Nucleostemin Expression Research Articles

Quantitative expression of pluripotency-related genes in parthenogenetically produced buffalo (Bubalus bubalis) embryos and in putative embryonic stem cells derived from them

Parthenogenetically produced embryos and embryonic stem (ES) cells derived from them offer a unique model for investigating the role of transcription factors in embryonic genome activation (EGA), pluripotent lineage specification and in pluripotency and self-renewal of ES cells because of the unique nature of these embryos. There is little information on the quantitative expression of important genes in parthenogenetically produced embryos and in ES cells derived from them. The present study examined the quantitative expression of some important genes in parthenogenetically produced buffalo embryos and in putative parthenogenetic ES cells (pES) cells. The quantitative expression of OCT-4, SOX-2, NANOG, REX-1, FOXD-3 and NUCLEOSTEMIN, which is very low in immature and mature oocytes, and in embryos at 2-, 4- and 8- to 16-cell stage, increases significantly at morula and blastocyst stage. The expression level of TELOMERASE, c-MYC and STAT-3, which is high in immature oocytes decreases during embryonic development followed by either an increase at the morula stage (TELOMERASE) or a low expression level maintained throughout development till blastocyst stage (c-MYC and STAT-3). There is a progressive decline in the expression level of OCT-4, SOX-2, c-MYC, REX-1, NUCLEOSTEMIN, TELOMERASE and STAT-3 during long term culture of pES cells.

Nucleostemin stabilizes ARF by inhibiting the ubiquitin ligase ULF.

Up-regulated expression of nucleolar GTPase Nucleostemin (NS) has been associated with increased cellular proliferation potential and tumor malignancy during cancer development. Recent reports attribute the growth regulatory effects of NS protein to its role in facilitating ribosome production. However, the oncogenic potential of NS remains unclear since imbalanced levels of NS have been reported to exert growth inhibitory effect by modulating p53 tumor suppressor activity. It also remains in questions if aberrant NS levels might play a p53-independent role in regulation of cell proliferation and growth. In this study, we performed affinity purification and mass spectrometry analysis to explore protein-protein interactions influencing NS growth regulatory properties independently of p53 tumor suppressor. We identified the Alternative Reading Frame (ARF) protein as a key protein associating with NS and further verified the interaction through in vitro and in vivo assays. We demonstrated that NS is able to regulate cell cycle progression by regulating the stability of the ARF tumor suppressor. Furthermore, overexpression of NS suppressed ARF polyubiquitination by its E3 ligase ULF and elongated its half-life, while knockdown of NS led to the decrease of ARF levels. Also, we found that NS can enhance NPM stabilization of ARF. Thus, we propose that in the absence of p53, ARF can be stabilized by NS and NPM to serve as an alternative tumor suppressor surveillance, preventing potential cellular transformation resulting from the growth inducing effects of NS overexpression.

Nucleostemin expression in invasive breast cancer

BackgroundRecently, the cancer stem cell hypothesis has become widely accepted. Cancer stem cells are thought to possess the ability to undergo self-renewal and differentiation, similar to normal stem cells. Nucleostemin (NS), initially cloned from rat neural stem cells, binds to various proteins, including p53, in the nucleus and is thought to be a key molecule for stemness. NS is expressed in various types of cancers; therefore, its role in cancer pathogenesis is thought to be important. This study was conducted to clarify the clinicopathological and prognostic impact of NS in invasive breast cancers.MethodThe correlation between NS immunoreactivity and clinicopathological parameters was examined in 220 consecutive surgically resected invasive breast cancer tissue samples by using tissue microarrays. The presence of nuclear NS and p53 immunoreactivity in 10% or more of cancer cells was considered as a positive result.ResultsAmong the 220 patients, 154 were hormone-receptor (HR)-positive, 22 HER2-positive/HR-negative, and 44 HR-negative/HER2-negative. One hundred and forty-two tumors (64.5%) showed NS positivity, and this positivity was significantly correlated with estrogen receptor (ER) (P = 0.050), human epidermal growth factor receptor 2 (HER2) (P = 0.021), and p53 (P = 0.031) positivity. The patients with NS-positive tumors showed significantly shorter disease-free survival than those with NS-negative tumors. Furthermore, the patient group with NS- and p53-positive tumors showed significantly poorer prognosis than other patient groups. Multivariate analysis showed that NS status was an independent prognostic indicator.ConclusionsNS may play a significant role in the determination of breast cancer progression in association with p53 alterations. The NS status of patients with luminal and HER2 type breast cancers may be a useful prognostic marker.

