Precartilaginous Stem Cells Research Articles

Rapid and simple isolation of precartilaginous stem cells: A novel approach without immunomagnetic bead sorting

ObjectiveThe present investigation was designed to devise a rapid and straightforward technique for the isolation of rat precartilaginous stem cells (PCSCs) that eschews the use of immunomagnetic bead sorting. MethodRat neonates within 24 h of birth were selected for this study. Microsurgical techniques were used to harvest the femur, tibia, and the musculature of the knee joint. The ring of LaCroix between the metaphysis of the femur and the epiphysis was excised and divided into fragments of approximately 1 mm³. Tissue sections were cultured in Dulbecco's modified Eagle's medium(DMEM)/F12 medium supplemented with 20 % fetal bovine serum and 1 % penicillin-streptomycin. Cell digestion and passaging were performed using trypsin when cells reached 70%–80 % confluence. Third-generation cells underwent immunofluorescence staining and flow cytometry to evaluate fibroblast growth factor receptor-3(FGFR-3) and proliferating cell nuclear antigen(PCNA) expression, while β-galactosidase staining was used to determine cellular senescence. ResultsWithin two days of isolation, numerous short spindle-shaped cells exhibiting distinct refractive properties were observed around the tissue fragments. These cells began to proliferate within 2–3 days and displayed ample cytoplasm. Adherent cells adopted various morphologies, including angular, triangular, and elongated spindles. By the fifth day, more than 80 % of the culture dish surface was covered with elongated cells, with some arranged in patterns reminiscent of whirlpools. Significant FGFR-3 and PCNA expression was confirmed via immunofluorescence in the third-generation cells. Additionally, flow cytometry identified that the proportion of cells positive for FGFR-3 and PCNA exceeded 98 %. Notably, the cells preserved their proliferative capacity through nine passages in vitro, with a marginal proportion showing senescence as indicated by β-galactosidase staining alone. ConclusionThe developed tissue adherence protocol was used to successfully isolate PCSCs with positive FGFR-3 and PCNA expression, rendering the immunomagnetic bead sorting superfluous. The expression of FGFR-3 and PCNA in the isolated cells persisted through the ninth passage in vitro with minimal senescence.

Osteogenesis of Iron Oxide Nanoparticles-Labeled Human Precartilaginous Stem Cells in Interpenetrating Network Printable Hydrogel.

Smart biomaterials combined with stem cell-based therapeutic strategies have brought innovation in the field of bone tissue regeneration. However, little is known about precartilaginous stem cells (PCSCs), which can be used as seed cells and incorporated with bioactive scaffolds for reconstructive tissue therapy of bone defects. Herein, iron oxide nanoparticles (IONPs) were employed to modulate the fate of PCSCs, resulting in the enhanced osteogenic differentiation potential both in vitro and in vivo. PCSCs were isolated from the ring of La-Croix extracted from polydactylism patient and identified through immunohistochemically staining using anti-FGFR-3 antibodies. Potential toxicity of IONPs toward PCSCs was assessed through cell viability, proliferation, and attachment assay, and the results demonstrated that IONPs exhibited excellent biocompatibility. After that, the effects of IONPs on osteogenic differentiation of PCSCs were evaluated and enhanced ALP activity, formation of mineralized nodule, and osteogenic-related genes expressions could be observed upon IONPs treatment. Moreover, in vivo bone regeneration assessment was performed using rabbit femur defects as a model. A novel methacrylated alginate and 4-arm poly (ethylene glycol)-acrylate (4A-PEGAcr)-based interpenetrating polymeric printable network (IPN) hydrogel was prepared for incorporation of IONPs-labeled PCSCs, where 4A-PEGAcr was the common component for three-dimensional (3D) printing. The implantation of IONPs-labeled PCSCs significantly accelerated the bone formation process, indicating that IONPs-labeled PCSCs could endow current scaffolds with excellent osteogenic ability. Together with the fact that the IONPs-labeled PCSCs-incorporated IPN hydrogel (PCSCs-hydrogels) was biosafety and printable, we believed that PCSCs-hydrogels with enhanced osteogenic bioactivity could enrich the stem cell-based therapeutic strategies for bone tissue regeneration.

