the first 8 days as indicated by increased expression of the chondrogenic genes Sox9, collagen type II, aggrecan, and COMP, suggesting that this is an appropriate time period to examine the ECM. Mass spectrometry showed that the P2 secretome (molecules released into the media) at 1 week had higher levels of collagen types I, III, and XII, and versican while type II collagen and COMP were found at higher levels in the P0 secretome. There was increased collagen synthesis and retention by P2 cells compared to P0 cells as early as 3 days of culture. Confocal microscopy showed that types XII, III, and II collagen, aggrecan, versican, and decorin were present in the ECM of P2 cells. In contrast, collagen types I, II, and III, aggrecan, and decorin were present in the ECM of P0 cells. As primary chondrocytes grown in serum-containing media, a condition that allows for the generation of cartilage tissue in vitro, also accumulate versican and collagen XII, this study suggests that these molecules may be necessary to provide a microenvironment that supports hyaline cartilage formation. Further study is required to determine if these molecules are also accumulated by passaged human chondrocytes and their role in promoting hyaline cartilage formation.

Serum- and growth-factor-free three-dimensional culture system supports cartilage tissue formation by promoting collagen synthesis via Sox9-Col2a1 interaction.

OBJECTIVE: One of the factors preventing clinical application of regenerative medicine to degenerative cartilage diseases is a suitable source of cells. Chondrocytes, the only cell type of cartilage, grown in vitro under culture conditions to expand cell numbers lose their phenotype along with the ability to generate hyaline cartilaginous tissue. In this study we determine that a serum- and growth-factor-free three-dimensional (3D) culture system restores the ability of the passaged chondrocytes to form cartilage tissue in vitro, a process that involves sox9. METHODS: Bovine articular chondrocytes were passaged twice to allow for cell number expansion (P2) and cultured at high density on 3D collagen-type-II-coated membranes in high glucose content media supplemented with insulin and dexamethasone (SF3D). The cells were characterized after monolayer expansion and following 3D culture by flow cytometry, gene expression, and histology. The early changes in signaling transduction pathways during redifferentiation were characterized. RESULTS: The P2 cells showed a progenitor-like antigen profile of 99% CD44(+) and 40% CD105(+) and a gene expression profile suggestive of interzone cells. P2 in SF3D expressed chondrogenic genes and accumulated extracellular matrix. Downregulating insulin receptor (IR) with HNMPA-(AM3) or the PI-3/AKT kinase pathway (activated by insulin treatment) with Wortmannin inhibited collagen synthesis. HNMPA-(AM3) reduced expression of Col2, Col11, and IR genes as well as Sox6 and -9. Co-immunoprecipitation and chromatin immunoprecipitation analyses of HNMPA-(AM3)-treated cells showed binding of the coactivators Sox6 and Med12 with Sox9 but reduced Sox9-Col2a1 binding. CONCLUSIONS: We describe a novel culture method that allows for increase in the number of chondrocytes and promotes hyaline-like cartilage tissue formation in part by insulin-mediated Sox9-Col2a1 binding. The suitability of the tissue generated via this approach for use in joint repair needs to be examined in vivo.

Modulation of annulus fibrosus cell alignment and function on oriented nanofibrous polyurethane scaffolds under tension.

BACKGROUND CONTEXT: Annulus fibrosus (AF), a component of the intervertebral disc (IVD), is always under tension in vivo, a condition that must be taken into consideration when tissue engineering an IVD. Loss of the tensile forces has been implicated in the pathogenesis of disc degeneration characterized by mechanical and structural breakdown of the AF. PURPOSE: In this study, we hypothesize that tensile forces modulate cellular and molecular behavior of AF cells grown on nanofibrous scaffolds in vitro. STUDY DESIGN/SETTING: Bovine AF cells were seeded onto strained electrospun-aligned nanofibrous polycarbonate urethane (PU) scaffolds. Tension was either maintained throughout the culture duration (monotonic) or removed after 24 hours (relaxed). METHODS: The effect of tension on AF cells cultured on PU scaffolds was evaluated over 7 days by scanning electron microscopy, biochemical assays, immunofluorescence microscopy, and quantitative polymerase chain reaction. RESULTS: Cells grown on the relaxed scaffold were significantly more proliferative, synthesized more collagen and had increased collagen type I and TGFß-1 gene expression; however these cells were not as aligned as were the cells and matrix on monotonic strained scaffolds. The alignment of AF cells grown on monotonic scaffolds correlated with significantly greater scaffold elastic modulus on day 7. Additionally, the cellular response to the change in strain was delayed by 3 to 5 days after tension release, which correlated with the time at which changes in scaffold length were detected. CONCLUSIONS: This study demonstrated that AF cells respond at the molecular and cellular level to the changes in matrix/scaffold tension. This suggests that it may be necessary to determine the optimal elastic modulus and applied tensile forces to tissue engineer an AF that mimics the native tissue. Furthermore, this study provides insight into how changes in tensile forces may lead to changes in the AF cell function.