Down-regulatory effect of Nucleostemin expression on signal molecule of PI3K/AKT/mTOR pathway in HL-60 cells

This study was purpose to explore the down-regulatory effect of nucleostemin (NS) expression on signal molecules of PI3K/AKT/mTOR pathway belonged to candidate ways of p53-independent signal pathway in the leukemia cells. The expression of NS was interfered by using recombinant lentivirus expression vector NS-RNAi-GV248 to transfect HL-60 cells of p53 deficiency. The expression of NS and signal molecules of PI3K/AKT/mTOR pathway were detected by using Real-time PCR. The results of showed that the HL-60 cells were transfected by recombinant lentivirus vector NS-RNAi-GV248 successfully and with transfection rate up to 80%. According to results of Real-time PCR detection, the inhibition rate of NS gene was 56.5% in HL-60 cells. And the expression levels of PI3K,AKT and GβL mRNA (0.491 ± 0.084,0.398 ± 0.164, 0.472 ± 0.097 respectively) were obviously down-regulated by silencing NS, and showed statistical difference (P < 0.05) in comparison with control (1.002 ± 0.171, 1.000 ± 0.411, 1.001 ± 0.206 respectively) . It is concluded that the changes of signal molecules of PI3K/AKT/mTOR pathway positively correlate with NS down-regulation, which provides evidence for confirming PI3K/AKT/mTOR signal pathway possible as a type of NS p53-independent pathway.

Comparison of nucleostemin gene expression in CD133+ and CD133- cell population in colon cancer cell line HT29.

Nucleostemin has been shown to be essential for proliferation and survival of colon cancer cells. In this study, we evaluate and comparing nucleostemin expression in CD133+ and CD133- colon cancer cell line HT29. After preparation and culturing of HT29 cell line, isolation was performed using magnetic cell separation system by CD133 MicroBeads and phycoerythrin conjugated to monoclonal anti-human CD133 monoclonal antibody and analyzed with flow cytometry. For quantitative expression of nucleostemin in HT29, CD133+ and CD133- cells used specific nucleostemin primer and glyceraldehyde 3-phosphate dehydrogenase primer as endogenous control. The results showed the percentage of CD133+ cells in HT29 colon cancer cell line ranged from 36.5% to 41.5%, whereas the percentage of CD133- cell ranged from 58.5% to 63.5%. The expression rate of nucleostemin in HT29, CD133+ and CD133- cells were 1.44 ± 0.78, 1.60 ± 0.70 and 1.00 ± 0.18 (respectively). The comparison of expression rate represents no significant difference in nucleostemin expression in CD133+, CD133- and HT29 colon cancer cells. It is concluded that nucleostemin expression could not be specific in a certain type of cells in colon cancer cell line HT29 and controlling strategies in colon cancer must not be focused on one certain type of colon cancer cells as main expressing nucleostemin gene.

Over expression of nucleostemin contributes to malignant phenotype and poor prognosis in oral squamous cell carcinoma

Background: Nucleostemin (NS) has been reported essential for maintenance of normal and cancer stem cell properties. However, the impacts of NS on malignant phenotype of OSCC and prognosis of patients with OSCC have not been clarified. The purpose of this study is to explore the impacts of NS in malignant phenotype and prognosis of OSCC.

