Asha Guttapalli

Differentiation of mesenchymal stem cells towards a nucleus pulposus-like phenotype in vitro: implications for cell-based transplantation therapy.

Objective. Because mesenchymal stem cells can differentiate into chondrocyte-like cells, we ask the question, can mesenchymal stem cells commit to the nucleus pulposus phenotype? Background. Back pain, a significant source of morbidity in our society, is linked to degenerative changes of the intervertebral disc. Absence of suitable graft tissue limits therapeutic approaches for repair of disc tissue. For this reason, there is considerable interest in developing “tissue engineering” strategies for the regeneration of the nucleus pulposus. Methods. Rat mesenchymal stem cells were immobilized in 3-dimensional alginate hydrogels and cultured in a medium containing transforming growth factor-&bgr;1 under hypoxia (2% O2) and normoxia (20% O2). Mesenchymal stem cells were examined by confocal microscopy to evaluate their viability and metabolic status after labeling with Celltracker green, a thiol sensitive dye, and Mitotracker red, a dye sensitive to the mitochondrial membrane potential. Flow cytometry, semiquantitative reverse transcription polymerase chain reaction and Western blot analysis were carried out to evaluate phenotypic and biosynthetic activities and the signaling pathways involved in the differentiation process. Results. Under hypoxic conditions, mesenchymal stem cells formed large aggregates and exhibited positive Celltracker and Mitotracker signals. Glucose transporter-3, matrix metalloproteinase-2, collagen type II and type XI, and aggrecan mRNA and protein expression was upregulated, whereas there was no change in the levels of decorin, biglycan, fibromodulin, and lumican. Hypoxia maintained the expression of CD44 (hyaluronan receptor), ALCAM (CD166), and endoglin (transforming growth factor-&bgr; receptor). Likewise, expression of &bgr;3 and &agr;2 integrin was upregulated. Transforming growth factor-&bgr; treatment increased MAPK activity and Sox-9, aggrecan, and collagen type II gene expression. Basal levels of the phosphorylated MAPK isoform ERK1/2, but not p38, were higher under hypoxic conditions than normoxia, and its activation was further augmented by treatment of cells with transforming growth factor-&bgr;. In hypoxia, transforming growth factor-&bgr; sustained phosphorylated p38 expression for an extended time period. Pharmacological inhibition of ERK1/2 and p38 enzymatic activity resulted in a decrease in Sox-9, aggrecan, and collagen type II mRNA levels. Conclusions. Our results indicate that hypoxia and transforming growth factor-&bgr; drive mesenchymal stem cell differentiation towards a phenotype consistent with that of the nucleus pulposus. Measurement of selected signaling molecules and response to specific inhibitors suggest involvement of MAPK signaling pathways. It is concluded that mesenchymal stem cells could be used to repopulate the damaged or degenerate intervertebral disc.

Evidence for skeletal progenitor cells in the degenerate human intervertebral disc.

Study Design. To identify and characterize endogenous progenitor cell population from intervertebral disc. Objective. To determine if progenitor cells exist in degenerate human discs. Summary of Background Data. Back pain, a significant source of morbidity in our society, is directly linked to the pathology of the intervertebral disc. Because disc disease is accompanied by a loss of cellularity, there is considerable interest in regeneration of cells of both the anulus fibrosus (AF) and nucleus pulposus (NP). Methods. To determine if skeletal progenitor cells are present in the disc, samples were obtained from the degenerate AF and NP of 5 patients (Thompson grade 2 and 3, mean age 34 ± 7.6 years) undergoing anterior cervical discectomy and fusion procedures as well as adult rat lumbar spine. Results. Cells isolated from degenerate human tissues expressed CD105, CD166, CD63, CD49a, CD90, CD73, p75 low affinity nerve growth factor receptor, and CD133/1, proteins that are characteristic of marrow mesenchymal stem cells. In osteogenic media, there was an induction of alkaline phosphatase activity and expression of alkaline phosphatase, osteocalcin, and Runx-2 mRNA. When maintained in adipogenic media, a small percentage of cells displayed evidence of adipogenic differentiation: accumulation of cytosolic lipid droplets and increased expression of peroxisome proliferator-activated receptor-&ggr;2 and lipoporotein lipase mRNA. AF- and NP-derived cells also evidenced chondrogenic differentiation. CD133 (+) cells in the AF were able to commit to either the chondrogenic or adipogenic lineages. The results of the human disc studies were confirmed using cell derived from the NP and AF tissue of the mature rat disc. Conclusion. The analytical data indicated that the pathologically degenerate human disc contained populations of skeletal progenitor cells. These findings suggest that these endogenous progenitors may be used to orchestrate the repair of the intervertebral disc.

