Chih-Chien Tsai

Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST.

Although low-density culture provides an efficient method for rapid expansion of human mesenchymal stem cells (MSCs), MSCs enriched by this method undergo senescence and lose their stem cell properties, which could be preserved by combining low-density and hypoxic culture. The mechanism was mediated through direct down-regulation of E2A-p21 by the hypoxia-inducible factor-1α (HIF-1α)-TWIST axis. Expansion under normoxia induced E2A and p21 expression, which were abrogated by overexpression of TWIST, whereas siRNA against TWIST up-regulated E2A and p21 in hypoxic cells. Furthermore, siRNA against p21 in normoxic cells enhanced proliferation and increased differentiation potential, whereas overexpression of p21 in hypoxic cells induced a decrease in proliferation and a loss of differentiation capacity. More importantly, MSCs expanded under hypoxic conditions by up to 100 population doublings, exhibited telomerase activity with maintained telomere length, normal karyotyping, and intact genetic integrity, and did not form tumors. These results support low-density hypoxic culture as a method for efficiently expanding MSCs without losing stem cell properties or increasing tumorigenicity.

Oct4 and Nanog Directly Regulate Dnmt1 to Maintain Self-Renewal and Undifferentiated State in Mesenchymal Stem Cells

The roles of Oct4 and Nanog in maintaining self-renewal and undifferentiated status of adult stem cells are unclear. Here, increase in Oct4 and Nanog expression along with increased proliferation and differentiation potential but decreased spontaneous differentiation were observed in early-passage (E), hypoxic culture (H), and p21 knockdown (p21KD) mesenchymal stem cells (MSCs) compared to late-passage (L), normoxic culture (N), and scrambled shRNA-overexpressed (Scr) MSCs. Knockdown of Oct4 and Nanog in E, H, and p21KD MSCs decreasedxa0proliferation and differentiation potential and enhanced spontaneous differentiation, whereas overexpression of Oct4 and Nanog in L, N, and Scr MSCs increased proliferation and differentiation potential and suppressed spontaneous differentiation. Oct4 and Nanog upregulate Dnmt1 through direct binding to its promoter, thereby leading to the repressed expression of p16 and p21 and genes associated with development and lineage differentiation. These data demonstrate the important roles of Oct4 and Nanog in maintaining MSC properties.

Mesenchymal stem cell‐conditioned medium facilitates angiogenesis and fracture healing in diabetic rats

The most critical factor for fracture union is the blood supply to the fracture site, which is usually impaired in patients with diabetes. Recently, mesenchymal stem cells‐derived conditioned medium (MSC‐CM) has shown significantly higher levels of angiogenic factors, such as VEGF and IL‐6. We demonstrate in this report that MSC‐CM delivered in gelatin sponges stimulates angiogenesis and promotes fracture healing in a diabetic rat model. Subcutaneous implantation of gelatin sponges soaked in MSC‐CM demonstrated better tissue ingrowth and higher capillary densities at 2 and 3u2009weeks than gelatin sponges in minimal essential medium (MEM) or 293 cell‐derived conditioned medium (293‐CM). Implantation of fibular defects with gelatin sponges soaked in MSC‐CM enhanced bone ingrowth and fracture healing rates compared to 293‐CM and MEM groups at 8u2009weeks. Micro‐computed tomography analysis further indicated a higher new bone volume in the MSC‐CM group compared to the other diabetic groups. Histological analysis with CD31 immunostaining also revealed that MSC‐CM increased endothelial cell counts compared to the other groups. Together, these results indicated that gelatin sponges used to deliver MSC‐CM promote angiogenesis and fracture healing in a diabetic model and may be an alternative strategy for treating fracture non‐union in patients with diabetes. Copyright

Knockdown of p21Cip1/Waf1 enhances proliferation, the expression of stemness markers, and osteogenic potential in human mesenchymal stem cells

Mammalian aging of many tissues is associated with a decline in the replicative and functional capacity of somatic stem cells. Understanding the basis of this decline is a major goal of aging research. Human bone marrow‐derived multipotent stromal cells (MSCs) have been applied in the treatment of fracture nonunion. Clinical application of MSCs requires abundant cells that can be overcome by ex vivo expansion of cells, but often at the expense of stemness and differentiation potentiality. We first demonstrated that late‐passage MSCs exhibited decreased proliferation capacity, reduced expression of stemness markers such as Oct‐4 and Nanog, and deterioration of osteogenic potential. Further, late‐passage MSCs showed increased expression of p21Cip1/Waf1 (p21), an inhibitor of the cyclin‐dependent kinase. Knockdown of p21 by lentivirus‐mediated shRNAs against p21 in late‐passage MSCs increased the proliferation capacity, the expression of Oct‐4 and Nanog, and osteogenic potential compared with cells transduced with control shRNA. More importantly, reduction in p21 expression in MSCs enhanced the bone repair capacity of MSCs in a rodent calvarial defect model. Knockdown of p21 in MSCs also increased the telomerase activity and telomere length, and did not show chromosomal abnormalities or acquire transformation ability. Therefore, these data successfully demonstrate the involvement of senescence gene in the expression of stemness markers and osteogenic potential of MSCs.

