Enhancement of Matrix Metalloproteinase-2 (MMP-2) as a Potential Chondrogenic Marker during Chondrogenic Differentiation of Human Adipose-Derived Stem Cells

Arai, Yoshie,Park, Sunghyun,Choi, Bogyu,Ko, Kyoung-Won,Choi, Won Chul,Lee, Joong-Myung,Han, Dong-Wook,Park, Hun-Kuk,Han, Inbo,Lee, Jong Hun,Lee, Soo-Hong MDPI 2016 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.17 No.6

<P>Human adipose-derived stem cells (hASCs) have a capacity to undergo adipogenic, chondrogenic, and osteogenic differentiation. Recently, hASCs were applied to various fields including cell therapy for tissue regeneration. However, it is hard to predict the direction of differentiation of hASCs in real-time. Matrix metalloproteinases (MMPs) are one family of proteolytic enzymes that plays a pivotal role in regulating the biology of stem cells. MMPs secreted by hASCs are expected to show different expression patterns depending on the differentiation state of hASCs because biological functions exhibit different patterns during the differentiation of stem cells. Here, we investigated proteolytic enzyme activity, especially MMP-2 activity, in hASCs during their differentiation. The activities of proteolytic enzymes and MMP-2 were higher during chondrogenic differentiation than during adipogenic and osteogenic differentiation. During chondrogenic differentiation, mRNA expression of MMP-2 and the level of the active form of MMP-2 were increased, which also correlated with Col II. It is concluded that proteolytic enzyme activity and the level of the active form of MMP-2 were increased during chondrogenic differentiation, which was accelerated in the presence of Col II protein. According to our findings, MMP-2 could be a candidate maker for real-time detection of chondrogenic differentiation of hASCs.</P>

Increase of chondrogenic potentials in adipose-derived stromal cells by co-delivery of type I and type II TGFβ receptors encoding bicistronic vector system

Kang, Sun-Woong,Do, Hyun-Jin,Han, In-Bo,Shin, Dong-Ah,Kim, Hyun Ok,Kim, Jae-Hwan,Lee, Soo-Hong Elsevier 2012 Journal of controlled release Vol.160 No.3

<P><B>Abstract</B></P><P>Stem cell therapy has been developing rapidly as a potential cure for repairing or regenerating the functions of diseased organs and tissues. Adipose-derived stromal cells (ASCs) are an attractive cell source for stem cell therapy because they can be isolated easily from fat tissue in significant numbers and exhibit multiple differentiation potential under appropriate <I>in vitro</I> culture conditions. However, ASCs derived from individual donors can show wide variations in differentiation potential. In addition, the regulatory mechanisms underlying stem cell differentiation remain unclear. Transforming growth factor β (TGFβ) is a well-known ASC chondrogenic differentiation factor that stimulates ASC signaling pathways by activating transmembrane type I and type II receptors. We hypothesized that the chondrogenic differentiation potential of ASCs is dependent upon the expression of TGFβ receptors and could be improved by the co-delivery of type I (TGFβRI) and type II (TGFβRII) TGFβ receptors. To prove this, heterogeneity within the chondrogenic potential of ASCs isolated from 10 donors was examined and their susceptibility to TGFβ during the process of chondrogenic differentiation investigated. In addition, the results showed that co-delivery of the TGFβRI and TGFβRII genes increased the expression of TGFβ receptor signaling in ASCs with low chondrogenic potential, resulting in increased chondrogenic differentiation. Monitoring and delivering TGFβRI and TGFβRII may, therefore, be a powerful tool for predicting the differentiation potential of stem cells and for enhancing their differentiation capacity prior to stem cell transplantation.</P> <P><B>Graphical abstract</B></P><P>We established an efficient prediction method and enhancement of chondrogenic potential through measuring the expression ratio of ALK1 and ALK5 signaling and by co-delivery TGFβ receptor genes prior to transplantation.<ce:figure id='f0005'></ce:figure></P>

Chondrogenic Differentiation of Bone Marrow Stromal Cells in Transforming Growth Factor-β1 Loaded Alginate Bead

Ki Suk Park,한창환,이해방,양윤선,김문석,Chae Moon Jin,Soon Hee Kim,John M. Rhee,강길선 한국고분자학회 2005 Macromolecular Research Vol.13 No.4

We developed alginate beads loaded with transforming growth factor-1 (TGF-1) to examine the possible application of the scaffold and cytokine carrier in tissue engineering. In this study, bone marrow stromal cells (BMSCs) and TGF-1 were uniformly encapsulated in the alginate beads and then cultured in vitro. The cell mor phology and shape of the alginate beads were observed using inverted microscope, scanning electron microscope (SEM), histological staining and RT-PCR to confirm chondrogenic differentiation. The amount of the TGF-1 released from the TGF-1 loaded alginate beads was analyzed for 28 days in vitro in a phosphate buffered saline (pH 7.4) at 37oC. We observed the release profile of TGF-1 from TGF-1 loaded alginate beads with a sustained release pattern for 35 days. Microscopic observation showed the open cell pore structure and abundant cells with a round morphology in the alginate beads. In addition, histology and RT-PCR results revealed the evidence of chondrogenic differentiation in the beads. In conclusion, these results confirmed that TGF-1 loaded alginate beads provide excel lent conditions for chondrogenic differentiation.

