Regulation of scleral cell contraction by transforming growth factor-β and stress competing roles in myopic growth

Reduced extracellular matrix accumulation in the sclera of myopic eyes leads to increased ocular extensibility and is related to reduced levels of scleral transforming growth factor-β (TGF-β). The current study investigated the impact of this extracellular environment on scleral cell phenotype and cellular biomechanical characteristics. Scleral cell phenotype was investigated in vivo in a mammalian model of myopia using the myofibroblast marker, α-smooth muscle actin (α-SMA). In eyes developing myopia α-SMA levels were increased, suggesting increased numbers of contractile myofibroblasts, and decreased in eyes recovering from myopia. To understand the factors regulating this change in scleral phenotype, the competing roles of TGF-β and mechanical stress were investigated in scleral cells cultured in three-dimensional collagen gels. All three mammalian isoforms of TGF-β altered scleral cell phenotype to produce highly contractile, α-SMA-expressing myofibroblasts (TGF-β3 > TGF-β2 > TGF-β1). Exposure of cells to the reduced levels of TGF-β found in the sclera in myopia produced decreased cell-mediated contraction and reduced α-SMA expression. These findings are contrary to the in vivo gene expression data. However, when cells were exposed to both the increased stress and the reduced levels of TGF-β found in myopia, increased α-SMA expression was observed, replicating in vivo findings. These results show that although reduced scleral TGF-β is a major contributor to the extracellular matrix remodeling in the myopic eye, it is the resulting increase in scleral stress that dominates the competing TGF-β effect, inducing increased α-SMA expression and, hence, producing a larger population of contractile cells in the myopic eye.

A study on small business enterprises in Bangladesh : searching for growth factors and obstacles

Small businesses are the heart of the market-based economy with their business operations in rural and urban areas of developed and developing countries. In Bangladesh, small business enterprises are playing a significant role by contributing to the production and services, employmnet and thereby to GDP. But these are found to face servere competition and different types of constraints. As a result, these have not achieved substantial growth. In view of this, the present study is primarily aimed at identifying factors and obstacles that influence the growth of SBEs. Finally, the paper suggests some policy measures which are expected to excel the growth of SBEs.

Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34+ hematopoietic progenitor cells

Background aims. Cord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters.

Methods. This is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34 hematopoietic cells utilizing clinically approvable reagents and methodologies throughout.

Results. Optimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes.

Conclusions. This study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.

TGF-Ý signalling in human skeletal muscle regeneration and diabetes

The TGF-Ý superfamily comprises a large group of proteins with many effects on muscle growth and maturation. The molecular regulation of skeletal muscle regeneration and metabolism in response to prominent superfamily members, myostatin and TGF-Ý1, were analysed, demonstrating the importance of this pathway in controlling how muscles grow and are regulated.

Biomechanics of the sclera in myopia: Extracellular and cellular factors

Excessive axial elongation of the eye is the principal structural cause of myopia. The increase in eye size results from active remodelling of the sclera, producing a weakened scleral matrix. The present study will detail the biomechanics of the sclera and highlight the matrix and cellular factors important in the control of eye size. Scleral elasticity (load vs. tissue extension) and creep rate (tissue extension vs. time) have been measured postmortem in human eyes. Animal models of myopia have allowed the direct relevance of scleral biomechanics to be investigated during myopia development. Recently, data on tissue matrices incorporating scleral fibroblasts have highlighted the role of cellular contraction in scleral biomechanics. Scleral elasticity is increased in eyes developing myopia, with a reduction in the failure load of the tissue. Scleral creep rate is increased in the sclera from eyes developing myopia, and reduced in eyes recovering from myopia. These changes in biomechanical properties of the sclera occur early in the development of myopia (within 24 h). Alterations in scleral biomechanics during myopia development have been attributed to changes in matrix constituents, principally reduced collagen content. Although the biochemical structure of the sclera plays a critical role in defining the mechanical properties, recent studies investigating the cellular mechanics of the sclera, implicate myofibroblasts in scleral biomechanics. Scleral myofibroblasts have the capacity to contract the matrix and are regulated by tissue stress and growth factors such as transforming growth factor-ß. Changes in these regulatory factors have been observed during myopia development, implicating cellular factors in the resultant weakened sclera. Changes in the biomechanical properties of the sclera are important in facilitating the increase in axial length that results in myopia. Understanding the matrix and cellular factors contributing to the weakened sclera may aid in the development of a clinically appropriate treatment for myopia.