Isolation of mesenchymal stem cells from shoulder rotator cuff: a potential source for muscle and tendon repair.

The self-healing potential of each tissue belongs to endogenous stem cells residing in the tissue; however, there are currently no reports mentioned for the isolation of human rotator cuff-derived mesenchymal stem cells (RC-MSCs) since. To isolate RC-MSCs, minced rotator cuff samples were first digested with enzymes and the single cell suspensions were seeded in plastic culture dishes. Twenty-four hours later, nonadherent cells were removed and the adherent cells were further cultured. The RC-MSCs had fibroblast-like morphology and were positive for the putative surface markers of MSCs, such as CD44, CD73, CD90, CD105, and CD166, and negative for the putative markers of hematopoietic cells, such as CD34, CD45, and CD133. Similar to BM-MSCs, RC-MSCs were demonstrated to have the potential to undergo osteogenic, adipogenic, and chondrogenic differentiation. Upon induction in the defined media, RC-MSCs also expressed lineage-specific genes, such as Runx 2 and osteocalcin in osteogenic induction, PPAR-γ and LPL in adipogenic differentiation, and aggrecan and Col2a1 in chondrogenic differentiation. The multipotent feature of RC-MSCs in the myogenic injury model was further strengthened by the increase in myogenic potential both in vitro and in vivo when compared with BM-MSCs. These results demonstrate the successful isolation of MSCs from human rotator cuffs and encourage the application of RC-MSCs in myogenic regeneration.

Isolation and characterization of mesenchymal stromal cells from human anterior cruciate ligament

BACKGROUNDnThe anterior cruciate ligament (ACL) is one of the most commonly injured ligaments of the knee. Because the torn ACL is always discarded during ACL reconstruction, it may be a potential source for isolating mesenchymal stromal cells (MSC).nnnMETHODSnTo characterize MSC from human ACL, cells were enzymatically released from the ACL of adult human donors and seeded in plastic dishes with serial passages at confluence. At different passages, ACL-derived cells were subjected to in vitro assays to investigate their multilineage potential. Upon treatment, the phenotypes of the cell cultures were analyzed by histo- and immunohistochemistry and semi-quantitative reverse transcription-polymerase chain reaction for the expression of lineage-specific genes.nnnRESULTSnSix independent cell lines from individual donors showed diversity in multilineage potential. Interestingly, five of the six lines displayed adipogenic potential, four had osteogenic and adipogenic potential, and only one cell line was tripotent. Both bone marrow (BM)- and ACL-derived MSC expressed marker genes for ligament fibroblasts, whereas the mRNA levels of collagen I and III were more abundant in ACL-derived MSC.nnnDISCUSSIONnOur study demonstrates that human MSC can be isolated from ACL with diversity in the potential to form bone, fat and cartilage and an increase as compared to BM MSC, in the potential to form ligament fibroblasts.

Functional roles of pluripotency transcription factors in mesenchymal stem cells.