The microRNA expression profiles of mouse mesenchymal stem cell during chondrogenic differentiation

( Bo Yang ),( Hong Feng Guo ),( Yu Lan Zhang ),( Shi Wu Dong ),( Da Jun Ying ) 생화학분자생물학회(구 한국생화학분자생물학회) 2011 BMB Reports Vol.44 No.1

MicroRNAs are potential key regulators in mesenchymal stem cells chondrogenic differentiation. However, there were few reports about the accurate effects of miRNAs on chondrogenic differentiation. To investigate the mechanisms of miRNAs-mediated regulation during the process, we performed miRNAs microarray in MSCs at four different stages of TGF-β3-induced chondrogenic differentiation. We observed that eight miRNAs were significantly up-regulated and five miRNAs were down- regulated. Interestingly, we found two miRNAs clusters, miR-143/145 and miR-132/212, kept on down-regulation in the process. Using bioinformatics approaches, we analyzed the target genes of these differentially expressed miRNAs and found a series of them correlated with the process of chondrogenesis. Furthermore, the qPCR results showed that the up-regulated (or down-regulated) expression of miRNAs were inversely associated with the expression of predicted target genes. Our results first revealed the expression profiles of miRNAs in chondrogenic differentiation of MSCs and provided a new insight on complicated regulation mechanisms of chondrogenesis. [BMB reports 2011; 44(1): 28-33]

Low-Intensity Ultrasound (LIUS) as an Innovative Tool for Chondrogenesis of Mesenchymal Stem Cells (MSCs).

Park, So Ra,Choi, Byung Hyune,Min, Byoung-Hyun LANDES BIOSCIENCE 2007 ORGANOGENESIS Vol.3 No.2

<P>Mesenchymal stem cells (MSCs) have a capacity to differentiate into the chondrogenic lineage and are a valuable allogenic source for cartilage tissue engineering. However, they still have critical limitations of relatively inefficient chondrogenic differentiation in vitro and of dedifferentiation and/or hypertrophic changes at late stages of differentiation. Numerous approaches using biochemical and mechanical factors have been tried but have so far failed to overcome these problems. Recent studies by other groups and ours have shown that low-intensity ultrasound (LIUS) is an efficient tool for promoting the chondrogenic differentiation of MSCs both in vitro and in vivo. A series of our experiments suggests that LIUS not only induces chondrogenic differentiation of MSCs but also has diverse additional activities that enhance the viability of MSCs, increase possibly the integrity of the differentiated tissues and delays hypertrophic changes during differentiation. Therefore, LIUS could be an innovative and versatile tool for chondrogenic differentiation of MSCs and for cartilage tissue engineering.</P>

The effect of serum types on Chondrogenic differentiation of adipose-derived stem cells

조혜란,Aeri Lee,김교범 한국생체재료학회 2018 생체재료학회지 Vol.22 No.1

Background: Fetal bovine serum (FBS) is the most essential supplement in culture media for cellular proliferation, metabolism, and differentiation. However, due to a limited supply and subsequently rising prices, a series of studies have investigated a biological feasibility of replaceable serums to substitute FBS. Along with the increasing interests to manufacture stem cell-based cellular products, optimizing the composition of culture media including serums and exogenous growth factors (GFs) is of importance. In this experiment, the effect of bovine serum (BS) and newborn calf serum (NCS) on proliferation and chondrogenic differentiation capacity of human adipose derived stem cells (ADSCs) was evaluated, especially in the chondrogenically supplemented culture condition. Methods: ADSCs were chondrogenically cultured with FBS, BS, and NCS for 14 days. For the acceleration of in vitro chondrogenesis, exogenous insulin-like growth factor and transforming growth factor-β3 were added. Viability and proliferation of ADSCs were evaluated using Live/Dead fluorescence staining and DNA amount, respectively. To investigate a chondrogenic differentiation, a series of assays were performed including a quantification of glycosaminoglycan deposition, alcian blue staining, and RT-PCR analysis for type II collagen, aggrecan and Sox-9 genes. Results: The results demonstrated that proliferation of ADSCs was facilitated in FBS condition as compared with other serum types. For chondrogenic marker gene expression, serum substitutes enhanced Sox-9 expression level on day 14. The deposition of glycosaminoglycan was more facilitated in BS condition regardless of additional chondrogenic GFs. Conclusion: It could be presumably speculated that serum types and exogenous supplements of GFs could also be important parameters to optimize culture media composition, especially in order to maintain the enhanced levels of both proliferation and chondrogenic differentiation of ADSCs during expansion.