Nucleostemin and ASPP2 expression is correlated with pituitary adenoma proliferation

Nucleostemin is a GTP-conjugated protein located in the nucleoli of stem cells and certain cancer cells, and maintains cellular self-renewal. The present study aimed to evaluate nucleostemin as a potential target for pituitary adenoma gene therapy by investigating nucleostemin and apoptosis-stimulating of p53 protein 2 (ASPP2) expression and their effect on pituitary adenoma cell proliferation. A total of 71 samples of pituitary adenomas were collected. Semi-quantitative PCR was used to detect the expression of nucleostemin and ASPP2 mRNA in the samples. Immunochemistry techniques were used to examine Ki-67 expression in the paraffin section of the samples. Coherent clinical data were also collected. Nucleostemin and ASPP2 were detectable in all the pituitary adenoma samples. Significant differences were observed in nucleostemin and ASPP2 expression between invasive pituitary adenoma and non-invasive pituitary adenomas (P<0.01) and the Ki-67 labeling index (LI; P>0.05). The difference in the Ki-67 LI between the recurrence and non-recurrence groups was significant (P<0.05). There was positive correlation between nucleostemin gene expression and the Ki-67 LI levels (P<0.05). The correlation between ASPP2 expression and the Ki-67 LI was negative (P<0.05). Negative correlation was demonstrated between nucleostemin and ASPP2 expression (P<0.01). The nucleostemin and ASPP2 genes were expressed in the human pituitary adenoma tissues. The differences in the expression of nucleostemin, ASPP2 and Ki-67 in the various pathological types of pituitary adenomas represented differences in molecular biological character and were associated with invasion. In the pituitary adenomas, the expression of nucleostemin and ASPP2 was correlated with tumor proliferation. Nucleostemin, ASPP2 and Ki-67 may serve as valid clinical detection markers for the invasion of pituitary adenomas.

Abundant Nucleostemin Expression Supports the Undifferentiated Properties of Germ Cell Tumors

Nucleostemin (NS) is a nucleolar GTP-binding protein that is involved in ribosomal biogenesis and protection of telomeres. We investigated the expression of NS in human germ cell tumors and its function in a mouse germ cell tumor model. NS was abundantly expressed in undifferentiated, but not differentiated, types of human testicular germ cell tumors. NS was expressed concomitantly with OCT3/4, a critical regulator of the undifferentiated status of pluripotent stem cells in primordial germ cells and embryonal carcinomas. To investigate the roles of NS in tumor growth invivo, we used a mouse teratoma model. Analysis of teratomas derived from embryonic stem cells in which the NS promoter drives GFP expression showed that cells highly expressing NS were actively proliferating and exhibited the characteristics of tumor-initiating cells, including the ability to initiate and propagate tumor cells invivo. NS-expressing cells exhibited higher levels of GTP than non-NS-expressing cells. Because NS protein is stabilized by intracellular GTP, metabolic changes may contribute to abundant NS expression in the undifferentiated cells. OCT3/4 deficiency in teratomas led to loss of NS expression, resulting in growth retardation. Finally, we found that teratomas deficient in NS lost their undifferentiated characteristics, resulting in defective tumor proliferation. These data indicate that abundant expression of NS supports the undifferentiated properties of germ cell tumors.

Nucleostemin in Injury-Induced Liver Regeneration

The high regenerative capacity of liver contributes to the maintenance of its size and function when injury occurs. Partial hepatectomy induces division of mature hepatocytes to maintain liver function, whereas severe injury stimulates expansion of undifferentiated hepatic precursor cells, which supply mature cells. Although several factors reportedly function in liver regeneration, the precise mechanisms underlying regeneration remain unclear. In this study, we analyzed expression of nucleostemin (NS) during development and in injured liver by using transgenic green fluorescent protein reporter (NS-GFP Tg) mice. In neonatal liver, the hepatic precursor cells that give rise to mature hepatocytes were enriched in a cell population expressing high levels of NS. In adult liver, NS was abundantly expressed in mature hepatocytes and rapidly upregulated by partial hepatectomy. Severe liver injury promoted by a diet containing 3,5-diethoxycarbonyl-1,4-dihydrocollidine induced the emergence of NS-expressing ductal epithelial cells as hepatic precursor cells. NS knockdown inhibited both hepatic colony formation in vitro and proliferation of hepatocytes in vivo. These data strongly suggest that NS plays a critical role in regeneration of both hepatic precursor cells and hepatocytes in response to liver injury.