Nucleus pulposus cells express HIF‐1α under normoxic culture conditions: A metabolic adaptation to the intervertebral disc microenvironment

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF‐1α, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF‐1α were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF‐1α subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF‐1α protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF‐1α protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF‐1α transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF‐1α is a metabolic adaptation of nucleus pulposus cells to a unique oxygen‐limited microenvironment. The study confirmed that HIF‐1α can be used as a phenotypic marker of nucleus pulposus cells. J. Cell. Biochem. 98: 152–159, 2006.

Toward an optimum system for intervertebral disc organ culture : TGF-β3 enhances nucleus pulposus and anulus fibrosus survival and function through modulation of TGF-β-R expression and ERK signaling

Study Design. Rat lumbar discs comprising nucleus pulposus, annulus fibrosus, and cartilaginous endplates were cultured for 1 week in a specialized media containing either TGF-β1 or TGF-β3. Role of TGF-β isoforms on cell function was evaluated. Objective. To develop an in vitro organ culture of rat intervertebral disc and evaluate effects of TGF-β3 on disc cell function. Summary of Background Data. An in vitro model system is of considerable value in understanding the cell biology of the intervertebral disc. Development of a useful organ culture model would enhance understanding of disc function in health and disease. Materials and Methods. Rat lumbar intervertebral discs were maintained in organ culture in media supplemented with TGF-β3 or TGF-β1 for 1 week. Tissue morphology was studied using routine histologic, histochemical and immunohistochemical techniques. Cell function was assessed by gene expression, sulfate incorporation, and Western blot analysis. Results. After 1 week in culture with TGF-β3 and TGF-β1, the gross morphology and tissue architecture of the disc were preserved. TUNEL analysis indicated that there was no evidence of cell death in the nucleus pulposus or the anulus fibrosus. The level of Alcian blue staining in the nucleus pulposus was similar to that of the freshly isolated disc. However, when compared with TGF-β1, TGF-β3 elevated the expression of critical matrix genes, enhanced [35S] incorporation into proteoglycans, preserved the expression of TGF-β receptors, and decreased aggrecan turnover. There was also increased activation (phosphorylation) of ERK, a critical signaling protein. Moreover, inhibition of ERK activity, in the presence TGF-β3, resulted in suppression of collagen Type II, aggrecan, TGF-β-RI, TGF-β-RII and TGF-β-RIII mRNA expression. Conclusions. TGF-β3 maintains the phenotype of disc cells in organ culture. It exerts this effect, in part, by elevating the levels of activated ERK1/2, which in turn regulates the expression of TGF-β-RI and TGF-β-RII.

MEK/ERK Signaling Controls Osmoregulation of Nucleus Pulposus Cells of the Intervertebral Disc by Transactivation of TonEBP/OREBP

Earlier studies have shown that intervertebral disc cells express TonEBP, a transcription factor that permits adaptation to osmotic stress and regulates aggrecan gene expression. However, the mechanism of hyperosmotic activation of TonEBP in disc cells is not known. Results of this study show that hypertonic activation of ERK signaling regulates transactivation activity of TonEBP, modulating its function.

TonEBP/OREBP is a regulator of nucleus pulposus cell function and survival in the intervertebral disc

The nucleus pulposus is an aggrecan-rich hydrated tissue that permits the intervertebral disc to resist compressive loads. Adaptation to loading is achieved through an elevation in disc osmolarity mediated by the numerous charged glycosoaminoglycan side chains of the aggrecan molecule. The goal of this investigation was to determine the functional role of the osmo-regulatory protein, TonEBP, in cells of the nucleus pulposus. We found that TonEBP and its downstream target genes were robustly expressed in the tissues of the disc. Above 330 mosmol/kg, cultured nucleus pulposus cells up-regulated target genes TauT, BGT-1, and SMIT; above 450 mosmol/kg, there was raised expression of HSP-70. In hypertonic media there was activation of TauT and heat shock protein-70 (HSP-70) reporter activity and increased binding of TonEBP to the TonE motif. When cells were transfected with the dominant-negative form of TonEBP (DN-TonEBP) there was suppression of TauT and HSP-70 reporter gene expression; pTonEBP enhanced reporter gene expression. Moreover, in hypertonic media, forced expression of DN-TonEBP induced apoptosis. We suppressed TonEBP using small interfering RNA technique and noted a decrease in TauT reporter activity in isotonic as well as hyperosmolar media. Finally, we report that the aggrecan promoter contains two conserved TonE motifs. To evaluate the importance of these motifs, we overexpressed DN-TonEBP and partially silenced TonEBP using small interfering RNA. Both approaches resulted in suppression of aggrecan promoter activity. It is concluded that TonEBP permits the disc cells to adapt to the hyperosmotic milieu while autoregulating the expression of molecules that generate the unique extracellular environment.