<i>Enterococcus faecium</i> L-15 Cell-Free Extract Improves the Chondrogenic Differentiation of Human Dental Pulp Stem Cells

Kim, Hyewon,Park, Sangkyu,Kim, Kichul,Ku, Seockmo,Seo, Jeongmin,Roh, Sangho MDPI AG 2019 INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES Vol.20 No.3

<P>Hyaline cartilage is a tissue of very low regenerative capacity because of its histology and limited nutrient supply. Cell-based therapies have been spotlighted in the regeneration of damaged cartilage. Dental pulp stem cells (DPSCs) are multipotent and are easily accessible for therapeutic purposes. In human gastrointestinal tracts, <I>Enterococcus faecium</I> is a naturally occurring commensal species of lactic acid bacteria. In this work, the human DPSCs were differentiated into chondrocytes using a chondrogenic differentiation medium with or without L-15 extract. We observed that chondrogenic differentiation improved in an <I>E. faecium</I> L-15 extract (L-15)-treated DPSC group via evaluation of chondrogenic-marker mRNA expression levels. In particular, we found that L-15 treatment promoted early-stage DPSC differentiation. Cells treated with L-15 were inhibited at later stages and were less likely to transform into hypertrophic chondrocytes. In L-15-treated groups, the total amount of cartilage extracellular matrix increased during the differentiation process. These results suggest that L-15 promotes chondrogenic differentiation, and that L-15 may be used for cartilage repair or cartilage health supplements. To our knowledge, this is the first report demonstrating the beneficial effect of L-15 treatment on chondrogenic differentiation.</P>

Comparison of the Infant and Adult Adipose-Derived Mesenchymal Stem Cells in Proliferation, Senescence, Anti-oxidative Ability and Differentiation Potential

Wu Szu-Hsien,Yu Jin-Huei,Liao Yu-Ting,Liu Kuo-Hao,Chiang En-Rung,Chang Ming-Chau,Wang Jung-pan 한국조직공학과 재생의학회 2022 조직공학과 재생의학 Vol.19 No.3

BACKGROUND: Infant adipose-derived mesenchymal stem cells (ADSCs) collected from excised polydactyly fat tissue, which was surgical waste, could be cultured and expanded in vitro in this study. In addition, the collecting process would not cause pain in the host. In this study, the proliferation, reduction of senescence, anti-oxidative ability, and differentiation potential in the infant ADSCs were compared with those in the adult ADSCs harvested from thigh liposuction to determine the availability of infant ADSCs. METHODS: Proliferation was determined by detecting the fold changes in cell numbers and doubling time periods. Senescence was analyzed by investigating the age-related gene expression levels and the replicative stress. The superoxide dismutase (SOD) gene expression, adipogenic, neurogenic, osteogenic, and tenogenic differentiation were compared by RTqPCR. The chondrogenic differentiation efficiency was also determined using RT-qPCR and immunohistochemical staining. RESULTS: The proliferation, SOD (SOD1, SOD2 and SOD3) gene expression, the stemness-related gene (c-MYC) and telomerase reverse transcriptase of the infant ADSCs at early passages were enhanced compared with those of the adults’. Cellular senescence related genes, including p16, p21 and p53, and replicative stress were reduced in the infant ADSCs. The adipogenic genes (PPARc and LPL) and neurogenic genes (MAP2 and NEFH) of the infant ADSC differentiated cells were significantly higher than those of the adults’ while the expression of the osteogenic genes (OCN and RUNX) and tenogenic genes (TNC and COL3A1) of both demonstrated opposite results. The chondrogenic markers (SOX9, COL2 and COL10) were enhanced in the infant ADSC differentiated chondrogenic pellets, and the expression levels of SODs were decreased during the differentiation process. CONCLUSION: Cultured infant ADSCs demonstrate less cellular senescence and replicative stress, higher proliferation rates, better antioxidant defense activity, and higher potential of chondrogenic, adipogenic and neurogenic differentiation.