MicroRNA-124-3p inhibits the differentiation of precartilaginous stem cells into nucleus pulposus-like cells via targeting FSTL1.

MicroRNA (miRNA/miR)-124-3p has been extensively studied in tumor biology and stem cells. However, little is known regarding its functional roles in the differentiation of precartilaginous stem cells (PSCs) into nucleus pulposus-like cells (NPLCs). In the present study, using miRNA microarray screening, it was demonstrated that the miRNA expression profiles differed between rat primary PSCs and TGF-β1-induced differentiated NPLCs, and that miR-124-3p was significantly differentially expressed during the differentiation of PSCs to NPLCs. Furthermore, RT-qPCR analysis verified that miR-124-3p expression was decreased during PSC differentiation, with the lowest levels being detected at the later stages. Subsequent experiments revealed that miR-124-3p overexpression significantly decreased the expression of the extracellular matrix proteins, aggrecan and collagen type II, which was accompanied by a significant decrease in follistatin-related protein 1 (FSTL1) expression levels. Moreover, bioinformatics analysis indicated that FSTL1 was a potential target of miR-124-3p, which was additionally verified using luciferase reporter assays. Taken together, these data revealed a specific regulatory pathway of miR-124-3p, which negatively regulated its target gene, FSTL1, during the differentiation of PSCs to NPLCs, and suggested a functional role for miR-124-3p in the differentiation of PSCs.

MiR-143 is implicated in growth plate injury by targeting IHH in precartilaginous stem cells.

Precartilaginous stem cells (PCSCs) are able to initiate chondrocyte and bone development. The present study aimed to investigate the role of miR-143 and the underlying mechanisms involved in PCSC proliferation. In a rat growth plate injury model, tissue from the injury site was collected and the expression of miR-143 and its potential targets was determined. PCSCs were isolated from the rabbits' distal epiphyseal growth plate. Cell viability, DNA synthesis, and apoptosis were determined with MTT, BrdU, and flow cytometric analysis, respectively. Real time PCR and western blot were performed to detect the mRNA and protein expression of the indicated genes. Indian hedgehog (IHH) was identified as a target gene for miR-143 with luciferase reporter assay. Decreased expression of miR-143 and increased expression of IHH gene were observed in the growth plate after injury. miR-143 mimics decreased cell viability and DNA synthesis and promoted apoptosis of PCSCs. Conversely, siRNA-mediated inhibition of miR-143 led to increased growth and suppressed apoptosis of PCSCs. Transfection of miR-143 decreased luciferase activity of wild-type IHH but had no effect when the 3'-UTR of IHH was mutated. Furthermore, the effect of miR-143 overexpression was neutralized by overexpression of IHH. Our study showed that miR-143 is involved in growth plate behavior and regulates PCSC growth by targeting IHH, suggesting that miR-143 may serve as a novel target for PCSC-related diseases.

MiR-132/212 promotes the growth of precartilaginous stem cells (PCSCs) by regulating Ihh/PTHrP signaling pathway.