Fibroblast growth factor-2 over-rides insulin-like growth factor-I induced proliferation and cell survival in human neuroblastoma cells

The insulin-like growth factor (IGF) system is a key regulator of cell growth, survival and differentiation, and these functions are co-modulated by other growth factors including fibroblast growth factor-2 (FGF-2). To investigate IGF/FGF interactions in neuronal cells, we employed neuroblastoma cells (SK-N-MC). In serum free conditions proliferation of the SK-N-MC cells was promoted by IGF-I (25 ng/ml), but blunted by FGF-2 (50 ng/ml). IGF-I-induced proliferation was abolished in the presence of FGF-2 even when IGF-I was used at 100 ng/ml. In addition to our previously described FGF-2 induced proteolytic cleavage of IGFBP-2, we found that FGF-2 increased IGFBP-6 levels in conditioned medium (CM) without affecting IGFBP-6 mRNA abundance. Modulation of IGFBP-2 and -6 levels were not significant mechanisms involved in the blockade of IGF-I action since the potent IGF-I analogues [QAYL]IGF-I and des(1-3)IGF-I (minimal IGFBP affinity) were unable to overcome FGF-2 inhibition of cell proliferation. FGF-2 treated cells showed morphological differentiation expressing the TUJ1 neuronal marker while cells treated with IGF-I alone showed no morphological change. When IGF-I was combined with FGF-2, however, cell morphology was indistinguishable from that seen with FGF-2 alone. FGF-2 inhibited proliferation and enhanced differentiation was also associated with a 70% increase in cell death. Although IGF-I alone was potently anti-apoptotic (60% decreased), IGF-I was unable to prevent apoptosis when administrated in combination with FGF-2. Gene-array analysis confirmed FGF-2 activation of the intrinsic and extrinsic apoptotic pathways and blockade of IGF anti-apoptotic signaling. FGF-2, directly and indirectly, overcomes the proliferative and anti-apoptotic activity of IGF-I by complex mechanisms, including enhancement of differentiation and apoptotic pathways, and inhibition of IGF-I induced anti-apoptotic signalling. Modulation of IGF binding protein abundance by FGF-2 does not play a significant role in inhibition of IGF-I induced mitogenesis.

Expression of the disintegrin metalloprotease ADAM-10 in prostate cancer and its regulation by dihydrotestosterone, insulin like growth factor -1 and epidermal growth factor in the prostate cell model LNCaP

Purpose: The disintegrin metalloprotease ADAM-10 is a multidomain metalloprotease that is potentially significant in tumor progression due to its extracellular matrix-degrading properties. Previously, ADAM-10 mRNA was detected in prostate cancer (PCa) cell lines; however, the presence of ADAM-10 protein and its cellular localization, regulation, and role have yet to be described. We hypothesized that ADAM-10 mRNA and protein may be regulated by growth factors such as 5α-dihydrotestosterone, insulin-like growth factor I, and epidermal growth factor, known modulators of PCa cell growth and invasion.

Experimental Design: ADAM-10 expression was analyzed by in situ hybridization and immunohistochemistry in prostate tissues obtained from 23 patients with prostate disease. ADAM-10 regulation was assessed using quantitative reverse transcription-PCR and Western blot analysis in the PCa cell line LNCaP.

Results: ADAM-10 expression was localized to the secretory cells of prostate glands, with additional basal cell expression in benign glands. ADAM-10 protein was predominantly membrane bound in benign glands but showed marked nuclear localization in cancer glands. By Western blot, the 100-kDa proform and the 60-kDa active form of ADAM-10 were synergistically up-regulated in LNCaP cells treated with insulin-like growth factor I plus 5α-dihydrotestosterone. Epidermal growth factor also up-regulated both ADAM-10 mRNA and protein.

