Regulating the fate of stem cells for regenerating the intervertebral disc degeneration

Lower back pain is a leading cause of disability and is one of the reasons for the substantial socioeconomic burden. The etiology of intervertebral disc (IVD) degeneration is complicated, and its mechanism is still not completely understood. Factors such as aging, systemic inflammation, biochemical mediators, toxic environmental factors, physical injuries, and genetic factors are involved in the progression of its pathophysiology. Currently, no therapy for restoring degenerated IVD is available except pain management, reduced physical activities, and surgical intervention. Therefore, it is imperative to establish regenerative medicine-based approaches to heal and repair the injured disc, repopulate the cell types to retain water content, synthesize extracellular matrix, and strengthen the disc to restore normal spine flexion. Cellular therapy has gained attention for IVD management as an alternative therapeutic option. In this review, we present an overview of the anatomical and molecular structure and the surrounding pathophysiology of the IVD. Modern therapeutic approaches, including proteins and growth factors, cellular and gene therapy, and cell fate regulators are reviewed. Similarly, small molecules that modulate the fate of stem cells for their differentiation into chondrocytes and notochordal cell types are highlighted.     

Effect of chromosome instability on the maintenance and differentiation of human embryonic stem cells in vitro and in vivo

The therapeutic potential of human embryonic stem cells (hESCs) has long been appreciated, and the recent FDA approval of hESC derivatives for cell-based therapy encourages the clinical application of hESCs. Here, using CHA3-hESCs with normal and abnormal karyotypes, we report the importance of maintaining normal chromosomes during in vitro culture and the differentiation of hESCs for minimization of posttransplantation complications. We found that undifferentiated CHA3-hESCs with trisomy chromosome 12 undergo abnormal cell division with multiple spindles in comparison to the bipolar cell division of the karyotypically normal CHA3-hESCs. Transplanted karyotypically abnormal CHA3-hESC derivatives formed a tumor-like tissue 6weeks after transplantation in two out of seven mice tested. Our results demonstrate that the preservation of normal chromosomes is indispensable for maintaining the true properties of hESCs in vitro and abolishing adverse effects posttransplantation. Thus, the development of optimized techniques for stabilizing the chromosome state during in vitro hESC culture is a prerequisite for the therapeutic application of hESCs.     

Disc cell senescence in intervertebral disc degeneration: Causes and molecular pathways

The accumulation of senescent disc cells in degenerative intervertebral disc (IVD) suggests the detrimental roles of cell senescence in the pathogenesis of intervertebral disc degeneration (IDD). Disc cell senescence decreased the number of functional cells in IVD. Moreover, the senescent disc cells were supposed to accelerate the process of IDD via their aberrant paracrine effects by which senescent cells cause the senescence of neighboring cells and enhance the matrix catabolism and inflammation in IVD. Thus, anti-senescence has been proposed as a novel therapeutic target for IDD. However, the development of anti-senescence therapy is based on our understanding of the molecular mechanism of disc cell senescence. In this review, we focused on the molecular mechanism of disc cell senescence, including the causes and various molecular pathways. We found that, during the process of IDD, age-related damages together with degenerative external stimuli activated both p53-p21-Rb and p16-Rb pathways to induce disc cell senescence. Meanwhile, disc cell senescence was regulated by multiple signaling pathways, suggesting the complex regulating network of disc cell senescence. To understand the mechanism of disc cell senescence better contributes to developing the anti-senescence-based therapies for IDD.     

Computation models simulating notochordal cell extinction during early ageing of an intervertebral disc

Lower back pain due to intervertebral disc (IVD) degeneration is a prevalent problem which drastically affects the quality of life of millions of sufferers. Healthy IVDs begin with high populations of notochordal cells in the nucleus pulposus, while by the second stage of degeneration, these cells will be replaced by chondrocyte-like cells. Because the IVD is avascular, these cells rely on passive diffusion of nutrients to survive. It is thought that this transition in cell phenotype causes the shift of the IVD's physical properties, which impede the flow of nutrients. Our computational model of the IVD illustrates its ability to simulate the evolving chemical and mechanical environments occurring during the early ageing process. We demonstrate that, due to the insufficient nutrient supply and accompanying changes in physical properties of the IVD, there was a resultant exponential decay in the number of notochordal cells over time.     