Precartilaginous stem cells (PCSCs) are adult stem cells that can initiate chondrocytes and bone development. In the present study, we explored whether miR-132/212 was involved in the proliferation of PCSCs via Hedgehog signaling pathway. PCSCs were isolated and purified with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Cell viability, DNA synthesis and apoptosis were measured using MTT, BrdU and flow cytometric analysis. The mRNA and protein expression were detected by real-time PCR and Western blot, respectively. The target gene for miR-132/212 was validated by luciferase reporter assay. Results showed that transfection with miR-132/212 mimic significantly increased cell viability and DNA synthesis, and inhibited apoptosis of PCSCs. By contrast, miR-132/212 inhibitor could suppress growth and promote apoptosis of PCSCs. Luciferase reporter assays indicated that transfection of miR-132/212 led to a marked reduction of luciferase activity, but had no effect on PTCH1 3′-UTR mutated fragment, suggesting that Patched1 (PTCH1) is a target of miR-132/212. Furthermore, treatment with miR-132/212 mimics obviously increased the protein expression of Indian hedgehog (Ihh) and parathyroid hormone related protein (PTHrP), which was decreased after treatment with Hedgehog signaling inhibitor, cyclopamine. We also found that inhibition of Ihh/PTHrP signaling by cyclopamine significantly suppressed growth and DNA synthesis, and induced apoptosis in PCSCs. These findings demonstrate that miR-132/212 promotes growth and inhibits apoptosis in PCSCs by regulating PTCH1-mediated Ihh/PTHrP pathway, suggesting that miR-132/212 cluster might serve as a novel target for bone diseases.

Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and β-catenin-related pathways.

Cartilage engineering strategies using mesenchymal stem cells (MSCs) could provide preferable solutions to resolve long-segment tracheal defects. However, the drawbacks of widely used chondrogenic protocols containing TGF-β3, such as inefficiency and unstable cellular phenotype, are problematic. In our research, to optimize the chondrogenic differentiation of human umbilical cord MSCs (hUCMSCs), kartogenin (KGN) preconditioning was performed prior to TGF-β3 induction. hUCMSCs were preconditioned with 1 μM of KGN for 3 d, sequentially pelleted, and incubated with TGF-β3 for 28 d. Then, the expression of chondrogenesis- and ossification-related genes was evaluated by immunohistochemistry and RT-PCR. The underlying mechanism governing the beneficial effects of KGN preconditioning was explored by phosphorylated kinase screening and validated in vitro and in vivo using JNK inhibitor (SP600125) and β-catenin activator (SKL2001). After KGN preconditioning, expression of fibroblast growth factor receptor 3, a marker of precartilaginous stem cells, was up-regulated in hUCMSCs. Furthermore, the KGN-preconditioned hUCMSCs efficiently differentiated into chondrocytes with elevated chondrogenic gene ( SOX9, aggrecan, and collagen II) expression and reduced expression of ossific genes (collagen X and MMP13) compared with hUCMSCs treated with TGF-β3 only. Phosphokinase screening indicated that the beneficial effects of KGN preconditioning are directly related to an up-regulation of JNK phosphorylation and a suppression of β-catenin levels. Blocking and activating tests revealed that the prochondrogenic effects of KGN preconditioning was achieved mainly by activating the JNK/Runt-related transcription factor (RUNX)1 pathway, and antiossific effects were imparted by suppressing the β-catenin/RUNX2 pathway. Eventually, tracheal patches, based on KGN-preconditioned hUCMSCs and TGF-β3 encapsulated electrospun poly( l-lactic acid-co-ε-caprolactone)/collagen nanofilms, were successfully used for restoring tracheal defects in rabbit models. In summary, KGN preconditioning likely improves the chondrogenic differentiation of hUCMSCs by committing them to a precartilaginous stage with enhanced JNK phosphorylation and suppressed β-catenin. This novel protocol consisting of KGN preconditioning and subsequent TGF-β3 induction might be preferable for cartilage engineering strategies using MSCs.-Jing, H., Zhang, X., Gao, M., Luo, K., Fu, W., Yin, M., Wang, W., Zhu, Z., Zheng, J., He, X. Kartogenin preconditioning commits mesenchymal stem cells to a precartilaginous stage with enhanced chondrogenic potential by modulating JNK and β-catenin-related pathways.

Transforming growth factor-β1 (TGF-β1) induces mouse precartilaginous stem cell differentiation through TGFRII-CK1ε-β-catenin signalling.