Effects of celecoxib on proliferation,apoptosis and Nucleostemin expression in human breast cancer cell line SKBR3

Objective To observe the effects of celecoxib on proliferation,apoptosis and Nucleostemin (NS) expression in human breast cancer cell line SKBR3.Methods SKBR3 cells were cultured in vitro. By using assays of methyl thiazol tetrazolium (MTT),morphology,Western blotting,Caspase-3 activity and flow cytometry,the effects of celecoxib on proliferation and apoptosis of SKBR3 cells,and the location and expression of NS were detected.Results The growth of SKBR3 cells was inhibited by celecoxib in a concentration-depeodent manner.Celecoxib ( 15,30,60,and 120 μmol/L) could upregulate the expression of NS protein (0.460 ± 0.094,0.695 ± 0.124,0.820 ± 0.161,1.058 ±0.196) and p53 protein (0.898 ±0.166,1.231 ±0.254,1.518 ±0.309,1.828 ±0.362),and simultaneously downregulate the expression of COX-2 protein (0.252 ±0.053,0.184 ± 0.032,0.131 ±0.028,0.099±0.019) and Cyclin D1 protein (0.829±0.178,0.661 ±10.112,0.524±0.108,0.378 ±0.075),respectively.Morphological observation revealed that celecoxib-treated cells showed several typical apoptotic features.Meanwhile the typical apoptosis peak was detected by flow cytometry.The changes in apoptosis rate and cell cycle were concentration-dependent and the cell percentage in G0/G1 phase was increased.Conclusion Celecoxib can concentration-dependently inhibit proliferation of SKBR3 cells and induce their aoootosis probably by up-regulating the expression of NS. Key words: Breast carcinoma; Celecoxib; Proliferation; Apoptosis

Expression of nucleostemin in proliferating and differentiating cells of the human retina during prenatal development

Expression of nucleostemin in proliferating and differentiating cells of the human retina during prenatal development

Expression of nucleostemin in synovial tissues and in normal and osteoarthritic chondrocytes

Expression of nucleostemin in synovial tissues and in normal and osteoarthritic chondrocytes

An Off-Target Nucleostemin RNAi Inhibits Growth in Human Glioblastoma-Derived Cancer Stem Cells

Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor.

GTP depletion synergizes the anti-proliferative activity of chemotherapeutic agents in a cell type-dependent manner

Mycophenolic acid (MPA) depletes intracellular GTP by blocking de novo guanine nucleotide synthesis. GTP is used ubiquitously for DNA/RNA synthesis and as a signaling molecule. Here, we made a surprising discovery that the anti-proliferative activity of MPA acts synergistically with specific chemotherapeutic agents in a cell type-dependent manner. In MDA-MB-231 cells, MPA shows an extremely potent synergy with 5-FU but not with doxorubicin or etoposide. The synergy between 5-FU and MPA works most effectively against the highly tumorigenic mammary tumor cells compared to the less tumorigenic ones, and does not work in the non-breast cancer cell types that we tested, with the exception of PC3 cells. On the contrary, MPA shows the highest synergy with paclitaxel but not with 5-FU in SCC-25 cells, derived from oral squamous cell carcinomas. Mechanistically, the synergistic effect of MPA on 5-FU in MDA-MB-231 cells can be recapitulated by inhibiting the RNA polymerase-I activity and requires the expression of nucleostemin. This work reveals that the synergy between MPA and anti-proliferative agents is determined by cell type-dependent factors.

Clinical Significance of Nucleostemin Expression and Its Correlation With Cyclin D1 Expression in Malignant Ovarian Tumors

To investigate the expression of the nucleostemin (NS) gene in ovarian tumors and its correlation with the expression of cyclin D1. The expression levels of nucleostemin and cyclin D1 proteins were measured by immunohistochemical staining in ovarian tumors and normal ovarian tissues, and the relationship between their levels was analyzed. Nucleostemin gene and cyclin D1 expressions were detected in 28 and 27 specimens of malignant ovarian tumors (93.7% and 90.0%), 4 and 8 specimens of ovarian borderline tumors (40.0% and 80.0%), and in both specimens of benign ovarian tumors, respectively. The expression of NS gene was seen in 5, 13, and 10 specimens of malignant ovarian tumors of high, moderate, and low grade, respectively. The expression of the nucleostemin gene was significantly related to the tumor grade (r = 0.786, P < 0. 05), and a significant relationship between nucleostemin and cyclin D1 expression was found (r = 0.834, P < 0.05). The expression of the nucleostemin gene was not detected in normal ovarian tissue. The expression of the nucleostemin gene in ovarian tumors is closely correlated with origination, progression, and grading of tumors and may serve as a marker in estimating the malignancy of ovarian tumors. The overexpression of cyclin D1 might be correlated with nucleostemin expression. Nucleostemin may have an impact on the passage of cells through the G1/S checkpoint, and thus cell cycle progress, by regulating cyclin D1 expression.