Computation models simulating notochordal cell extinction during early ageing of an intervertebral disc

Lower back pain due to intervertebral disc (IVD) degeneration is a prevalent problem which drastically affects the quality of life of millions of sufferers. Healthy IVDs begin with high populations of notochordal cells in the nucleus pulposus, while by the second stage of degeneration, these cells will be replaced by chondrocyte-like cells. Because the IVD is avascular, these cells rely on passive diffusion of nutrients to survive. It is thought that this transition in cell phenotype causes the shift of the IVD's physical properties, which impede the flow of nutrients. Our computational model of the IVD illustrates its ability to simulate the evolving chemical and mechanical environments occurring during the early ageing process. We demonstrate that, due to the insufficient nutrient supply and accompanying changes in physical properties of the IVD, there was a resultant exponential decay in the number of notochordal cells over time.     

Notochordal cells protect nucleus pulposus cells from degradation and apoptosis: implications for the mechanisms of intervertebral disc degeneration

Introduction

The relative resistance of non-chondrodystrophic (NCD) canines to degenerative disc disease (DDD) may be due to a combination of anabolic and anti-catabolic factors secreted by notochordal cells within the intervertebral disc (IVD) nucleus pulposus (NP). Factors known to induce DDD include interleukin-1 beta (IL-1ß) and/or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal cell conditioned medium (NCCM) to protect NP cells from IL-1ß and IL-1ß +FasL-mediated cell death and degeneration.

Methods

We cultured bovine NP cells with IL-1ß or IL-1ß+FasL under hypoxic serum-free conditions (3.5% O₂) and treated the cells with either serum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow cytometry to evaluate cell death and real-time (RT-)PCR to determine the gene expression of aggrecan, collagen 2, and link protein, mediators of matrix degradation ADAMTS-4 and MMP3, the matrix protection molecule TIMP1, the cluster of differentiation (CD)44 receptor, the inflammatory cytokine IL-6 and Ank. We then determined the expression of specific apoptotic pathways in bovine NP cells by characterizing the expression of activated caspases-3, -8 and -9 in the presence of IL-1ß+FasL when cultured with NCCM, conditioned medium obtained using bovine NP cells (BCCM), and basal medium all supplemented with 2% FBS.

Results

NCCM inhibits bovine NP cell death and apoptosis via suppression of activated caspase-9 and caspase-3/7. Furthermore, NCCM protects NP cells from the degradative effects of IL-1ß and IL-1ß+Fas-L by up-regulating the expression of anabolic/matrix protective genes (aggrecan, collagen type 2, CD44, link protein and TIMP-1) and down-regulating matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which encodes a protein for aggrecan remodeling, is increased. NCCM also protects against IL-1+FasL-mediated down-regulation of Ank expression. Furthermore, NP cells treated with NCCM in the presence of IL-1ß+Fas-L down-regulate the expression of IL-6 by almost 50%. BCCM does not mediate cell death/apoptosis in target bovine NP cells.

Conclusions

Notochordal cell-secreted factors suppress NP cell death by inhibition of activated caspase-9 and -3/7 activity and by up-regulating genes contributing anabolic activity and matrix protection of the IVD NP. Harnessing the restorative powers of the notochordal cell could lead to novel cellular and molecular strategies in the treatment of DDD.     

Bone tissue formation from human embryonic stem cells in vivo

Although the use of embryonic stem cells in the assisted repair of musculoskeletal tissues holds promise, a direct comparison of this cell source with adult marrow-derived stem cells has not been undertaken. Here we have compared the osteogenic differentiation potential of human embryonic stem cells (hESC) with human adult-derived stem cells in vivo. hESC lines H7, H9, the HEF-1 mesenchymal-like, telomerized H1 derivative, the human embryonic kidney epithelial cell line HEK293 (negative control), and adult human mesenchymal stem cells (hMSC) were either used untreated or treated with osteogenic factors for 4 days prior to injection into diffusion chambers and implantation into nude mice. After 11 weeks in vivo chambers were removed, frozen, and analyzed for evidence of bone, cartilage, and adipose tissue formation. All hESCs, when pretreated with osteogenic (OS) factors gave rise exclusively to bone in the chambers. In contrast, untreated hESCs (H9) formed both bone and cartilage in vivo. Untreated hMSCs did not give rise to bone, cartilage, or adipose tissue in vivo, while pretreatment with OS factors engendered both bone and adipose tissue. These data demonstrate that hESCs exposed to OS factors in vitro undergo directed differentiation toward the osteogenic lineage in vivo in a similar fashion to that produced by hMSCs. These findings support the potential future use of hESC-derived cells in regenerative medicine applications.     