Conclusions: This study describes for the first time the expression, regulation, and cellular localization of ADAM-10 protein in PCa. The regulation and membrane localization of ADAM-10 support our hypothesis that ADAM-10 has a role in extracellular matrix maintenance and cell invasion, although the potential role of nuclear ADAM-10 is not yet known.

Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells.

Using two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2-D SDS-PAGE) of 32P-labeled cytosolic and membrane extracts, we identified a 21.5 kDa phosphoprotein with an isoelectric point of 6.0 in NFS-60 cells that was phosphorylated maximally at 15 min by treatment with granulocyte-colony stimulating factor (G-CSF) but not with interlevkin-3 (IL-3) or colony-stimulating factor-1 (macrophage-colony stimulating factor (CSF-1 (M-CSF)). The phosphorylation of this protein, designated 21.5/6.0, was unaffected by a series of antiproliferative agents [32]. These findings suggested that the 21.5/6.0 phosphoprotein may be involved in specific G-CSF-mediated biological responses such as activation and/or differentiation. We sought to characterize this 21.5/6.0 by a novel combination of 2-D SDS-PAGE and hydroxyapatite (HTP)-chromatography. Amino acid sequence determination of 21.5/6.0 revealed it to share a high level of homology with copper/zinc superoxide dismutase (Cu/Zn-SOD), indicating that a Cu/Zn-SOD is phosphorylated following treatment with G-CSF. This is the first report of the phosphorylation and possible involvement of Cu/Zn-SOD protein in granulocyte activation/differentiation events. In addition, Cu/Zn-SOD levels and activity were diminished by G-CSF but not IL-3 treatment. This new protocol combining 2-D SDS-PAGE and HTP-chromatography allows the characterization of low abundance phosphoproteins involved in the cellular responses to G-CSF and presumably to other cytokines/growth factors.

Exercise-induced activation of STAT3 signaling is increased with age

Activation of the transcription factor signal transducers and activators of transcription (STAT) 3 is common to many inflammatory cytokines and growth factors, with recent evidence of involvement in skeletal muscle regeneration. The purpose of this study was to determine whether STAT3 signaling activation is regulated differentially, at rest and following intense resistance exercise, in aged human skeletal muscle. Skeletal muscle biopsies were harvested from healthy younger (n = 11, 20.4 ± 0.8 years) and older men (n = 10, 67.4 ± 1.3 years) under resting conditions and 2 h after the completion of resistance exercise. No differences were evident at rest, whereas the phosphorylation of STAT3 was significantly increased in old (23-fold) compared to young (5-fold) subjects after exercise. This correlated with significantly higher induction of the STAT3 target genes including; interleukin-6 (IL-6), JUNB, c-MYC, and suppressor of cytokine signaling (SOCS) 3 mRNA in older subjects following exercise. Despite increased SOCS3 mRNA, cellular protein abundance was suppressed. SOCS3 protein is an important negative regulator of STAT3 activation and cytokine signaling. Thus, in aged human muscle, elevated responsiveness of the STAT3 signaling pathway and suppressed SOCS3 protein are evident following resistance exercise. These data suggest that enhanced STAT3 signaling responsiveness to proinflammatory factors may impact on mechanisms of muscle repair and regeneration.

Suppressors of cytokine signaling : functions in normal biology and roles in disease

Suppressor of cytokine signaling (SaCS) proteins have been identified as key negative regulators of cytokine and growth factor signaling. Therefore, given the diverse roles played by cytokines and growth factors in development and disease, it is not surprising that the sacs proteins themselves possess equally diverse and important functions, such as the control of hematopoiesis, immune function, growth and placental development. Significantly, more recent studies are increaSingly highlighting the crucial roles played by SOCS proteins in disease, particularly their tumor suppressor and anti-infammatory functions. Collectively, this research has served to confirm the importance of this class of proteins and suggests that therapeutic strategies for modulating SOCS proteins might be relevant for a range of diseases.