Nucleostemin affects the proliferation but not differentiation of oral squamous cell carcinoma cells

Nucleostemin (NS) has been reported as essential for stem and cancer cell proliferation. To investigate the significance of NS in oral squamous cell carcinomas (OSCCs), we examined NS expression in neoplastic tissue of the tongue and in OSCC cell lines. Nucleostemin expression in the histological samples showed positive correlation with Ki-67 expression. Furthermore, NS expression was associated with cellular proliferation in OSCC cell lines using siRNA, which upregulated p27, a cyclin-dependent kinase inhibitor. Regarding OSCC differentiation, NS expression did not influence cornification or oral epithelial differentiation markers such as involucrin and cytokeratin19. Thus, NS is widely expressed in normal and neoplastic oral epithelial tissues, and is likely a marker of proliferation.

311 EXPRESSION OF PLURIPOTENT MARKER NUCLEOSTEMIN IN BUFFALO (BUBALUS BUBALIS) EMBRYOS AND EMBRYONIC STEM CELLS GENERATED THROUGH PARTHENOGENETIC ACTIVATION

Nucleostemin is a newly found putative GTPase protein that binds to P53 and exists mainly in the nucleoli and at a very low level in nucleoplasm of undifferentiated embryonic stem cells (ESC) and myeloid stem cells but is not expressed in committed and terminally differentiated cells. Embryonic stem cells are pluripotent cells derived from the inner cell mass (ICM) of blastocysts. The ICM and ESC express a number of transcription factors, and their expression is used as a pluripotency marker in the ESC of many species. The present study was undertaken to identify expression of the nucleostemin gene in different developmental stages of buffalo embryos and cultured ESC. Parthenogenetic activation is a process by which an oocyte can be developed up to blastocyst without fertilization. The parthenotes were produced by following protocol. Briefly, immature oocytes were aspirated from slaughterhouse buffalo ovaries and subjected to in vitro maturation for 24 h in a CO2 incubator (5% O2, 5% CO2, 90–95% relative humidity) at 38.5°C. After 24 h of in vitro maturation, oocytes were activated by exposure to 7% ethanol for 7 min, followed by incubation with 2 mM 6-dimethyl aminopurine in CR2 medium for 3.5 h, and they were then subjected to in vitro culture. The activated embryos were cultured for 8 days in CR2 medium containing 0.6% BSA and 10% FBS to obtain different stages (immature and mature oocytes 2-, 4-,8–16-cell, morula, and blastocyst) of embryos. A total of 23 blastocysts were produced parthenogenetically, of which 5 blastocysts were used for nucleostemin expression and the rest were used for ICM isolation. The isolated ICM were subsequently cultured on mitomycin-C (10 μg mL–1) treated buffalo fetal fibroblast feeder layer in DMEM medium supplemented with 20% fetal bovine serum, 1 000 IU mL–1 of mouse leukemia inhibitory factor, 1% nonessential amino acids, 2 mM L-glutamine, and 50 μg mL–1 gentamycin. These ESC were cultured up to 5 passages. The 5 embryos of different developmental stages and a clump of ESC were used for nucleostemin expression. The total RNA was isolated and transcribed using Cell-to-cDNA-II (Ambion, Austin, TX, USA) according to manufacturer protocol. To amplify the nucleostemin gene, the PCR cycle was carried out and included heating to 94°C for 5 min, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 40 s. The expressions of nucleostemin transcript were observed in all the developmental stages including immature and mature oocytes. The transcript was highly expressed in the 2-cell stage, blastocysts, and ESC, but immature oocytes and 8–16-cell stage showed lower expression. The experiment was repeated, and the same result was found. To our knowledge this is the first report in buffalo. It is concluded that the transcript was expressed in all the early stages of parthenogenetically derived buffalo embryos from immature oocytes to blastocysts and continued to be expressed in ESC. This work was funded by NAIP, C-420678075, India.