인간 제대혈 유래 간엽줄기세포의 연골세포 분화

정미현(Mee Hyun Jung),양성은(Sung-Eun Yang),진혜진(Hae Jin Jin),이만경(Man Kyoung Lee),송호선(Ho Sun Song),양정윤(Jung Yoon Yang),양윤선(Yoon Sun Yang),하철원(Chul-Won Ha) 대한정형외과학회 2004 대한정형외과학회지 Vol.39 No.6

목적: 인간 제대혈로부터 간엽성 섬유세포양 세포를 분리, 배양함으로써 제대혈 내의 순환성 간엽줄기세포의 존재여부를 확인하고, 이의 연골세포 분화능을 증명하고자 하였다. 대상 및 방법: 총 50 유니트의 제대혈로부터 분리된 유착성 세포군의 세포형태 및 면역표현형을 분석하였다. BMP-6을 첨가하였거나 또는 첨가하지 않은 연골분화배양액을 이용하여 펠렛 배양의 형태로 연골분화를 유도 배양하였다. 연골세포로의 분화를 관찰하기 위해 역전사 중합효소연쇄반응을 수행하였으며, Safranin-O 염색과 제2형 콜라겐에 관한 면역염색을 시행하였다. 결과: 제대혈 단핵세포로부터 균질성의 섬유세포양 형태의 유착성 세포군이 배양되었다. 이 세포군은 간엽줄기세포 관련 항원 양성, 조혈모세포항원 음성, 조직적합항원 음성, 내피세포 및 파골세포 관련 항원 음성이었다. 연골분화 펠렛 배양 시 BMP-6을 첨가한 군에서 펠렛의 크기 및 연골세포관련 인자의 발현이 증가하였고, Safranin-O 염색과 제2형 콜라겐의 면역염색도 강양성이었다. 결론: 제대혈 내에 순환성 비조혈성 연골형성 간엽줄기세포가 존재함을 확인하였고, 연골분화 배지에 BMP-6을 첨가함으로써 제대혈 유래 비조혈성 간엽줄기세포의 시험관 내 연골분화의 효율을 향상시킬 수 있었다. Purpose: The aim of this study was to demonstrate the existence of circulating mesenchymal stem cells (MSC) in the human umbilical cord blood (hUCB) and to evaluate the chondrogenic differentiation potential of hUCB-derived MSC in vitro. Materials and Methods: Fifty hUCB harvests were cultured in media supplemented with 10% fetal bovine serum. The adherent fibroblast-like cells were characterized by immunophenotyping and induced to differentiate into chondrocytes in the pellet culture with and without BMP-6. This study performed RTPCR of the chondrogenic markers, Safranin-O stain and type Ⅱ collagen immunohistochemical stain. Results: The mononuclear cells isolated from hUCB formed adherent colonies with an attached well-spread fibroblast-like morphology. The cells positively expressed the MSC-related antigens, but did not express the hematopoietic, HLA-DR, endothelial, or osteoclast antigens and could be induced to differentiate into chondrocytes under proper stimulation. BMP-6 increased the size of the pellet and the mRNA levels for aggrecan, type Ⅱ collagen and type Ⅸ collagen and enhanced the levels of proteoglycan synthesis during chondrogenic differentiation. Conclusion: The homogenous fibroblast-like cells developed in cultures from hUCB with chondrogenic differentiation potential were considered to be MSC. Furthermore, it was found that BMP-6 enhanced chondrogenic differentiation of the hUCB-derived MSC in the pellet culture.

Chondrogenic Differentiation of Bone Marrow Stromal Cells in Transforming Growth $Factor-{\beta}_{1}$ Loaded Alginate Bead

Park, Ki-Suk,Jin Chae-Moon,Kim, Soon-Hee,Rhee John M.,Khang Gil-Son,Han, Chang-Whan,Yang, Yoon-Sun,Kim, Moon-Suk,Lee, Hai-Bang The Polymer Society of Korea 2005 Macromolecular Research Vol.13 No.4

We developed alginate beads loaded with transforming growth $factor-{\beta}_{1}(TGF-{\beta}_{1})$ to examine the possible application of the scaffold and cytokine carrier in tissue engineering. In this study, bone marrow stromal cells (BMSCs) and $TGF{\beta}_{1}$ were uniformly encapsulated in the alginate beads and then cultured in vitro. The cell morphology and shape of the alginate beads were observed using inverted microscope, scanning electron microscope (SEM), histological staining and RT-PCR to confirm chondrogenic differentiation. The amount of the $TGF{\beta}_{1}$ released from the $TGF-{\beta}_{1}$ loaded alginate beads was analyzed for 28 days in vitro in a phosphate buffered saline (pH 7.4) at $37^{\circ}C$. We observed the release profile of $TGF-{\beta}_{1}$ from $TGF-{\beta}_{1}$ loaded alginate beads with a sustained release pattern for 35 days. Microscopic observation showed the open cell pore structure and abundant cells with a round morphology in the alginate beads. In addition, histology and RT-PCR results revealed the evidence of chondrogenic differentiation in the beads. In conclusion, these results confirmed that $TGF-{\beta}_{1}$ loaded alginate beads provide excellent conditions for chondrogenic differentiation.