Hypoxia Activates Mapk Activity in Rat Nucleus Pulposus Cells: Regulation of Integrin Expression and Cell Survival

Objective. The aim of the present study was to investigate whether activation of MAPK subtypes ERK and p38 influences integrin expression and promotes nucleus pulposus cell survival in hypoxia. Summary of Background Data. We have recently shown that in a low oxygen environment, rat nucleus pulposus cells activate phosphatidylinositol 3-kinase/Akt (PI3K/Akt) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways. However, the role of these signaling pathways in regulating cell matrix interactions is not known. Methods. Rat nucleus pulposus cells were cultured in hypoxia (2% O2) or normoxia (20% O2). Western blotting and kinase assay were used to analyze expression of MAPK signaling molecules. Cell attachment to collagen was studied using an adhesion assay, whereas flow cytometry and RT-PCR were performed to quantify integrin receptor expression. Results. In a hypoxic environment (2% O2), rat nucleus pulposus cells showed a persistent phosphorylation of p38 and ERK proteins; pERK catalyzed the phosphorylation of Elk1-Gst fusion protein. When ERK activity was blocked, cell adhesion to Type II collagen, one of the major extracellular matrix proteins in the nucleus pulposus tissue, was impaired. A similar inhibitory effect on collagen adhesion was observed when nucleus pulposus cells were treated with an antibody to &agr;2 integrin. Furthermore, when ERK activity was inhibited, there was a decrease in &agr;2 integrin mRNA expression. In contrast to ERK, inhibition of p38 activity did not modulate &agr;2 integrin subunit mRNA expression. Likewise, inhibition of ERK, but not p38, resulted in downregulation of nucleus pulposus &agr;2 integrin protein levels and blocked cell survival in hypoxia. Conclusions. Hypoxia activated MAPK signaling pathway activity in nucleus pulposus cells. ERK, but not p38, regulated &agr;2 integrin expression and cell survival.

Osteogenic potential of adult human stem cells of the lumbar vertebral body and the iliac crest.

Study Design. Marrow was aspirated from the vertebral body (VB) and iliac crest (IC) of patients undergoing lumbar spinal surgery, following an approved protocol. Progenitor cells were isolated using standard culture conditions and their osteogenic potential evaluated. Objective. To evaluate the osteogenic potential of mesenchymal stem cells (MSCs) isolated from the bone marrow of the human VB. Summary of Background Data. IC marrow grafting during cervical discectomy and fusion procedure is associated with donor site morbidity. Since the VB contains marrow cells, it may be possible to circumvent this problem by using this tissue for osseous graft supplementation. However, there is paucity of information concerning the osteogenic potential of non-IC-derived progenitor cells. Herein, we address this issue. Methods. Marrow samples from VB of patients undergoing lumbar spinal surgery were collected; marrow was also harvested from the IC. Progenitor cells were isolated and the number of colony forming unit-fibroblastic (CFU-F) determined. The osteogenic potential of the cells was characterized using biochemical and molecular biology techniques. Results. Both the VB and IC marrow generated small, medium, and large sized CFU-F. Higher numbers of CFU-F were obtained from the VB marrow than the IC (P < 0.05). Progenitor cells from both anatomic sites expressed comparable levels of CD166, CD105, CD49a, and CD63. Moreover, progenitor cells from the VB exhibited an increased level of alkaline phosphatase activity. MSCs of the VB and the IC displayed similar levels of expression of Runx-2, collagen Type I, CD44, ALCAM, and ostecalcin. The level of expression of bone sialoprotein was higher in MSC from the IC than the VB. VB and IC cells mineralized their extracellular matrix to a similar extent. Conclusions. Our studies show that CFU-F frequency is higher in the marrow of the VB than the IC. Progenitor cells isolated from both sites respond in a similar manner to an osteogenic stimulus and express common immunophenotypes. Based on these findings, we propose that progenitor cells from the lumbar vertebral marrow would be suitable candidate for osseous graft supplementation in spinal fusion procedures. Studies must now be conducted using animal models to ascertain if cells of the VB are as effective as those of the IC for the fusion applications.