Identification of novel genes differentially expressed in haemopoietic progenitor cells

The biochemical and molecular processes that maintain the stem cell pool, and govern the proliferation and differentiation of haemopoietic stem/progenitor cells (HSPCs) have been widely investigated but are incompletely understood. The purpose of this study was to identify and characterise novel genes that may play a part in regulating the mechanisms that control the proliferation, differentiation and self-renewal of human HSPCs. Reverse transcription differential display polymerase chain reaction (dd-PCR) was used to identify differences in gene expression between a HSPC population defined by expression of the CD34 phenotype, and the more mature CD34 depleted populations. A total of 6 differentially expressed complementary deoxyribonucleic acid (cDNA) sequences were identified. Four of these transcripts were homologous to well characterised genes, while two (band 1 and band 20) were homologous to unknown and uncharacterised partial gene sequences on the GenBank database and were thus chosen for further investigation. The partial cDNA sequences for band 1 and band 20 were designated ORP-3 and MERP-1 (respectively) due to homologies with other well-characterised gene families. Differential expression of the ORP-3 and MERP-1 genes was confirmed using Taqman™ real-time polymerase chain reaction (PCR) with 3 - 4-fold and 4-10 -fold higher levels in the CD34+ fractions of haemopoietic cells compared to CD34- populations respectively. Additionally, expression of both these genes was down regulated with proliferation and differentiation of CD34+ cells further confirming higher expression in a less differentiated subset of haemopoietic cells. The full coding sequences of ORP-3 and MERP-1 were elucidated using bioinformatics, rapid amplification of cDNA ends (RACE) and PCR amplification. The MERP-1 cDNA is 2600 nucleotides (nt) long, and localizes by bioinformatics to chromosome 7.. It consists of three exons and 2 introns spanning an entire length of 31.4 kilobases (kb). The MERP-1 open reading frame (ORF) codes for a putative 344 amino acid (aa) type II transmembrane protein with an extracellular C-terminal ependymin like-domain and an intracellular N-terminal sequence with significant homology to the cytoplasmic domains of members of the protocadherin family of transmembrane glycoproteins. Ependymins and protocadherins are well-characterised calcium-dependant cell adhesion glycoproteins. Although the function of MERP-1 remains to be elucidated, it is possible that MERP-1 like its homologues plays a role in calcium dependent cell adhesion. Differential expression of the MERP-1 gene in haemopoietic cells suggests a role in haemopoietic stem cell proliferation and differentiation, however, its broad tissue distribution implies that it may also play a role in many cell types. Characterization of the MERP-1 protein is required to elucidate these possible roles. The ORP-3 cDNA is 6631nt long, and localizes by bioinformatics to chromosome 7pl5-p21. It consists of 23 exons and 22 introns spanning an entire length of 183.5kb. The ORP-3 ORF codes for a putative 887aa protein which displays the consensus sequence for a highly conserved oxysterol-binding domain. Other well-characterised proteins expressing these domains have been demonstrated to bind oxysterols (OS) in a dose dependant fashion. OS are hydroxylated derivatives of cholesterol Their biological activities include inhibition of cholesterol biosynthesis and cell proliferation in a variety of cell types, including haemopoietic cells. Differential expression of the ORP-3 gene in haemopoietic cells suggests a possible role in the transduction of OS effects on haemopoietic cells, however, its broad tissue distribution implies that it may also play a role in many cell types. Further investigation of ORP-3 gene expression demonstrates a significant correlation with CD34+ sample purity, and 2-fold higher expression in a population of haemopoietic cells defined by the CD34+38- phenotype compared to more mature CD34+38+ cells. This finding, taken together with the previous observation of