Characterization of Dental Pulp Stem Cells from Impacted Third Molars Cultured in Low Serum-Containing Medium

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-β1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.

Continual Expression Throughout the Cell Cycle and Downregulation upon Adipogenic Differentiation Makes Nucleostemin a Vital Human MSC Proliferation Marker

Nucleostemin (NS) is a nucleolar protein expressed in stem and cancer cells. In combination with nuclear/nucleolar proteins, NS has been demonstrated to be involved in cell-cycle regulation and telomere maintenance. NS expression reflects the cell's proliferation state indicating that the cell is active in the cell cycle, whereas NS signals disappear upon differentiation. This study analyzes the spatio-temporal (nucleolar/nuclear localization during interphase and M-phase) NS remodeling in two distinct human mesenchymal stem cell (MSC) populations to discriminate the NS differences, if any, throughout their stem cell and differentiation states. Beside its prominent multilobular nucleolar localization in interphase cells, coexistence of NS with chromosome arms during mitosis was also observed. Disruption of mitotic microtubules induced dissociation of NS from the chromosome arms and scattered it into the cytoplasm. Compared to deciduous dental pulp MSCs, NS mRNA expression gradually decreased upon aging in umbilical cord stroma-derived MSCs as culture time increased. Following adipogenic differentiation of the latter, NS signals gradually disappeared in both dividing and non-dividing cells, even before the morphological and functional signs of adipogenic transformation appeared. Quantitative NS mRNA measurements showed that MSCs from two sources exhibit a strong nucleostemin expression similar to embryonic stem cells. In conclusion, apart from its novel chromosomal localization shown in this study, nucleolar NS can be considered as a marker that indicates the proliferation/differentiation states in human MSCs. Moreover, differences in the relative NS protein and mRNA levels may reflect the degree of proliferation and can be used to characterize in vitro expansion capabilities.

Platelet-Rich Plasma Releasate Promotes Differentiation of Tendon Stem Cells into Active Tenocytes

Background Platelet-rich plasma (PRP) has been used to enhance tendon healing in clinical settings. However, the cellular mechanisms underlying PRP treatment of injured tendons remain unclear. The aim of this study was to determine the effects of PRP, in the form of PRP-clot releasate (PRCR), on tendon stem cells (TSCs), a newly discovered cell population in tendons. Hypothesis The PRCR treatment promotes differentiation of TSCs into tenocytes that are activated to proliferate quickly and increase collagen production. Study Design Controlled laboratory study. Methods After PRCR treatment, cell morphology, expression of stem/progenitor cell marker nucleostemin, and population doubling time were examined. In addition, gene and protein analyses were performed using reverse transcription-polymerase chain reaction, immunocytochemistry, and Western blot to characterize the type of cells that had differentiated after PRCR treatment. Results The TSCs without PRCR treatment were small and exhibited an irregular shape, whereas with increasing PRCR dosage, TSCs became large, well spread, and highly elongated with downregulation of nucleostemin expression. The PRCR treatment also markedly enhanced TSC proliferation, tenocyte-related gene and protein expression, and total collagen production, all of which indicated that PRCR treatment induced differentiation of TSCs into activated tenocytes. Conclusion The PRCR treatment promotes differentiation of TSCs into active tenocytes exhibiting high proliferation rates and collagen production capability. Clinical Relevance The findings of this study suggest that PRP treatment of injured tendons is “safe” as it promotes TSC differentiation into tenocytes rather than nontenocytes, which would compromise the structure and function of healing tendons by formation of nontendinous tissues. Moreover, they suggest that PRP treatment can enhance tendon healing because tenocytes induced to differentiate by PRP are activated to proliferate quickly and produce abundant collagen to repair injured tendons that have lost cells and matrix.