Pluripotency, the capacity of a cell to give rise to differentiated derivatives that represent each of the three primary germ layers, belongs to the cells that are located within the inner cell mass (ICM) of the developing blastocyst. Functional studies have identified a group of transcription factors, the pluripotency transcription factors that affect the pluripotent capacity.1 Within this group, the transcription factors Oct4 (Pou5f1), Nanog and Sox2 are crucial for the efficient maintenance of pluripotent cell identity.1 During mouse development, the specification of pluripotent cell identity requires the embryonic genome to express Oct4 and Nanog,1 but not Sox2,2 perhaps owing to the presence of long-lived maternal Sox2 protein. Pluripotency transcription factors regulate stem cell pluripotency and differentiation via the colocalization and the cooperation with each other, polycomb repressive complexes (PRC) and microRNAs in the transcriptional and epigenetic regulation of key stem cell genes.3 n nStem cell is a specific cell population with the abilities of self-renewal and multipotent differentiation. According to the origin and potentiality of the cells, mammalian stem cells could be classified into two groups: one is embryonic stem cells (ESCs), which are isolated from ICM of blastocysts; the other is adult stem cells, such as hematopoietic stem cells, neural stem cells and mesenchymal stem cells (MSCs), which are found in adult tissues. In addition, it has been demonstrated that somatic cell can be reprogrammed to pluripotent-like stem cells, otherwise known as induced pluripotent stem-like cells (iPS) by overexpressing specific pluripotency transcription factors, including Oct4, Sox2, c-Myc and Klf4, or Oct4, Nanog, Sox2 and Lin28.4 n nExpression of pluripotency transcription factors is restricted to pluripotent cells and is downregulated upon differentiation.1 In mouse ESCs, downregulation of Oct4 induces the differentiation into trophoblast lineage, while reduction in Nanog induces differentiation into extra-embryonic endoderm.5 However, the roles of these pluripotency transcription factors in maintaining ESC self-renewal and differentiation are not the same across human and mouse. Z. Wang and his colleagues investigated the roles of Oct4, Nanog and Sox2 in human ESCs using the Lentiviral-knockdown system.6 They identified that high levels of Oct4 and Nanog, rather than Sox2, are indispensable for maintaining self-renewal of human ESCs. Moreover, they found Oct4 directly inhibits the BMP4 signaling pathway, which activates mesoderm and extraembryonic ectoderm/endoderm differentiation, while Nanog acts as a repressor of neural crest and neuroectoderm lineage (Fig.xa01A). n n n nFigurexa01. Schematization of the potential signaling pathways that Oct4 and Nanog mediate to regulate self-renewal and differentiation of human ESCs and MSCs. (A) In human ESCs, Oct4 inhibits the BMP4 signaling pathway, which activates mesoderm ... n n n nMSCs have somewhat similar stem cell properties to ESCs with regards to their maintenance and differentiation potential. Many studies also demonstrated the expression of pluripotency transcription factors in MSCs and their involvement in regulation of stem cell properties.7 However, the expression and the roles of these pluripotent genes in adult stem cells have been controversial, since knockout of Oct4 did not affect the ability of MSCs in colony formation and differentiation into bone, fat and cartilage.8 To clarify the roles of pluripotency transcription factors in MSC maintenance, we first demonstrate that MSCs expanded under normoxic conditions underwent significant changes in cell proliferation rate, differentiation potential and expression of developmental markers and tissue-specific genes and also decreased in the expression of Oct4 and Nanog.9,10 Our recent studies further demonstrated that the expression of Oct4 and Nanog was higher in MSCs at early passage (E), in hypoxic culture (H) and with p21 knockdown (p21KD) compared with MSCs at late passage (L), in normoxic culture (N) and with scrambled shRNA-overexpression (Scr), respectively.10 The expression of Oct4 and Nanog was not only localized in the nucleus, but associated with a less methylated pattern in the CpG regions of their promoters. Knockdown of Oct4 and Nanog in E, H and p21KD MSCs reduced cell proliferation rate and differentiation potential but induced the expression of higher levels of various developmental markers and tissue-specific genes, while overexpression of Oct4 and Nanog in L, N and Scr MSCs increased cell proliferation rate as well as differentiation potential and inhibited spontaneous differentiation. These data suggest that Oct4 and Nanog are not only essential for the maintenance of pluripotency in ESCs, but are also essential for maintaining MSC properties. n nOur studies further demonstrated that Oct4 and Nanog directly bind to the promoter of Dnmt1 and enhance its expression, which, in turn, downregulates the expression of cell cycle regulators such as p16 and p21 as well as development and lineage genes.10 Meanwhile, the binding sites for Oct4 and Nanog were only 38 bases apart, suggesting that Oct4 and Nanog in MSCs, like in ESCs, work together to regulate downstream genes.3 Moreover, MSCs, when treated with inhibitor of DNA methylation or transfected with shRNA against Dnmt1, had decreased proliferation rate and differentiation potential, but increased expression of genes associated with senescence and developmental regulators. These data suggest MSCs like ESCs undergo changes in methylation of pluripotency genes upon expansion in culture, and Oct4 and Nanog cooperatively induce Dnmt1 expression to regulate the proliferative and undifferentiated states of MSCs (Fig.xa01B). n nSince our study is focused on the roles of Oct4 and Nanog in maintaining MSC properties, we have not checked whether the same phenomenon occurred in other stem cells or somatic cells. However, it would be interesting to know whether the regulation of Dnmt1 by Oct4 and Nanog is also found in other adult stem cells or pluripotent stem cells. Notably, overexpression of Dnmt1 also rescues somatic cells from serum starvation-induced upregulation of p16 and p21 as well as cell cycle arrest.10 This finding argues that Oct4 and Nanog control their downstream genes through indirectly controlling their expression by binding to the Dnmt1 promoter.