Precartilaginous stem cells (PSCs) are adult stem cells which could self-renew or differentiate into chondrocytes to promote bone growth. In this study, we aimed to understand the role of transforming growth factor-β1 (TGF-β1) in precartilaginous stem cell (PSC) differentiation and to study the mechanisms that underlie this role. We purified PSCs from the neonatal murine perichondrial mesenchyme using immunomagnetic beads, and primary cultured them. Their phenotype was confirmed by the PSC marker fibroblast growth factor receptor-3 (FGFR-3) overexpression. TGF-β1 was added to induce PSCs differentiation. TGF-β1 increased mRNA expression of chondrogenesis-related genes (collagen type II, Sox 9 and aggrecan) in the cultured PSCs. This was abolished by TGF-β receptor II (TGFRII) and Casein kinase 1 epsilon (CK1ε) lentiviral shRNA depletion. Meanwhile, we found that TGF-β1 induced CK1ε activation, glycogen synthase kinase-3β (GSK3β) phosphorylation and β-catenin nuclear translocation in the mouse PSCs, which was almost completely blocked by TGFRII and CK1ε shRNA knockdown. Based on these results, we suggest that TGF-β1 induces CK1ε activation to promote β-catenin nuclear accumulation, which then regulates chondrogenesis-related gene transcription to eventually promote mouse PSC differentiation.

Cell therapy of a knee osteoarthritis rat model using precartilaginous stem cells.

To explore the effect and mechanism of precartilaginous stem cells (PSCs) engraftment-inducing tissue repair in a knee osteoarthritis (OA) rat model. Knee osteoarthritis (OA) model was constructed in Sprague Dawley (SD) rats by partial removal of the medial meniscus of the right knee. PSCs were engrafted by injecting precartilaginous stem cells (PSCs) into the right knee cavity. At 4 and 8 weeks after model construction, the serum levels of interleukine (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6 were assessed using enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was performed to assess the histopathology of synovial membrane and cartilage. Western blot analysis was used to assess Notch1, Bcl-2 and Bax levels in the articular cartilage. At 4 and 8 weeks, OA rats demonstrated significantly higher IL-1β, TNF-α, and IL-6 levels than normal rats (p < 0.05), whereas PSCs treatment prominently attenuated IL-1β upregulation (p < 0.05). In OA rats, the number of chondrocytes dramatically decreased over time in OA rats, with disruption of chondrocytes organization and cell layers. PSCs alleviated the deterioration of cartilage, as evidenced by the relatively smooth articular surface, distinct tidemark and clear cell layers. The model and treatment groups demonstrated substantially higher Notch1 expression. The Bcl-2/Bax value in the OA rats was lower than the control group, while PSCs treatment led to increase in Bcl-2/Bax value. PSCs treatment downregulated the expression of inflammatory cytokines, alleviating osteoarthritis in the knee of rats. Notch1 signaling pathway plays an important role in this ameliorating effect of PSCs treatment.

Transforming growth factor-β1 (TGF-β1) induces mouse precartilaginous stem cell proliferation through TGF-β receptor II (TGFRII)-Akt-β-catenin signaling.

Precartilaginous stem cells (PSCs) could self-renew or differentiate into chondrocytes to promote bone growth. In the current study, we aim to understand the role of transforming growth factor-β1 (TGF-β1) in precartilaginous stem cell (PSC) proliferation, and to study the underlying mechanisms. We successfully purified and primary-cultured PSCs from the neonate mice’ perichondrial mesenchyme, and their phenotype was confirmed by the PSC marker fibroblast growth factor receptor-3 (FGFR-3) overexpression. We found that TGF-β1 induced Akt-glycogen synthase kinase-3β (GSK3β) phosphorylation and β-catenin nuclear translocation in the mouse PSCs, which was almost blocked by TGF-β receptor-II (TGFRII) shRNA knockdown. Further, perifosine and MK-2206, two Akt-specific inhibitors, suppressed TGF-β1-induced GSK3β phosphorylation and β-catenin nuclear translocation. Akt inhibitors, as well as β-catenin shRNA knockdown largely inhibited TGF-β1-stimulated cyclin D1/c-myc gene transcription and mouse PSC proliferation. Based on these results, we suggest that TGF-β1 induces Akt activation to promote β-catenin nuclear accumulation, which then regulates cyclin D1/c-myc gene transcription to eventually promote mouse PSC proliferation.

