Scalable mesenchymal stem cell enrichment from bone marrow aspirate using deterministic lateral displacement (DLD) microfluidic sorting.

The growing interest in regenerative medicine has opened new avenues for novel cell therapies using stem cells. Bone marrow aspirate (BMA) is an important source of stromal mesenchymal stem cells (MSCs). Conventional MSC harvesting from BMA relies on archaic centrifugation methods, often leading to poor yield due to osmotic stress, high centrifugation force, convoluted workflow, and long experimental time (∼2-3 hours). To address these issues, we have developed a scalable microfluidic technology based on deterministic lateral displacement (DLD) for MSC isolation. This passive, label-free cell sorting method capitalizes on the morphological differences between MSCs and blood cells (platelets and RBCs) for effective separation using an inverted L-shaped pillar array. To improve throughput, we developed a novel multi-chip DLD system that can process 2.5 mL of raw BMA in 20 ± 5 minutes, achieving a 2-fold increase in MSC recovery compared to centrifugation methods. Taken together, we envision that the developed DLD platform will enable fast and efficient isolation of MSCs from BMA for effective downstream cell therapy in clinical settings.

Critical attributes of human early mesenchymal stromal cell-laden microcarrier constructs for improved chondrogenic differentiation.

BACKGROUND: Microcarrier cultures which are useful for producing large cell numbers can act as scaffolds to create stem cell-laden microcarrier constructs for cartilage tissue engineering. However, the critical attributes required to achieve efficient chondrogenic differentiation for such constructs are unknown. Therefore, this study aims to elucidate these parameters and determine whether cell attachment to microcarriers throughout differentiation improves chondrogenic outcomes across multiple microcarrier types. METHODS: A screen was performed to evaluate whether 1) cell confluency, 2) cell numbers, 3) cell density, 4) centrifugation, or 5) agitation are crucial in driving effective chondrogenic differentiation of human early mesenchymal stromal cell (heMSC)-laden Cytodex 1 microcarrier (heMSC-Cytodex 1) constructs. RESULTS: Firstly, we found that seeding 10 × 103 cells at 70% cell confluency with 300 microcarriers per construct resulted in substantial increase in cell growth (76.8-fold increase in DNA) and chondrogenic protein generation (78.3- and 686-fold increase in GAG and Collagen II, respectively). Reducing cell density by adding empty microcarriers at seeding and indirectly compacting constructs by applying centrifugation at seeding or agitation throughout differentiation caused reduced cell growth and chondrogenic differentiation. Secondly, we showed that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes since critically defined heMSC-Cytodex 1 constructs developed larger diameters (2.6-fold), and produced more DNA (13.8-fold), GAG (11.0-fold), and Collagen II (6.6-fold) than their equivalent cell-only counterparts. Thirdly, heMSC-Cytodex 1/3 constructs generated with cell-laden microcarriers from 1-day attachment in shake flask cultures were more efficient than those from 5-day expansion in spinner cultures in promoting cell growth and chondrogenic output per construct and per cell. Lastly, we demonstrate that these critically defined parameters can be applied across multiple microcarrier types, such as Cytodex 3, SphereCol and Cultispher-S, achieving similar trends in enhancing cell growth and chondrogenic differentiation. CONCLUSIONS: This is the first study that has identified a set of critical attributes that enables efficient chondrogenic differentiation of heMSC-microcarrier constructs across multiple microcarrier types. It is also the first to demonstrate that cell attachment to microcarriers throughout differentiation improves cell growth and chondrogenic outcomes across different microcarrier types, including biodegradable gelatin-based microcarriers, making heMSC-microcarrier constructs applicable for use in allogeneic cartilage cell therapy.

Expansion in microcarrier-spinner cultures improves the chondrogenic potential of human early mesenchymal stromal cells.