Specification of chondrocytes and cartilage tissues from embryonic stem cells.

Osteoarthritis primarily affects the articular cartilage of synovial joints. Cell and/or cartilage replacement is a promising therapy, provided there is access to appropriate tissue and sufficient numbers of articular chondrocytes. Embryonic stem cells (ESCs) represent a potentially unlimited source of chondrocytes and tissues as they can generate a broad spectrum of cell types under appropriate conditions in vitro. Here, we demonstrate that mouse ESC-derived chondrogenic mesoderm arises from a Flk-1(-)/Pdgfrα(+) (F(-)P(+)) population that emerges in a defined temporal pattern following the development of an early cardiogenic F(-)P(+) population. Specification of the late-arising F(-)P(+) population with BMP4 generated a highly enriched population of chondrocytes expressing genes associated with growth plate hypertrophic chondrocytes. By contrast, specification with Gdf5, together with inhibition of hedgehog and BMP signaling pathways, generated a population of non-hypertrophic chondrocytes that displayed properties of articular chondrocytes. The two chondrocyte populations retained their hypertrophic and non-hypertrophic properties when induced to generate spatially organized proteoglycan-rich cartilage-like tissue in vitro. Transplantation of either type of chondrocyte, or tissue generated from them, into immunodeficient recipients resulted in the development of cartilage tissue and bone within an 8-week period. Significant ossification was not observed when the tissue was transplanted into osteoblast-depleted mice or into diffusion chambers that prevent vascularization. Thus, through stage-specific manipulation of appropriate signaling pathways it is possible to efficiently and reproducibly derive hypertrophic and non-hypertrophic chondrocyte populations from mouse ESCs that are able to generate distinct cartilage-like tissue in vitro and maintain a cartilage tissue phenotype within an avascular and/or osteoblast-free niche in vivo.

Hyaline cartilage tissue is formed through the co-culture of passaged human chondrocytes and primary bovine chondrocytes.

To circumvent the problem of a sufficient number of cells for cartilage engineering, the authors previously developed a two-stage culture system to redifferentiate monolayer culture-expanded dedifferentiated human articular chondrocytes by co-culture with primary bovine chondrocytes (bP0). The aim of this study was to analyze the composition of the cartilage tissue formed in stage 1 and compare it with bP0 grown alone to determine the optimal length of the co-culture stage of the system. Biochemical data show that extracellular matrix accumulation was evident after 2 weeks of co-culture, which was 1 week behind the bP0 control culture. By 3 to 4 weeks, the amounts of accumulated proteoglycans and collagens were comparable. Expression of chondrogenic genes, Sox 9, aggrecan, and collagen type II, was also at similar levels by week 3 of culture. Immunohistochemical staining of both co-culture and control tissues showed accumulation of type II collagen, aggrecan, biglycan, decorin, and chondroitin sulfate in appropriate zonal distributions. These data indicate that co-cultured cells form cartilaginous tissue that starts to resemble that formed by bP0 after 3 weeks, suggesting that the optimal time to terminate the co-culture stage, isolate the now redifferentiated cells, and start stage 2 is just after 3 weeks.

Proteoglycan and collagen accumulation by passaged chondrocytes can be enhanced through side-by-side culture with primary chondrocytes.

Identifying a source of sufficient numbers of chondrocytes for cartilage tissue engineering is a major factor limiting its use clinically. Previously we demonstrated that combined coculture of passaged dedifferentiated articular chondrocytes with primary bovine chondrocytes will induce their redifferentiation. In this study we determine whether these two cell types have to be in contact, whether human chondrocytes respond similarly, and whether the ability of primary cells to influence passaged cells depends on the age of the donor. Coculture of primary and passaged bovine chondrocytes grown on filter inserts placed in the same culture well but not in direct contact resulted in the passaged cells accumulating matrix rich in proteoglycans and type II collagen. There was upregulation of type II collagen and Sox9 and decrease in type I collagen gene expression in the passaged cells, to levels not significantly different from those of primary chondrocytes. Passaged chondrocytes obtained from older animals responded similarly to cells from younger animals. Further, passaged human chondrocytes were also induced to form cartilage tissue when placed in side-by-side culture with bovine chondrocytes; these data suggest that a soluble factor(s) may be responsible for redifferentiation of passaged chondrocytes and that it is not species specific. The responsiveness of human chondrocytes to this factor(s) suggests that this approach may be suitable to overcome the problem of limited chondrocyte numbers for cartilage tissue engineering.