Transforming growth factor-β (TGF-β) induces the expression of chondrogenesis-related genes through TGF-β receptor II (TGFRII)–AKT–mTOR signaling in primary cultured mouse precartilaginous stem cells

Precartilaginous stem cells (PSCs) are adult stem cells which could initiate chondrocytes and bone growth. In the current study, we purified PSCs from the neonate mice’ perichondrial mesenchyme through immunomagnetic beads with the fibroblast growth factor receptor-3 (FGFR-3) antibody. Mouse PSCs were seeded and cultured, and their phenotype was confirmed by FGFR-3 over-expression. Transforming growth factor-β (TGF-β) was added to induce PSCs differentiation. TGF-β increased mRNA expression of chondrogenesis-related genes (collagen type II, Sox 9, and aggrecan) in the cultured PSCs, which was abolished by TGF-β receptor II (TGFRII) lentiviral shRNA depletion. TGF-β induced AKT activation in mouse PSCs, while the PI3K/AKT inhibitor (LY294002) and the AKT specific inhibitors (perifosine and MK-2206) largely suppressed TGF-β-induced collagen II, Sox 9, and aggrecan mRNA expression. Meanwhile, the mTOR complex 1 (mTORC1) blocker RAD001 or the mTORC1/2 dual inhibitor AZD-2014 also alleviated TGF-β-induced chondrogenesis-associated genes expression. Further, lentiviral shRNA depletion of SIN1 (a mTORC2 component) or mTOR inhibited TGF-β’s effect in the mouse PSCs. In conclusion, our evidence suggests that TGF-β induces the expression of chondrogenesis-related genes through TGFRII–AKT–mTOR signaling in cultured mouse PSCs.

Chondrogenic effect of precartilaginous stem cells following NLS-TAT cell penetrating peptide-assisted transfection of eukaryotic hTGFβ3

Cell penetrating peptides (CPPs) are a series of promising carriers for delivering exogenous DNA to living cells. Among them, the combination of the human immunodeficiency virus TAT protein (TAT) with the SV40 large T protein nuclear localization signal (NLS) to form NLS-TAT performs well. In the present study, we took advantage of this new carrier to deliver transforming growth factor-beta 3 (TGFβ3) genes. TGFβ3 was expressed by the pEGFP-N1 vector following transfection of rat precartilaginous stem cells (PSCs), which promoted hTGFβ3 protein self-expression. At 24, 48, 72, and 120 h after transfection, the expression levels of hTGFβ3 were found to be elevated as compared with the control. The expression of hTGFβ3 was found to mediate the chondrogenic effect of PSCs. Thus, we determined the expression of the chondrogenesis-related genes type II collagen, Sox 9, and aggrecan in PSCs at 24, 48, 72, and 120 h after transfection. We found that their transcription and translation was augmented, which indicated a trend of active chondrogenesis in the PSCs. Our results demonstrated that NLS-TAT had the ability to deliver exogenous DNA into rat PSCs and could be actively expressed. This process successfully promoted PSC chondrogenesis. Additionally, PSC, may represent a new type of stem cells, and thus show great potential in regenerative repair following cartilage injury.

Enhancing chondrogenic differentiation in precartilaginous stem cells with 620 nm red light