down-regulation of ORP-3 expression with proliferation and differentiation of CD34+ cells, indicates that ORP-3 expression may be higher in a less differentiated subset of cells with a higher proliferative capacity. This hypothesis is supported by the observation that expression of the ORP-3 gene is approximately 2-fold lower in differentiated HL60 promyelocytic cells compared to control, undifferentiated cells. ORP-3 expression in HL60 cells during normal culture conditions was also found to vary with expression positively correlated with cell number. This indicates a possible cell cycle effect on ORP-3 gene expression with levels highest when cell density, and therefore the percentage of cells in G(0)/G(1) phase of the cell cycle is highest. This observation also correlates with the observation of higher ORP-3 expression in CD34+38-cells, and in CD34+ and HL60 cells undergoing OS induced and camptothecin induced apoptosis that is preceded by cell cycle arrest at G(0)/G(1). Expression of the ORP-3 gene in CD34+ HSPCs from UCB was significantly decreased to approximately half the levels observed in control cells after 24 hours incubation in transforming growth factor beta-1 (TGFâl). As ≥90% of these cells are stimulated into cell cycle entry by TGFâl, this observation further supports the hypothesis that ORP-3 expression is highest when cells reside in the G(0)/G(1) phase of the cell cycle. Data obtained from investigation of ORP-3 gene expression in synchronised HL60 cells however does not support nor disprove this hypothesis. Culture of CD34+ enriched HSPCs and HL60 cells with 25-OHC significantly increased ORP-3 gene expression to approximately 1.5 times control levels. However, as 25-OHC treatment also increased the percentage of apoptotic cells in these experiments, it is not valid to make any conclusions regarding the regulation of ORP-3 gene expression by OS. Indeed, the observation that camptothecin induced apoptosis also increased ORP-3 gene expression in HL60 cells raises the possibility that up-regulation of ORP-3 gene expression is also associated with apoptosis, Taken together, expression of the ORP-3 gene appears to be regulated by differentiation and apoptosis of haemopoietic progenitors, and may also be positively associated with proliferative and G(0)/G(1) cell cycle status indicating a possible role in all of these processes. Given the important regulatory role of apoptosis in haemopoiesis and differential expression of the ORP-3 gene in haemopoietic progenitors, final investigations were conducted to examine the effects OS on human HSPCs. Granulocyte/macrophage colony forming units (CFU-GM) generated from human bone marrow (ABM) and umbilical cord blood (UCB) were grown in the presence of varying concentrations of three different OS - 7keto-cholesterol (7K-C), 7beta-hydroxycholesterol (7p-OHC) and 25-hydroxycholesterol (25-OHC). Similarly, the effect of OS on HL60 and CD34+ cells was investigated using annexin-V staining and flow cytometry to measure apoptosis. Reduction of nitroblue tetrazolium (NBT) was used to assess differentiative status of HL60 cells. CFU-GM from ABM and HL60 growth was inhibited by all three OS tested, with 25-OHC being the most potent. 25-OHC inhibited ≥50% of bone marrow CFU-GM and ≥95% of HL60 cell growth at a level of 1 ug/ml. Compared to UCB, CFU-GM derived from ABM were more sensitive to the effects of all OS tested. Only 25-OHC and 7(5-OHC significantly inhibited growth of UCB derived CFU-GM. OS treatment increased the number of annexin-V CD34+ cells and NBT positive HL60 cells indicating that OS inhibition of CFU-GM and HL60 cell growth can be attributed to induction of apoptosis and differentiation. From these studies, it can be concluded that dd-PCR is an excellent tool for the discovery of novel genes expressed in human HSPCs. Characterisation of the proteins encoded by the novel genes ORP-3 and MERP-1 may reveal a regulatory role for these genes in haemopoiesis. Finally, investigations into the effects of OS on haemopoietic progenitor cells has revealed that OS are a new class of inhibitors of HSPC proliferation of potential relevance in vivo and in vitro.