BACKGROUND AIMS: Cartilage tissue engineering with human mesenchymal stromal cells (hMSC) is promising for allogeneic cell therapy. To achieve large-scale hMSC propagation, scalable microcarrier-based cultures are preferred over conventional static cultures on tissue culture plastic. Yet it remains unclear how microcarrier cultures affect hMSC chondrogenic potential, and how this potential is distinguished from that of tissue culture plastic. Hence, our study aims to compare the chondrogenic potential of human early MSC (heMSC) between microcarrier-spinner and tissue culture plastic cultures. METHODS: heMSC expanded on either collagen-coated Cytodex 3 microcarriers in spinner cultures or tissue culture plastic were harvested for chondrogenic pellet differentiation with empirically determined chondrogenic inducer bone morphogenetic protein 2 (BMP2). Pellet diameter, DNA content, glycosaminoglycan (GAG) and collagen II production, histological staining and gene expression of chondrogenic markers including SOX9, S100ß, MMP13 and ALPL, were investigated and compared in both conditions. RESULTS: BMP2 was the most effective chondrogenic inducer for heMSC. Chondrogenic pellets generated from microcarrier cultures developed larger pellet diameters, and produced more DNA, GAG and collagen II per pellet with greater GAG/DNA and collagen II/DNA ratios compared with that of tissue culture plastic. Moreover, they induced higher expression of chondrogenic genes (e.g., S100ß) but not of hypertrophic genes (e.g., MMP13 and ALPL). A similar trend showing enhanced chondrogenic potential was achieved with another microcarrier type, suggesting that the mechanism is due to the agitated nature of microcarrier cultures. CONCLUSIONS: This is the first study demonstrating that scalable microcarrier-spinner cultures enhance the chondrogenic potential of heMSC, supporting their use for large-scale cell expansion in cartilage cell therapy.

Up-regulation of microRNA-190b plays a role for decreased IGF-1 that induces insulin resistance in human hepatocellular carcinoma.

BACKGROUND & AIMS: Insulin-like growth factor, (IGF)-1, is produced mainly by the liver and plays important roles in promoting growth and regulating metabolism. Previous study reported that development of hepatocellular carcinoma (HCC) was accompanied by a significant reduction in serum IGF-1 levels. Here, we hypothesized that dysregulation of microRNAs (miRNA) in HCC can modulate IGF-1 expression post-transcriptionally. METHODS: The miRNAs expression profiles in a dataset of 29 HCC patients were examined using illumina BeadArray. Specific miRNA (miR)-190b, which was significantly up-regulated in HCC tumor tissues when compared with paired non-tumor tissues, was among those predicted to interact with 3'-untranslated region (UTR) of IGF-1. In order to explore the regulatory effects of miR-190b on IGF-1 expression, luciferase reporter assay, quantitative real-time PCR, western blotting and immunofluorecence analysis were performed in HCC cells. RESULTS: Overexpression of miR-190b in Huh7 cells attenuated the expression of IGF-1, whereas inhibition of miR-190b resulted in up-regulation of IGF-1. Restoration of IGF-1 expression reversed miR-190b-mediated impaired insulin signaling in Huh7 cells, supporting that IGF-1 was a direct and functional target of miR-190b. Additionally, low serum IGF-1 level was associated with insulin resistance and poor overall survival in HCC patients. CONCLUSIONS: Increased expression of miR-190 may cause decreased IGF-1 in HCC development. Insulin resistance appears to be a part of the physiopathologic significance of decreased IGF-1 levels in HCC progression. This study provides a novel miRNA-mediated regulatory mechanism for controlling IGF-1 expression in HCC and elucidates the biological relevance of this interaction in HCC.

Downregulation of alpha-fetoprotein expression by LHX4: a critical role in hepatocarcinogenesis.

LHX4 is a member of the LIM-homeobox family and plays a critical role in pituitary development and differentiation. Several lines of evidences have reported their aberrant expression in cancers. However, the exact roles of LHX4 in carcinogenesis remain unclear. In this study, LHX4 expression was analyzed in tumor and paired non-tumor tissues obtained from patients with hepatocellular carcinoma (HCC) using western blotting and immunohistochemistry. LHX4 was found to be downregulated in tumor tissues and negatively correlated with differentiation grade and alpha-fetoprotein (AFP) levels in 66 HCC patients. To clarify the biological functions of LHX4, transient or stable transfectants overexpressing LHX4 were generated in human hepatoma cells (Huh7 and HepG2). LHX4 overexpression in Huh7 and HepG2 cells induced a more differentiated phenotype by reducing AFP expression. Using in silico analysis, the evolutionary conserved region within the AFP promoter containing LHX4-binding site was identified, implying that AFP is a putative target for LHX4. Moreover, ectopic LHX4 overexpression attenuated Huh7 and HepG2 proliferation. Importantly, the growth-inhibitory effect of LHX4 was reversed by replenishing AFP to the LHX4-overexpressing cells, providing a functional relevance between LHX4 and AFP. Finally, we analyzed expressions of LHX4 and AFP during normal liver development. Hepatic LHX4 expression increased in adult liver in a manner that parallel AFP repression. In conclusion, these data indicate that LHX4 may act as a potential tumor suppressor in hepatocarcinogenesis, suggesting that targeting LHX4 to downregulate AFP might have therapeutic implications.