Objective To investigate the effect of 620 nm red light on chondrogenic differentiation in rat precartilaginous stem cells (PSCs). Methods Rats' PSCs were isolated and purified using magnetically activated cell sorting and cultured in vitro.The PSCs were exposed once to 620 nm wavelength red light from a light-emitting diode (LED) with an irradiation energy of 0.5 J/cm2,1 J/cm2,2 J/cm2 or 4 J/cm2.Any effect was confirmed by Alcian blue staining,immunohistochemistry and observing histomorphological changes under a light microscope,as well as detection using a reverse transcription polymerase chain reaction (RT-PCR). Results After being induced for 14 d,the PSCs exhibited polygonal and round shapes. Alcian blue and type Ⅱ collagen immunohistoehemistry staining showed positive results,but the control group had no significant change.RT-PCR showed that the mRNA expression of Sox9 and type Ⅱ collagen increased significantly compared with the control group. Conclusion Low energy 620 nm red light can enhance chondrogenic differentiation in PSCs significantly. Key words: Chondrocytes ; Cell differentiation ; Precartilaginous stem cells; Red light

Construction of eukaryotic expression plasmid of hTGF-β3 and its inducing effect on differentiation of precartilaginous stem cells into chondroblasts

This study examined the construction of eukaryotic expression plasmid of human transforming growth factor-β3 (hTGF-β3) and its inducing effect on the differentiation of precartilaginous stem cells (PSCs) into chondroblasts. hTGF-β3 gene was amplified by using polymerase chain reaction (PCR) and then inserted into the eukaryotic expression plasmid pcDNA3.1 to construct the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3. Rat PSCs were isolated and purified by employing an immunomagnetic cell sorting system. pcDNA3.1(+)-hTGF-β3 was transfected into purified PSCs with the use of linear polyamines. The expression of TGF-β3 and cartilage-specific extracellular matrix (ECM) components was detected after transfection by real-time quantitative PCR, ELISA, immunochemistry and Western blotting, respectively. The results showed that the eukaryotic expression plasmid pcDNA3.1(+)-hTGF-β3 was successfully established as identified by enzyme digestion and DNA sequencing. Real-time quantitative PCR and ELISA revealed that hTGF-β3 was strongly expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs. Real-time quantitative PCR, immunochemistry and Western blotting showed that the cartilage-specific ECM markers, i.e., cartilage oligomeric matrix protein (COMP), Aggrecan, collagen type X and II were intensely expressed in the pcDNA3.1(+)-hTGF-β3-transfected cells. It was concluded that hTGF-β3 could be stably expressed in pcDNA3.1(+)-hTGF-β3-transfected PSCs and induce the differentiation of PSCs into chondroblasts.

Chondrogenesis of precartilaginous stem cells in KLD-12 self-assembling peptide nanofiber scaffold loading TGF-β3 gene

The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells (PSCs) into chondrocytes was studied. KLD-12 was synthesized by solid-state method. After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold, DNA release ability was investigated. PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold, and MTT assay was performed to investigate the cell proliferation, and ELASA assay was used to investigate the expression of TGF-β3. Specific cartilage matrix was examined by quantitative real-time PCR, immunohistochemistry and Alcian Blue staining. Compared with control group, DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, and maintained this high level within 2 weeks. MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group (P<0.05). Quantitative realtime PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group (P<0.01). ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group’s PSCs, reached the peak after 72 h and then declined a little to the plateau phase. Compared with the control group, the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P<0.01). The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid, which provided a fresh approach to cartilage tissue engineering.

Ectopic chondrogenesis of composite of enhanced green fluorescent protein gene-modified precartilaginous stem cells and bone matrix gelatin

Objective To evaluate the chondrogenesis potential of composite of enhanced green fluorescent protein (EGFP) gene-modified precartilaginous stem cells (PSCs) and bone matrix gelatin (BMG) ectopically. Methods PSCs were transfected with pEGFP by means of lipofectamine. The composite of EGFP gene-modified PSCs and BMG was cultured in vitro, then implanted into the right thigh of athymic mice, and BMG implanted into the left thigh as control group. The harvested implants were weighed, and the number of cells and the expression of GFP were examined. The expression of type Ⅱ collegen was detected by using immunohistochemistry. Results EGFP gene-modified PSCs had high expression of GFP in vitro 6 weeks after purification. The composite had high expression of GFP from 1 to 6 weeks after implantation, and high expression of type Ⅱ collagen from 3 to 6 weeks. The mass of the composite was (0.71±0.07), (1.35±0.32), (1.76±0.48), (1.96±0.54), (1.89±0.13) g from 2 to 6 weeks, respectively, which was significantly greater than that in control group (P<0.01). The total number of cells in the composite was (40.0±2.1)×106, (67.0±5.6)×106, (139.0±11.2)×106, (169.0±2.9)×106, (173.0±13.5)×106, (169.0±6.9)×106 from 1 to 6 weeks, respectively, which was significantly greater than that in control group (P<0.01). Conclusion The composite of EGFP gene-modified PSCs and BMG could generate ectopic cartilaginous tissue. Key words: Precartilainous stem cells; Gene transfectection; Bone matrix; Gelatin