Effects of pulsed electromagnetic field on intervertebral disc cell apoptosis in rats

Despite numerous studies on pulsed electromagnetic field (PEMF) application, its effects of PEMF on intervertebral disc (IVD) have not yet been investigated in vivo. Accordingly, the effects of PEMF upon IVD in rats were evaluated through molecular surveys. Rats were divided into six groups: Group I and II were exposed to low and high frequency of PEMF (LF and HF, respectively). Group III and IV underwent induced disc degeneration and were exposed to low and high frequency of PEMF (LF/IDD and HF/IDD, respectively). Group V underwent induced disc degeneration (IDD), and group VI was control. The values of caspase 3, Bax, Bcl-2 and β-actin band density, as cell apoptotic markers, were obtained from band densitometry. Our results showed that the value of cleaved caspase-3 of cells and Bax/Bcl-2 ratio in IDD group increased significantly compared to the control group (p?p?相似文献   

Emerging role and therapeutic implication of mTOR signalling in intervertebral disc degeneration

Intervertebral disc degeneration (IDD), an important cause of chronic low back pain (LBP), is considered the pathological basis for various spinal degenerative diseases. A series of factors, including inflammatory response, oxidative stress, autophagy, abnormal mechanical stress, nutritional deficiency, and genetics, lead to reduced extracellular matrix (ECM) synthesis by intervertebral disc (IVD) cells and accelerate IDD progression. Mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine/threonine kinase that plays a vital role in diverse degenerative diseases. Recent studies have shown that mTOR signalling is involved in the regulation of autophagy, oxidative stress, inflammatory responses, ECM homeostasis, cellular senescence, and apoptosis in IVD cells. Accordingly, we reviewed the mechanism of mTOR signalling in the pathogenesis of IDD to provide innovative ideas for future research and IDD treatment.     

Modulating Notochordal Differentiation of Human Induced Pluripotent Stem Cells Using Natural Nucleus Pulposus Tissue Matrix

Human induced pluripotent stem cells (hiPSCs) can differentiate into notochordal cell (NC)-like cells when cultured in the presence of natural porcine nucleus pulposus (NP) tissue matrix. The method promises massive production of high-quality, functional cells to treat degenerative intervertebral discs (IVDs). Based on our previous work, we further examined the effect of cell-NP matrix contact and culture medium on the differentiation, and further assessed the functional differentiation ability of the generated NC-like. The study showed that direct contact between hiPSCs and NP matrix can promote the differentiation yield, whilst both the contact and non-contact cultures can generate functional NC-like cells. The generated NC-like cells are highly homogenous regarding the expression of notochordal marker genes. A culture medium containing a cocktail of growth factors (FGF, EGF, VEGF and IGF-1) also supported the notochordal differentiation in the presence of NP matrix. The NC-like cells showed excellent functional differentiation ability to generate NP-like tissue which was rich in aggrecan and collagen type II; and particularly, the proteoglycan to collagen content ratio was as high as 12.5–17.5 which represents a phenotype close to NP rather than hyaline cartilage. Collectively, the present study confirmed the effectiveness and flexibility of using natural NP tissue matrix to direct notochordal differentiation of hiPSCs, and the potential of using the generated NC-like cells for treating IVD degeneration.     

Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment

Background  

We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays.     

Pinacidil enhances survival of cryopreserved human embryonic stem cells

Human embryonic stem cells (hESCs) can be maintained as undifferentiated cells in vitro and induced to differentiate into a variety of somatic cell types. Thus, hESCs provide a source of differentiated cell types that could be used to replace diseased cells of a tissue. The efficient cryopreservation of hESCs is important for establishing effective stem cell banks, however, conventional slow freezing methods usually lead to low rates of recovery after thawing cells and their replating in culture. We have established a method for recovering cryopreserved hESCs using pinacidil and compared it to a method that employs the ROCK inhibitor Y-27632. We show that pinacidil is similar to Y-27632 in promoting survival of hESCs after cryopreservation. The cells exhibited normal hESC morphology, retained a normal karyotype, and expressed characteristic hESC markers (OCT4, SSEA3, SSEA4 and TRA-1-60). Moreover, the cells retained the capacity to differentiate into derivatives of all three embryonic germ layers as demonstrated by differentiation through embryoid body formation. Pinacidil has been used for many years as a vasodilator drug to treat hypertension and its manufacture and traceability are well defined. It is also considerably cheaper than Y-27632. Thus, the use of pinacidil offers an efficient method for recovery of cryopreserved dissociated human ES cells.     