Human T-lymphotropic virus tax activates human cytomegalovirus major-immediate early promoter and improves production of recombinant proteins in HEK293 cells.

The human cytomegalovirus (CMV) major immediate-early (MIE) promoter is widely used in mammalian cells for production of recombinant proteins. It is of great interest to further enhance protein production driven by the CMV promoter. Here, we report that the Tax protein of human T-lymphotropic virus stimulates the transgene expression under the control of CMV MIE promoter in HEK293 cells. At least threefold increases in transient production of recombinant proteins, including luciferase and two biopharmaceutical proteins (erythropoietin and interferon-γ), were detected. Furthermore, cyclic adenosine monophosphate (AMP)-response element binding protein 2 (CREB2) was identified as a cellular cofactor, which might be responsible for Tax transactivation of the CMV MIE promoter. Our results not only demonstrate the potential use of this novel expression strategy for improvement of recombinant protein production in HEK293 cells but also provide the molecular mechanism for Tax-mediated activation of CMV MIE promoter.

PCAF interacts with XBP-1S and mediates XBP-1S-dependent transcription.

X-box binding protein 1 (XBP-1) is a key regulator required for cellular unfolded protein response (UPR) and plasma cell differentiation. In addition, involvement of XBP-1 in host cell-virus interaction and transcriptional regulation of viruses, such as human T-lymphotropic virus type 1 (HTLV-1), has been revealed recently. Two XBP-1 isoforms, XBP-1U and XBP-1S, which share an identical N-terminal domain, are present in cells. XBP-1S is a transcription activator while XBP-1U is the inactive isoform. Although the transactivation domain of XBP-1S has been identified within the XBP-1S-specific C-terminus, molecular mechanism of the transcriptional activation by XBP-1S still remains unknown. Here we report the interaction between p300/CBP-associated factor (PCAF) and XBP-1S through the C-terminal domain of XBP-1S. No binding between XBP-1U and PCAF is detected. In a cell-based reporter assay, overexpression of PCAF further stimulates the XBP-1S-mediated cellular and HTLV-1 transcription while knockdown of PCAF exhibits the opposite effect. Expression of endogenous XBP-1S cellular target genes, such as BiP and CHOP, is significantly inhibited when PCAF is knocked down. Furthermore, PCAF is recruited to the promoters of XBP-1S target genes in vivo, in a XBP-1S-dependent manner. Collectively, our results demonstrate that PCAF mediates the XBP-1S-dependent transcription through the interaction with XBP-1S.

The 3'UTR of HIC mRNA improves the production of recombinant proteins in Chinese hamster ovary cells.

Positive transcription elongation factor b (P-TEFb) is an important transcriptional regulator which controls 70-80% of RNA polymerase II transcription. It has been reported that the human I-mfa (inhibitor of MyoD family a) domain-containing protein (HIC) interacts with P-TEFb and that expression of HIC cDNA stimulates P-TEFb-dependent transcription. Interestingly, our recent study shows that transcriptional stimulation by HIC is predominately due to the 3' untranslated region (3'UTR) of HIC mRNA rather than its coding region. In this report, we investigate the effects of HIC 3'UTR on recombinant protein expression in mammalian cells. In transient transfections, overexpression of HIC 3'UTR stimulates transgene expression in several mammalian cell lines and significantly increases the production of human erythropoietin and interferon-gamma in Chinese hamster ovary (CHO) cells. This is the first report that demonstrates the improvement of expression of biopharmaceutical proteins by overexpressing a non-coding 3'UTR in CHO cells.