Isolation of mesenchymal stem cells from human ligamentum flavum: implicating etiology of ligamentum flavum hypertrophy.

Study Design. To demonstrate the existence of mesenchymal stem cells (MSCs) in ligamentum flavum (LF) and their pathogenic role in LF hypertrophy. Objective. To isolate and characterize LF-derived MSCs and their response to transforming growth factor-beta 1 (TGF-&bgr;1) and trichostatin A (TSA), a histone deacetylase inhibitor (HDACi). Summary of Background Data. LF is a connective tissue, of which hypertrophic changes induce spinal stenosis. The pathogenic role of TGF-&bgr;1 in spinal stenosis has been implicated. TSA has been shown to suppress TGF-&bgr;1–induced alpha-smooth muscle actin (&agr;-SMA), type I and III collagen synthesis in a variety of cells. MSCs have been isolated from a variety of adult tissues, except LF. Whether MSCs exist in LF and their response to TGF-&bgr;1 and TSA is not clear. Methods. The MSCs from LF were isolated and cultured. Their phenotypic character, linage differentiation potential, and response to TGF-&bgr;1 and TSA were analyzed. Results. LF-derived MSCs have the similar profile of surface markers as bone marrow MSCs. They were demonstrated to have the potential to be differentiated into osteoblasts, adipocytes, and chondrocytes. Administration of TGF-&bgr;1 stimulated cell proliferation, enhanced the gene expression of type I and III collagen, and increased the gene expression and protein level of &agr;-SMA. TSA blocked the fibrogenic effects of TGF-&bgr;1. Conclusion. The current results demonstrated the isolation of MSCs from LF. The cellular response to TGF-&bgr;1 implied that these cells might play an important role in the pathogenesis of LF hypertrophy. TSA, which blocks the effects of TGF-&bgr;1, may be a potent therapeutic choice for inhibiting LF hypertrophy.

Scale-up of MSC under hypoxic conditions for allogeneic transplantation and enhancing bony regeneration in a rabbit calvarial defect model†

To realize the therapeutic potential of mesenchymal stem cells (MSCs), we aimed to develop a method for isolating and expanding New Zealand rabbit MSCs in a great scale. Rabbit MSCs expanded under hypoxic and normoxic conditions were compared in terms of replication capacity, differentiation potential, and the capacity for allogeneic transplantation in a calvarial defect model. The cells from all tested rabbits were expanded more rapidly when plated at low‐density under hypoxic conditions compared to under normoxic conditions. Moreover, cells expanded under hypoxic conditions increased in the potential of osteoblastic, adipocytic, and chondrocytic differentiation. More importantly, radiographic analysis and micro‐CT measurement of bone volume revealed the hypoxic cells when transplanted in the calvarial defects of another rabbit increased in the ability to repair bone defect compared to the normoxic cells. Six weeks after allogeneic transplantation of hypoxic MSCs, histological analysis revealed a callus spanned the length of the defect, and several bone tissues spotted in the implant. At 12 weeks, new bone had formed throughout the implant. Using BrdU labeling to track the transplanted cells, the hypoxic cells were more detected in the newly formed bone compared to the normoxic cells. For defects treated with allogeneic MSCs, no adverse host response could be detected at any time‐point. In conclusion, we have developed a robust method for isolation and expansion of rabbit MSCs by combining low‐density with hypoxic culture, which can be applied for the design of clinical trials in allogeneic transplantation of MSCs for bone healing.

Efficient expansion of mesenchymal stem cells from mouse bone marrow under hypoxic conditions

To realize the therapeutic potential of mesenchymal stem cells (MSCs), a large number of high‐quality MSCs isolated from different species, such as mouse, were acquired for preclinical animal studies. Surprisingly, isolation and purification of mouse MSCs (mMSCs) is arduous because of the low frequency of MSCs and contamination of haematopoietic cells in culture. We have developed a method based on low density and hypoxic culture to isolate and expand mMSCs from different strains, including BALB/c, C57BL/6J, FVB/N and DBA/2. The cells from all of the strains expanded more rapidly when plated at low density in hypoxic culture compared with normoxic culture. These cells expressed CD44, CD105, CD29 and Sca‐1 markers but not CD11b, CD34, CD45 and CD31 markers. Moreover, they were able to differentiate along osteoblastic, adipocytic and chondrocytic lineages. In conclusion, we have developed a robust method for isolation and expansion of mMSCs by combining low‐density culture with hypoxic culture. Copyright