The weakest link: A new paradigm for stabilizing the integrin–actin connection

Human embryonic stem cells (hESCs) are to be considered as a valuable source for regenerative medicine because of their capacity to differentiate into all cell types. We have developed an efficient culture system to differentiate hECSs into endothelial cells without the formation of embryoid bodies Establishing appropriate culture conditions with a cocktail of growth factors allowed us to differentiate hESCs directly to endothelial primary culture with about 50% efficiency. CD31 immunomagnetic cell sorting was used to purify derived endothelium from the primary culture of hESCs. Isolated endothelial cells expressed immunological markers (vWF, CD105), specific genes (VE-cadherin, KDR, GATA-2, GATA-3, eNOS), and formed cord-like structures on collagen matrix and in Matrigel assay. During differentiation to endothelial lineage promoter regions of the genes involved in specific cell fate determination and homeostasis (GATA-2,-3, and eNOS) underwent intensive hypomethylation which correlated with the gene expression. Overall our data demonstrate that direct differentiation of hESCs leads to endothelial cells that acquire epigenetic patterning similar to the functional endothelial cells of the organism.     

Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

Introduction

Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods

Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results

Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions

Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.     

An in vitro study investigating the survival and phenotype of mesenchymal stem cells following injection into nucleus pulposus tissue

Introduction  

The decreased disc height characteristic of intervertebral disc (IVD) degeneration has often been linked to low back pain, and thus regeneration strategies aimed at restoring the disc extracellular matrix and ultimately disc height have been proposed as potential treatments for IVD degeneration. One such therapy under investigation by a number of groups worldwide is the use of autologous mesenchymal stem cells (MSCs) to aid in the regeneration of the IVD extracellular matrix. To date, however, the optimum method of application of these cells for regeneration strategies for the IVD is unclear, and few studies have investigated the direct injection of MSCs alone into IVD tissues. In the present article, we investigated the survival and phenotype of human MSCs, sourced from aged individuals, following injection into nucleus pulposus (NP) tissue explant cultures.     

Variable allelic expression of imprinted genes in human pluripotent stem cells during differentiation into specialized cell types in vitro

Genomic imprinting is an epigenetic phenomenon by which a subset of genes is asymmetrically expressed in a parent-of-origin manner. However, little is known regarding the epigenetic behaviors of imprinted genes during human development. Here, we show dynamic epigenetic changes in imprinted genes in hESCs during in vitro differentiation into specialized cell types. Out of 9 imprinted genes with single nucleotide polymorphisms, mono-allelic expression for three imprinted genes (H19, KCNQ1OT1, and IPW), and bi- or partial-allelic expression for three imprinted genes (OSBPL5, PPP1R9A, and RTL1) were stably retained in H9-hESCs throughout differentiation, representing imprinting stability. Three imprinted genes (KCNK9, ATP10A, and SLC22A3) showed a loss and a gain of imprinting in a lineage-specific manner during differentiation. Changes in allelic expression of imprinted genes were observed in another hESC line during in vitro differentiation. These findings indicate that the allelic expression of imprinted genes may be vulnerable in a lineage-specific manner in human pluripotent stem cells during differentiation.     

Long non‐coding RNAs in nucleus pulposus cell function and intervertebral disc degeneration

Intervertebral disc degeneration (IDD) is the major cause of low back pain which incurs a significant public‐health and economic burden. The aetiology of IDD is complex, with developmental, genetic, biomechanical and biochemical factors contributing to the disease development. Deregulated phenotypes of nucleus pulposus cells, including aberrant differentiation, apoptosis, proliferation and extracellular matrix deposition, are involved in the initiation and progression of IDD. Non‐coding RNAs, including long non‐coding RNAs (lncRNAs), have recently been identified as important regulators of gene expression. Research into their roles in IDD has been very active over the past 5 years. Our review summarizes current research regarding the roles of deregulated lncRNAs (eg, RP11‐296A18.3, TUG1, HCG18) in modulating nucleus pulposus cell functions in IDD. These exciting findings suggest that specific modulation of lncRNAs or their downstream signalling pathways might be an attractive approach for developing novel therapeutics for IDD.     

Programmed cell death in intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is largely a process of destruction and failure of the extracellular matrix (ECM), and symptomatic IVD degeneration is thought to be one of the leading causes of morbidity or life quality deterioration in the elderly. To date, however, the mechanism of IVD degeneration is still not fully understood. Cellular loss from cell death in the process of IVD degeneration has long been confirmed and considered to contribute to ECM degradation, but the causes and the manners of IVD cell death remain unclear. Programmed cell death (PCD) is executed by an active cellular process and is extensively involved in many physiological and pathological processes, including embryonic development and human degenerative diseases. Thus, the relationship between PCD and IVD degeneration has become a new research focus of interest in recent years. By reviewing the available literature concentrated on PCD in IVD and discussing the methodology of detecting PCD in IVD cells, its inducing factors, the relationship of cell death to ECM degradation, and the potential therapy for IVD degeneration by modulation of PCD, we conclude that IVD cells undergo PCD via different signal transduction pathways in response to different stimuli, that PCD may play a role in the process of IVD degeneration, and that modulation of PCD might be a potential therapeutic strategy for IVD degeneration.