Nucleophosmin interacts with HEXIM1 and regulates RNA polymerase II transcription.

Hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) was identified earlier as an inhibitor of positive transcription elongation factor b (P-TEFb), which is a key transcriptional regulator of RNA polymerase II (Pol II). Studies show that more than half of P-TEFb in cells is associated with HEXIM1, which results in the inactivation of P-TEFb. Here, we identify a nucleolar protein, nucleophosmin (NPM), as a HEXIM1-binding protein. NPM binds to HEXIM1 in vitro and in vivo, and functions as a negative regulator of HEXIM1. Over-expression of NPM leads to proteasome-mediated degradation of HEXIM1, resulting in activation of P-TEFb-dependent transcription. In contrast, an increase in HEXIM1 protein levels and a decrease in transcription are detected when NPM is knocked down. We show that a cytoplasmic mutant of NPM, NPMc+, associates with and sequesters HEXIM1 in the cytoplasm resulting in higher RNA Pol II transcription. Correspondingly, cytoplasmic localization of endogenous HEXIM1 is detected in an acute myeloid leukemia (AML) cell line containing the NPMc+ mutation, suggesting the physiological importance of HEXIM1-NPMc+ interaction. Over-expression of NPM has been detected in tumors of various histological origins and our results may provide a possible molecular mechanism for the proto-oncogenic function of NPM. Furthermore, considering that 35% of AML patients are diagnosed with NPMc+ mutation, our findings suggest that in some cases of AML, RNA Pol II transcription may be disregulated by the malfunction of NPM and the mislocation of HEXIM1.

XBP-1, a novel human T-lymphotropic virus type 1 (HTLV-1) tax binding protein, activates HTLV-1 basal and tax-activated transcription.

X-box binding protein 1 (XBP-1), a basic leucine zipper transcription factor, plays a key role in the cellular unfolded protein response (UPR). There are two XBP-1 isoforms in cells, spliced XBP-1S and unspliced XBP-1U. XBP-1U has been shown to bind to the 21-bp Tax-responsive element of the human T-lymphotropic virus type 1 (HTLV-1) long terminal repeat (LTR) in vitro and transactivate HTLV-1 transcription. Here we identify XBP-1S as a transcription activator of HTLV-1. Compared to XBP-1U, XBP-1S demonstrates stronger activating effects on both basal and Tax-activated HTLV-1 transcription in cells. Our results show that both XBP-1S and XBP-1U interact with Tax and bind to the HTLV-1 LTR in vivo. In addition, elevated mRNA levels of the gene for XBP-1 and several UPR genes were detected in the HTLV-1-infected C10/MJ and MT2 T-cell lines, suggesting that HTLV-1 infection may trigger the UPR in host cells. We also identify Tax as a positive regulator of the expression of the gene for XBP-1. Activation of the UPR by tunicamycin showed no effect on the HTLV-1 LTR, suggesting that HTLV-1 transcription is specifically regulated by XBP-1. Collectively, our study demonstrates a novel host-virus interaction between a cellular factor XBP-1 and transcriptional regulation of HTLV-1.

Cell-specific effects of human cytomegalovirus unique region on recombinant protein expression.

The major immediate-early (MIE) promoter of human cytomegalovirus (CMV) is widely used to express recombinant proteins in mammalian cells. CMV MIE promoter contains a strong enhancer and an AT-rich unique region (UR). The UR can function as an insulator or a negative element of CMV MIE promoter, depending on the cellular proteins associated with it. To examine the effects of UR on recombinant protein expression in mammalian cells, we constructed two CMV MIE promoter-based expression plasmids for comparison to the conventional CMV MIE promoter by removing or adding UR. Addition of UR enhances transgene expression in HEK293 stable cells while removal of UR increases both transient and stably integrated expression in HeLa cells. Our results further demonstrate that the cell-specific effect of UR depends on the protein levels of UR-binding proteins, pancreatic-duodenal homeobox factor-1, special AT-rich sequence binding protein 1, and CCAAT displacement protein, in these cells. Collectively, these modified CMV expression plasmids can be utilized to improve recombinant protein production in specific mammalian cell lines.