Passaged human chondrocytes accumulate extracellular matrix when induced by bovine chondrocytes.

A source of sufficient number of cells is a major limiting factor for cartilage tissue engineering. To circumvent this problem, we developed a co-culture method to induce redifferentiation in bovine articular chondrocytes, which had undergone dedifferentiation following serial passage in monolayer culture. In this study we determine whether human osteoarthritic (OA) and non-diseased passaged dedifferentiated chondrocytes will respond similarly. Human passaged chondrocytes were co-cultured for 4 weeks with primary bovine chondrocytes and their redifferentiation status was determined. Afterwards the cells were cultured either independently or in co-culture with cryopreserved passaged cells for functional analysis. The co-culture of passaged cells with primary chondrocytes resulted in reversion of their phenotype towards articular chondrocytes, as shown by increased gene expression of type II collagen and COMP, decreased type I collagen expression and extracellular matrix formation in vitro. Furthermore, this redifferentiation was stable, as those cells not only formed hyaline-like cartilage tissue when grown on their own but also they could induce redifferentiation of passaged chondrocytes in co-culture. These data suggest that it may be possible to use autologous chondrocytes obtained from osteoarthritic cartilage to form tissue suitable to use for cartilage repair.

Gene and protein expression profile of naive and osteo-chondrogenically differentiated rat bone marrow-derived mesenchymal progenitor cells.

Adult mesenchymal progenitor cells (MPCs) are adherent stromal cells of non-haematopoietic origin derived from bone marrow and other tissues. Upon limited in vitro expansion, they retain their self-renewal capacity as well as their potential to differentiate into tissues of mesenchymal lineage, such as bone, cartilage, muscle, tendon and connective tissues. Amongst these tissues, cartilage is the only one with insufficient self-renewal capacity, thus MPCs would qualify as an excellent tool for therapeutic regeneration of focal cartilage lesions. However, optimal in vitro manipulation of MPCs is a prerequisite; identification and a better understanding of the molecular mechanisms regulating their differentiation pathways are needed. Despite wide usage of rats as a mammalian experimental model for preclinical fracture healing and orthopaedic tissue regeneration studies, basal gene and protein expression profiles of the osteo-chondrogenic differentiation lineages of adult rat MPCs have rarely been investigated. Therefore, this study was carried out for a quantitative RT-PCR based time-course profiling of osteo- and chondrogenesis related gene expression in undifferentiated and differentiated rat adult MPCs. In addition, with an antibody array analysis TIMP-1, MCP-1 and VEGFalpha-164 were detected in the culture supernatant and CINC-2 and beta-NGF in the cell lysate of MPCs according to their differentiation commitment. Identification of differentially expressed genes and proteins along the osteo-chondrogenic lineage provides a foundation for a more reproducible and reliable quality and differentiation control of rat bone marrow-derived MPCs used for osteochondrogenic differentiation studies.

Cartilage tissue formation using redifferentiated passaged chondrocytes in vitro.

Articular cartilage has limited ability for repair when damaged by trauma or degenerative disease, such as osteoarthritis, which can result in pain and compromised quality of life. Biological surface replacements developed using tissue engineering methods are a promising approach for cartilage repair, which would avoid the need for total joint replacement with the synthetic implants used currently. A basic requirement of in vitro tissue generation is a supply of sufficient number of cells, which are difficult to acquire from sparsely cellular cartilage tissue. Previously, we have shown that coculture of in vitro-expanded dedifferentiated chondrocytes (P2) with small numbers of primary chondrocytes (P0) induces redifferentiation in passaged (P2) cells. In this study we show that this redifferentiation is not a transient change. After 4 weeks of coculture, the P0 and P2 cells were separated by flow-associated cell sorting, and the redifferentiated P2 (dP2) were cultured alone for a further 4 weeks. The redifferentiated dP2 cells formed thicker cartilage tissue compared to the tissue generated by P2 cells. The newly formed tissue contained type II collagen as demonstrated by immunohistochemical staining and accumulated more proteoglycan per cell than the tissue formed by P2 cells. The dP2 cells also exhibited higher type II collagen and lower type I collagen gene expression than the P2 cells. Interestingly, dP2 cells were able to exert the same effect as P0 cells when cocultured with P2 cells. In conclusion, under proper culture conditions, redifferentiated passaged chondrocytes behave similarly to primary chondrocytes. This coculture system approach can be used to increase the number of differentiated chondrocytes that can be obtained by classical monolayer cell expansion and represents a novel way to acquire sufficient cell numbers for cartilage tissue engineering.