Identification and characterisation of novel genes involved in skeletal muscle hypertrophy

There is mounting evidence in support of the view that skeletal muscle hypertrophy results from the complex and coordinated interaction of numerous signalling pathways. Well characterised components integral to skeletal muscle adaptation include the transcriptional activity of the members of the myogenic regulatory factors, numerous secreted peptide growth factors, and the regenerative potential of satellite cells. Whilst studies investigating isolated components or pathways have enhanced our current understanding of skeletal muscle hypertrophy, our knowledge of how all of these components react in concert to a common stimulus remains limited. The broad aim of this thesis was to identify and characterise novel genes involved in skeletal muscle hypertrophy. We have created a customised human skeletal muscle specific microarray which contains ∼11,000 cDNA clones derived from a normalised human skeletal muscle cDNA library as well as 270 genes with known functional roles in human skeletal muscle. The first aspect of this thesis describes the production of the microarray and evaluates the robustness and reproducibility of this analytical technique. Study one aimed to use this microarray in the identification of genes that are differentially expressed during the forced differentiation of human rhabdomyosarcoma cells, an in vitro model of skeletal muscle development. Firstly using this unique model of aberrant myogenic differentiation we aimed to identify genes with previously unidentified roles in myogenesis. Secondly, the data from this study permitted the examination of the performance of the microarray in detecting differential gene expression in a biological system. We identified several new genes with potential roles in the myogenic arrest of rhabdomyosarcoma and further characterised the expression of muscle specific genes in rhabdomyosarcoma differentiation. In study two, the molecular responses of cell cycle regulators, muscle regulatory factors, and atrophy related genes were mapped in response to a single bout of resistance exercise in human skeletal muscle. We demonstrated an increased expression of MyoD, myogenin and p21, whilst the expression of myostatin was decreased. The results of this study contribute to the existing body of knowledge on the molecular regulation skeletal muscle to a hypertrophic stimulus. In study three, the muscle samples collected in study two were analysed using the human skeletal muscle specific microarray for the identification of novel genes with potential roles in the hypertrophic process. The analysis uncovered four interesting genes (TXNIP, MLP, ASB5, FLJ 38973) that have not previously been examined in human skeletal muscle in response to resistance exercise. The functions of these genes and their potential roles in skeletal muscle are discussed. In study four, the four genes identified in study three were examined in human primary skeletal muscle cell cultures during myogenic differentiation. Human primary skeletal muscle cells were derived from the vastus lateralis muscle of 8 healthy volunteers (6 males and 2 females). Cell cultures were differentiated using serum withdrawal and serum withdrawal combined with IGF-1 supplementation. Markers of the cell proliferation, cell cycle arrest and myogenic differentiation were examined to assess the effectiveness of the differentiation stimulus. Additionally, the expressions of TXNIP, MLP, ASB5 and FLJ 38973 measured in an attempt to characterise further their roles in skeletal muscle. The expression of TXNIP changed markedly in response to both differentiation stimuli, whilst the expression of the remaining genes were not altered. Therefore it was suggested that expression of these genes might be responsive to the mechanical strain or contraction induced by the resistance exercise. In order to examine whether these novel genes responded specifically to resistance type exercise, their expression was examined following a single bout of endurance exercise. The expression of TXNIP, MLP, and FLJ 38973 remained unchanged whilst ASB5 increased 30 min following the cessation of the exercise.

Early inflammatory and myogenic responses to resistance exercise in the elderly

Introduction and Methods: This study compared changes in myokine and myogenic genes following resistance exercise (3 sets of 12 repetitions of maximal unilateral knee extension) in 20 elderly men (67.8 ± 1.0 years) and 15 elderly women (67.2 ± 1.5 years). Results: Monocyte chemotactic protein (MCP)-1, macrophage inhibitory protein (MIP)-1β, interleukin (IL)-6 and MyoD mRNA increased significantly (P < 0.05), whereas myogenin and myostatin mRNA decreased significantly after exercise in both groups. Macrophage-1 (Mac-1) and MCP-3 mRNA did not change significantly after exercise in either group. MIP-1β, Mac-1 and myostatin mRNA were significantly higher before and after exercise in men compared with women. In contrast, MCP-3 and myogenin mRNA were significantly higher before and after exercise in the women compared with the men. Conclusions: In elderly individuals, gender influences the mRNA expression of certain myokines and growth factors, both at rest and after resistance exercise. These differences may influence muscle regeneration following muscle injury.

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS-5 stromal cell line

Lineage-specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bonemarrowfailure and immunodeficiencies. The currentmethods forHSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell-free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell- free matrices from a murine bone marrow stromal cell line (MS-5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O2 tensions. These acellularmatrices were used as biological scaffolds for the lineage-specific expansion of magnetically sorted CD34+ cells and the results were evaluated by flow cytometry and colony-forming assays. We could get up to 80-fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering-based ex vivo HSPC expansion, and itmay also provide insights into the constitution of the niche in which these cells reside in the bone marrow.