Fibroblast growth factor-2 maintains the differentiation potential of nucleus pulposus cells in vitro: implications for cell-based transplantation therapy.

Study Design. To investigate effects of FGF-2 on nucleus pulposus cell growth and differentiation. Objectives. To elucidate the phenotypic changes that occur during expansion of nucleus pulposus cells in monolayer culture, and to investigate the effects of fibroblast growth factor (FGF)-2 on cell growth and differentiation. Summary of Background Data. Nucleus pulposus cells would have a limited application for autologous cell transplantation if phenotypic dedifferentiation takes place during culture expansion. FGF-2 has been shown to retain the differentiation potential of monolayer expanded chondrocytic cells. However, its effect on nucleus pulposus cells is not known. Methods. Bovine nucleus pulposus cells were serially passaged in the presence or absence of FGF-2 (1 and 10 ng/mL). After passage numbers 1 and 7, cells were immobilized in alginate beads and treated with transforming growth factor (TGF)-&bgr;1 for 1 week to assess their differentiation. Results. During culture expansion in monolayer, nucleus pulposus cells maintained the expression of aggrecan messenger ribonucleic acid (mRNA). However, mRNA levels of collagen type I, collagen type II, Sox-9, and versican decreased with increasing passage number for both control (untreated) cells and FGF-2 treated cells. When grown in alginate with TFG-&bgr;1, passage 7 cells that received FGF-2 during culture expansion restored the mRNA expression of type II collagen, Sox-9, COMP, chondroadherin, and fibromodulin. Moreover, FGF-2 treatment resulted in increased sulfated proteoglycan synthesis and lower aggrecan turnover compared to untreated controls under identical culture conditions. FGF-2 treated cells continued to express HIF-1&agr; protein till passage 7, while MMP-2 expression was evident in cells treated with TGF-&bgr;1. In addition, cells pretreated with FGF-2 showedhigher induction of phospho ERK1/2 after treatment withTGF-&bgr;1. Also, FGF-2 maintained smad 2/smad 3 mediatedsignaling in cells after TGF-&bgr; treatment. FGF-2 action resulted in reduced actin stress fiber formation and migratory cell morphology, with no effect on cell proliferation. Conclusions. The presence of FGF-2 during culture expansion of nucleus pulposus cells in monolayer can sustain a differentiated cell phenotype by maintaining responsiveness to TGF-&bgr;1. Our results suggest that FGF-2 should be tested for its ability to maintain the reactivity of the nucleus pulposus cells to other morphogenic factors that may be used for cell-based transplantation therapy.

Galectin-3 expression in the intervertebral disc: a useful marker of the notochord phenotype?

Study Design. Galectin-3 expression in rat intervertebral disc at different stages in postnatal life is evaluated. Objective. To determine if galectin-3 expression is confined to cells of the nucleus pulposus in the postnatal rat intervertebral disc. Summary of Background Data. During embryonic development, the anulus fibrosus is derived from the sclerotome, whereas the nucleus pulposus is notochordal. Many authorities opine that in the postnatal disc, notochordal cells play a central role in controlling the development of degenerative disc disease. Surprisingly, unequivocal evidence supporting the existence of notochordal cells in the nucleus pulposus in postnatal life has yet to be demonstrated. Since the expression of galectin-3 is commonly used to identify notochordal cells, we evaluated its expression in tissues of the rat disc and in cultured cells. Methods. Galectin-3 expression was studied in the nucleus pulposus and anulus fibrosus tissue of rat discs (2 days, 9 weeks, and 10 months old), and cultured cells using different biochemical and molecular biology methods. Rat sternal cartilage and cultured sternal chondrocytes were used as controls. Results. Immunohistochemical studies indicated that galectin-3 was present in the nucleus pulposus and anulus fibrosus. In both discal tissues and cultured cells, studies confirmed that there was a robust expression of galectin-3 messenger ribonucleic acid and protein. Protein expression patterns were similar in neonatal, young, and mature rats. There was also evidence of intracellular and membrane expression of galectin-3 in the cultured disc cells. Finally, significant levels of galectin-3 were evident in rat sternal cartilage and cultured sternal chondrocytes. Conclusions. Results of the study indicate that galectin-3 is expressed in the neonatal, young, and mature rat disc, and its expression is not restricted to the cells of the nucleus pulposus. Because of its ubiquitous expression, this protein cannot be used as a marker of notochordal cells in the postnatal rat disc.