Cellular homeoproteins, SATB1 and CDP, bind to the unique region between the human cytomegalovirus UL127 and major immediate-early genes.

An AT-rich region of the human cytomegalovirus (CMV) genome between the UL127 open reading frame and the major immediate-early (MIE) enhancer is referred to as the unique region (UR). It has been shown that the UR represses activation of transcription from the UL127 promoter and functions as a boundary between the divergent UL127 and MIE genes during human CMV infection [Angulo, A., Kerry, D., Huang, H., Borst, E.M., Razinsky, A., Wu, J., Hobom, U., Messerle, M., Ghazal, P., 2000. Identification of a boundary domain adjacent to the potent human cytomegalovirus enhancer that represses transcription of the divergent UL127 promoter. J. Virol. 74 (6), 2826-2839; Lundquist, C.A., Meier, J.L., Stinski, M.F., 1999. A strong negative transcriptional regulatory region between the human cytomegalovirus UL127 gene and the major immediate-early enhancer. J. Virol. 73 (11), 9039-9052]. A putative forkhead box-like (FOX-like) site, AAATCAATATT, was identified in the UR and found to play a key role in repression of the UL127 promoter in recombinant virus-infected cells [Lashmit, P.E., Lundquist, C.A., Meier, J.L., Stinski, M.F., 2004. Cellular repressor inhibits human cytomegalovirus transcription from the UL127 promoter. J. Virol. 78 (10), 5113-5123]. However, the cellular factors which associate with the UR and FOX-like region remain to be determined. We reported previously that pancreatic-duodenal homeobox factor-1 (PDX1) bound to a 45-bp element located within the UR [Chao, S.H., Harada, J.N., Hyndman, F., Gao, X., Nelson, C.G., Chanda, S.K., Caldwell, J.S., 2004. PDX1, a Cellular Homeoprotein, Binds to and Regulates the Activity of Human Cytomegalovirus Immediate Early Promoter. J. Biol. Chem. 279 (16), 16111-16120]. Here we demonstrate that two additional cellular homeoproteins, special AT-rich sequence binding protein 1 (SATB1) and CCAAT displacement protein (CDP), bind to the human CMV UR in vitro and in vivo. Furthermore, CDP is identified as a FOX-like binding protein and a repressor of the UL127 promoter, while SATB1 has no effect on UL127 expression. Since CDP is known as a transcription repressor and a nuclear matrix-associated region binding protein, CDP may have a role in the regulation of human CMV transcription.

Clonal amniotic fluid-derived stem cells express characteristics of both mesenchymal and neural stem cells.

Recent evidence has shown that amniotic fluid may be a novel source of fetal stem cells for therapeutic transplantation. We previously developed a two-stage culture protocol to isolate a population of amniotic fluid-derived mesenchymal stem cells (AFMSCs) from second-trimester amniocentesis. AFMSCs maintain the capacity to differentiate into multiple mesenchymal lineages and neuron-like cells. It is unclear whether amniotic fluid contains heterogeneous populations of stem cells or a subpopulation of primitive stem cells that are similar to marrow stromal cells showing the behavior of neural progenitors. In this study, we showed a subpopulation of amniotic fluid-derived stem cells (AF-SCs) at the single-cell level by limiting dilution. We found that NANOG- and POU5F1 (also known as OCT4)-expressing cells still existed in the expanded single cell-derived AF-SCs. Aside from the common mesenchymal characteristics, these clonal AF-SCs also exhibit multiple phenotypes of neural-derived cells such as NES, TUBB3, NEFH, NEUNA60, GALC, and GFAP expressions both before and after neural induction. Most importantly, HPLC analysis showed the evidence of dopamine release in the extract of dopaminergic-induced clonal AF-SCs. The results of this study suggest that besides being an easily accessible and expandable source of fetal stem cells, amniotic fluid will provide a promising source of neural progenitor cells that may be used in future cellular therapies for neurodegenerative diseases and nervous system injuries.

Pregnancy outcomes in unselected singleton pregnant women with an increased risk of first-trimester Down's syndrome.