Transforming growth factor β induces chondrogenesis of precartilaginous stem cells

Objective To investigate the possibility of transforming growth factor β (TGF-p) inducing chondrogenesis of precartilaginous stem cells (PSCs) and discuss expression mechanism of extracel-luar matrix. Methods PSCs were induced into a chondrogenic pathway in alginate bead culture in the absence of serum and in the presence of TGF-β1, β2, or-β3. The temporal pattern of expression of cartilage-specific extracellular matrix during chondrogenesis were analyzed by immunocytochemistry, immunoflu-orescence, RT-PCR, immunoprecipitation, Western blot and spectrophotometer. Results Type Ⅱ collagen staining was positive at days 7, 14 and 21 in alginate bead culture, showing most intense staining in the TGF-p3-treated culture. Expression of type Ⅱ collagen was increased in TGF-β3 group. Immunocytochemi-cal analysis of a number of other extracellular matrix components showed widespread expressions of aggre-can, fibromodulin and COMP in alginate bead culture that presented TGF-p3 for 21 days. The expressions of Aggrcan, fibromodulin, type Ⅰ and ⅹ collagen, and COMP were detected by RT-PCR in TGF-β3 group within 8 days, while type Ⅱ collagen began expression at days 8-21. COMP or type X collagen was present in TGF-β3 group at days 7, 14 and 21 by immunoprecipitation or Western blot analysis respectively. The extracted glycosaminoglycan content or the glycosaminoglycan/DNA rate in TGF-βl group was significantly lower than those in TGF-β2 group or TGF-β3 group at days 14 and 21 (P <0.01). Conclusions TGF-β can evocate chondrogenesis of PSCs, when rapid deposition of cartilage-specific extracellular matrix is involved. The sequential events in this pathway leading from the undifferentiated stem cells to mature chon-drocytes can be investigated by analysis of key matrix elements. Key words: Stem cells; Transforming growth factor β; Extracellular matrix; Chondrocytes; Chondrogenesis

Common Skeletal Growth Retardation Disorders Resulting from Abnormalities within the Mesenchymal Stem Cells Reservoirs in the Epiphyseal Organs Pertaining to the Long Bones

Among the objectives in writing the current chapter were the curiosity and the interest in allocating the sites and routes of migration of the reservoirs of the mesenchymal precartilaginous stem cells of the developing limbs in health and in disease. We chose to emphasize the events believed to initiate in these regions of stem cells, which may lead to growth retardation disorders. Thus, this narrow niche touches an enlarged scope of developmental biology angles and fields. The enclosed coverage sheds light on part of the musculoskeletal system, skeletogenesis, organogenesis of mobile structures and organs, the limbs, joints and digits (arthrology). It appears that the key role of the cartilage-bone regions is their responsibility to replenish the physis with committed chondrocytes, during the developmental, maturation and puberty periods. We shall start by outlining the framework of normal limb formation, the modalities, signals and the agents participating in this biological creation and regulation, illustrating potential sites that might deviate from normal development during the growth periods.