Soluble signalling factors derived from differentiated cartilage tissue affect chondrogenic differentiation of rat adult marrow stromal cells.

BACKGROUND: Chondral defects show lack of proper regeneration whereas osteochondral lesions display limited regeneration capacity. Latter is probably due to immigration of chondroprogenitor cells from the subchondral bone. Known chondroprogenitor cells for cartilage tissues are multi-potent adult marrow stromal or mesenchymal stem cells (MSCs). In vitro chondrogenic differentiation of these precursor cells usually require cues from growth and signalling factors provided in vivo by surrounding tissues and cells. We hypothesise that signalling factors secreted by differentiated cartilage tissue can initiate and maintain chondrogenic differentiation status of MSCs. METHODS: To study such paracrine communication between allogenic rat articular cartilage and rat MSCs embedded in alginate beads a novel coculture system without addition of external growth factors has been established. RESULTS: Impact of cartilage on differentiating MSCs was observed at two different time points. Firstly, sustained expression of Sox9 was observed at an early stage which indicated induction of chondrogenic differentiation. Secondly, late stage repression of collagen X indicated pre-hypertrophic arrest of differentiation. In the culture supernatant we have identified vascular endothelial growth factor alpha (VEGF-164 alpha), matrix metalloproteinase (MMP) -13 and tissue inhibitors of MMPs (TIMP-1 and TIMP-2) which could be traced back either to the cartilage explant or to the MSCs under the influence of cartilage. CONCLUSION: The identified factors might be involved in regulation of collagen X gene and protein expression and therefore, may have an impact on the control and regulation of MSCs differentiation.

Influence of cellular microenvironment and paracrine signals on chondrogenic differentiation.

Articular cartilage disorders and injuries often result in life long chronic pain and compromised quality of life, thus regeneration of articular cartilage is a persistent challenge to medical science. One of the most promising therapeutic approaches is cell based tissue engineering which provides a healthy population of cells to the injured site and requires differentiated chondrocytes from the uninjured site as base material. Use of healthy chondrocytes has several limitations and an excellent alternative cell population could be adult marrow stromal cells/mesenchymal stem cells (MSCs) which are known to possess extensive proliferation potential and proven capability to differentiate into chondrocytes. Both, in vivo and in vitro pliability of MSCs and chondrocytes greatly depends on their microenvironment. Gene and protein expression profiles of both the cell types can be altered by soluble factors from surrounding tissue/ cells or by direct cellular contact. For MSC or chondrocyte-based cartilage repair, inhibition of hypertrophy and stabilization of the cartilaginous phenotype in the implant is a prerequisite for success and long lasting vitality of the repaired tissue.

CD45-positive cells of haematopoietic origin enhance chondrogenic marker gene expression in rat marrow stromal cells.

Adult mesenchymal stem cells (MSCs) can be readily isolated from bone marrow, expanded in culture and subsequently subjected to differentiation into various connective tissue lineages. In general, for animal studies separation of MSCs from other bone marrow-derived cells is achieved by sole adherence to plastic surface of tissue culture flasks; however, this procedure produces a heterogeneous cell population containing CD45-positive haematopoietic cells (HCs) and haematopoietic stem cells (HSCs). It is known, that mixed cell cultures consisting of cocultures of differentiated somatic cells with adult stem cells promote differentiation towards specific cell lineages. For determining the effect of the CD45-positive cell population on the differentiation potential of MSCs, we sorted out the bone marrow-derived adherent cells by immunomagnetic technique (MACS) to attain a subpopulation of CD45-depleted cells. Herein, we show that the presence of adherent CD45-positive HCs not only promote expression of the chondrogenic marker genes Col2a1, COMP and Sox9, but also of Col1a1, Col10a1 and to a certain degree Cbfa1 in MSCs when cultured in an appropriate three-dimensional environment. These observations constitute a step towards unravelling the influence of haematopoietic cells on chondrogenic differentiation of MSCs.