BACKGROUND: The purpose of this study was to assess outcomes in pregnancies with a positive screen of first-trimester combined test (nuchal translucency, pregnancy-associated plasma protein-A and free beta-human chorionic gonadotropin). METHODS: Using a cut-off level of 1 in 270, 216 (7.1%) women had a positive screen. Among them, 187 delivered their babies in our hospital and the adverse outcomes, such as spontaneous abortion, intrauterine fetal demize, preterm prelabor rupture of the membranes, preterm labor, intrauterine growth restriction, gestational hypertensive disorders, placenta previa, chromosomal abnormalities and fetal structural anomalies, were identified and compared with the 2097 women who screened negative for Down's syndrome. RESULTS: Pregnancies with a positive screen had a significantly higher risk of adverse outcomes than those with negative screens (30.5% versus 15.3%; odds ratio 2.4; p < 0.001), especially for those complicated by spontaneous abortion (odds ratio 11.4; p < 0.05) and placenta previa (odds ratio 4.3; p < 0.05). CONCLUSIONS: Besides fetal chromosomal abnormalities and structural abnormalities, pregnancies with a positive screen for Down's syndrome in the first-trimester had a significantly higher incidence of subsequent adverse obstetric outcomes.

Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol.

BACKGROUND: The aim of this study was to isolate mesenchymal stem cells (MSCs) from amniotic fluid obtained by second-trimester amniocentesis. METHODS: A novel two-stage culture protocol for culturing MSCs was developed. Flow cytometry, RT-PCR and immunocytochemistry were used to analyse the phenotypic characteristics of the cultured MSCs. Von Kossa, Oil Red O and TuJ-1 stainings were used to assess the differentiation potentials of MSCs. RESULTS: Amniotic fluid-derived MSCs (AFMSCs) were successfully isolated, cultured and enriched without interfering with the routine process of fetal karyotyping. Flow cytometry analyses showed that they were positive for SH2, SH3, SH4, CD29, CD44 and HLA-ABC (MHC class I), low positive for CD90 and CD105, but negative for CD10, CD11b, CD14, CD34, CD117, HLA-DR, DP, DQ (MHC class II) and EMA. Importantly, a subpopulation of Oct-4-positive cells was detectable in our cultured AFMSCs. Under specific culture conditions, AFMSCs could be induced to differentiate into adipocytes, osteocytes and neuronal cells. CONCLUSIONS: We demonstrate that human multipotent MSCs are present in second-trimester amniotic fluid. Considering the great potential of cellular therapy using fetal stem cells and the feasibility of intrauterine fetal tissue engineering, amniotic fluid may provide an excellent alternative source for investigation of human MSCs.

Effects of malonate C60 derivatives on activated microglia.

Activated microglia in acute and chronic neurodegenerative disease of the central nervous system (CNS) can produce large amounts of free radicals, such as reactive oxygen species (ROS), which subsequently contribute to neuropathogenesis. Thus, it is believed that the induction of microglial deactivation can reduce neuronal injury. Buckminsterfullerene (C60) derivatives that possess free radical scavenging properties have been demonstrated to prevent neuronal cell death caused by excitotoxic insult. In this study, we investigated the biological role of two malonic acid C60 derivatives referred as trans-2 and trans-3 on microglia in the presence of the endotoxin lipopolysaccharide (LPS). Treatment of LPS-activated microglia with trans-2 and trans-3 induced a significant degree of transformation of amoeboid microglia to the ramified phenotype. To understand the mechanism underlying this C60 mediated microglial morphological transformation, we examined the production of proinflammatory cytokines, interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), as well as the final NO products (nitrate and nitrite) in the microglial culture supernatant. Although inducible nitric oxide (iNOS) mRNA and protein expression in LPS-activated microglia were slightly decreased by trans-2 and trans-3, levels of nitrate and nitrite were unaffected. Paradoxically, trans-2 and trans-3 were found to increase the release of IL-1beta in the activated microglial culture. However, trans-2 and trans-3 improved the activity of the antioxidant enzyme, superoxide dismutase (SOD) in LPS-treated microglia. Therefore, our results suggest that the C60 derivatives might increase microglial SOD enzymatic activity which causes microglial morphological transformation from the activated amoeboid phenotype to the resting ramified form.