Construction of Sox9 gene eukaryotic expression vector and its inductive effects on directed differentiation of bone marrow stromal cells into precartilaginous stem cells in rats

Sox9 gene was cloned from immortalized precartilaginous stem cells and its eukaryotic expression vector constructed in order to explore the possibility of bone marrow-derived stromal cells differentiation into precartilaginous stem cells induced by Sox9. A full-length fragment of Sox9 was obtained by RT-PCR, inserted into pGEM-T Easy clone vector, and ligated with pEGFP-IRES2 expression vector by double digestion after sequencing. The compound plasmid was transfected into born marrow-derived stromal cells by Lipofectamine 2000, and the transfection efficacy and the expression of Sox9 and FGFR-3 were observed. Flow cytometry was used to identify the cell phenotype, and MTT was employed to assay proliferative viability of cells. Sequencing, restrictive endonuclease identification and RT-PCR confirmed that the expansion of Sox9 and construction of Sox9 expression vector were successful. After transfection of the recombinant vector into bone marrow-derived stromal cells, the expression of Sox9 and FGFR-3 was detected, and proliferative viability was not different from that of precartilaginous stem cells. It was concluded that Sox9 gene eukaryotic expression vector was successfully constructed, and the transfected bone marrow-derived stromal cells differentiated into the precartilaginous stem cells.

The effect of pulsed electromagnetic fields on the proliferation of immunomagnetically separated human pre-cartilaginous stem cells

Objective To study the biological effects of pulsed electromagnetic fields (PEMFs) on the pro-liferation of immunomagnetically separated human precartilaginous stem cells (PSCs) in vitro. Methods The cells from an aborted fetus's metaphysis were digested using collagenase. The PSCs were isolated by magnetic cell sorting (MACS), then subcultured and amplified. Flow cytometry, immunohistochemistry, immunofluorescence and RT-PCR analysis were performed to identify the purified PSCs. The PSCs were stimulated by PEMFs at 50 Hz frequency and 1 mT intensity. Cell proliferation was measured at different time points using methyl thiazolyl tetrazolium ( MTT), and the cell growth curve was plotted. Flow cytometry was applied to measure the cell cycle and apoptosis. Results The PSCs were successfully cultured. There was fibroblast growth factor receptor-3 (FGFR-3) on their sur-face. Cell proliferation was promoted after 4 and 6 days of PEMF stimulation. The percentage of cells at the S phase was higher than in a control group. Early, late and total rates of apoptosis in the experimental group decreased signifi-cantly. Conclusion PEMFs can enhance the proliferation and inhibit the apoptosis of human PSCs, and it is possi-ble to cultivate the high density human PSCs in vitro. Key words: Precartilaginous stem cells; Fibroblast growth factor receptor-3 ; Cell culture ; Pulsed elec-tromagnetic fields

Proliferation and differentiation of precartilaginous stem cells in response to stable expression of hTGF-β3

Objective To study the effect of stable expression of reconstructed human transforming growth factor-β3 ( hTGF-β3) on proliferation of precartilaginous stem cells ( PSCs) and their differentiation into cartilage chondrocytes.Methods After isolated and purified by immunological microbeads,PSCs of rats were transfected with pcDNA3.1 ( + )-hTGF-β3.MTT reduction assay ( MTT) and flow cytometry were performed to investigate the effect of transduction on proliferation and DNA synthesis.Biosynthesis of hTGF-β3 and expressions of cartilage associated genes and proteins were examined by qRT-PCR,immunohistology and Western blot.Results hTGF-β3 was expressed in PSCs stably.Compared with non-transfection group,PSCs' DNA synthesis level and proliferation rate were significantly increased after transfection.Quantitative real-time PCR and immunological investigation suggested up-regulated expression of specific genes and proteins of chondrocyte and increase of deposition of chondrocyte typical extracellular matrices proteoglycan and collagen type Ⅱ .Conclusions Gene enhanced PSCs can stably express hTGF-β3 protein to promote proliferation of PSCs and induce differentiation of PSCs in to chondrocytes,which provides a fresh approach to cartilage tissue engineering. Key words: Transforming growth factor β; Stem cells; Transfection; Cell differentiation