Denis Evseenko

Mapping the first stages of mesoderm commitment during differentiation of human embryonic stem cells

Our understanding of how mesodermal tissue is formed has been limited by the absence of specific and reliable markers of early mesoderm commitment. We report that mesoderm commitment from human embryonic stem cells (hESCs) is initiated by epithelial-to-mesenchymal transition (EMT) as shown by gene expression profiling and by reciprocal changes in expression of the cell surface proteins, EpCAM/CD326 and NCAM/CD56. Molecular and functional assays reveal that the earliest CD326−CD56+ cells, generated from hESCs in the presence of activin A, BMP4, VEGF, and FGF2, represent a multipotent mesoderm-committed progenitor population. CD326−CD56+ progenitors are unique in their ability to generate all mesodermal lineages including hematopoietic, endothelial, mesenchymal (bone, cartilage, fat, fibroblast), smooth muscle, and cardiomyocytes, while lacking the pluripotency of hESCs. CD326−CD56+ cells are the precursors of previously reported, more lineage-restricted mesodermal progenitors. These findings present a unique approach to study how germ layer specification is regulated and offer a promising target for tissue engineering.

Independent regulation of apical and basolateral drug transporter expression and function in placental trophoblasts by cytokines, steroids, and growth factors.

Placental ATP binding cassette (ABC) transporters protect placental and fetal tissues by effluxing xenobiotics and endogenous metabolites. We have investigated the effects of cytokines and survival/growth factors, implicated in various placental pathologies, on ABC transporter expression and function in primary placental trophoblast cells. Treatment of primary term trophoblasts in vitro with tumor necrosis factor-α (TNF-α) or interleukin (IL)-1β decreased mRNA and protein expression of apical transporters ABCB1/multidrug resistance gene product 1 (MDR1) and ABCG2/breast cancer resistance protein (BCRP) protein by 40 to 50% (P < 0.05). In contrast, IL-6 increased mRNA and protein expression of the basolateral transporter ABCB4/MDR3 (P < 0.05), whereas ABCC1/MRP1 expression was unaltered. Pretreatment of trophoblasts with TNF-α over 48 h resulted in significantly decreased BCRP efflux activity (increased mitoxantrone accumulation) with minimal changes in MDR1/3 activity. Epidermal growth factor (EGF) and insulin-like growth factor II, on the other hand, significantly increased BCRP expression at the mRNA and protein level (P < 0.05); EGF treatment also increased BCRP functional activity. Estradiol stimulated BCRP, MDR1, and MDR3 mRNA and protein expression by 40 to 60% and increased MDR1/3 functional activity (P < 0.05). Progesterone had modest positive effects on MRP1 mRNA and MDR1 protein expression (P < 0.05). In conclusion, this study shows that proinflammatory cytokines, sex steroids, and growth factors exert independent effects on expression of apical and basolateral placental ABC transporters in primary trophoblast. Such changes could alter placental drug disposition, increase fetal susceptibility to toxic xenobiotics, and impact on placental viability and function.

TGF-β1 conjugated chitosan collagen hydrogels induce chondrogenic differentiation of human synovium-derived stem cells

BackgroundUnlike bone tissue, articular cartilage regeneration has not been very successful and has many challenges ahead. We have previously developed injectable hydrogels using photopolymerizable chitosan (MeGC) that supported growth of chondrocytes. In this study, we demonstrate a biofunctional hydrogel for specific use in cartilage regeneration by conjugating transforming growth factor-β1 (TGF-β1), a well-documented chondrogenic factor, to MeGC hydrogels impregnating type II collagen (Col II), one of the major cartilaginous extracellular matrix (ECM) components.ResultsTGF-β1 was delivered from MeGC hydrogels in a controlled manner with reduced burst release by chemically conjugating the protein to MeGC. The hydrogel system did not compromise viability of encapsulated human synovium-derived mesenchymal stem cells (hSMSCs). Col II impregnation and TGF-β1 delivery significantly enhanced cellular aggregation and deposition of cartilaginous ECM by the encapsulated cells, compared with pure MeGC hydrogels.ConclusionsThis study demonstrates successful engineering of a biofunctional hydrogel with a specific microenvironment tailored to promote chondrogenesis. This hydrogel system can provide promising efficacious therapeutics in the treatment of cartilage defects.

The ABC transporter BCRP/ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction

The efflux pump ATP binding cassette superfamily member G2 (ABCG2)/breast cancer resistance protein (BCRP) is highly expressed in human placenta. We have investigated the role of BCRP in the protection of the human placental trophoblasts from apoptosis and its expression in idiopathic fetal growth restriction, a condition associated with abnormal pla‐cental apoptosis. Inhibition of BCRP activity with the selective inhibitor Ko143 augmented cytokine (tumor necrosis factor‐α/interferon‐γ)‐induced apoptosis and phosphatidylserine externalization in primary tropho‐blast and trophoblast‐like BeWo cells. Silencing of BCRP expression in BeWo cells significantly increased their sensitivity to apoptotic injury in response to cytokines and exogenous C6 and C8 ceramides. BCRP silencing also increased intracellular ceramide levels after cytokine exposure but did not affect cellular protoporphyrin IX concentrations or sensitivity to activators of the intrinsic apoptotic pathway. BCRP expression in placentas from pregnancies complicated by idiopathic fetal growth restriction was decreased compared with controls, suggesting reduced transport of its substrates from the placenta. We conclude that BCRP may play a hitherto unrecognized survival role in the placenta, protecting the trophoblast against cytokine‐induced apoptosis and possibly other extrinsic activators via modulation of ceramide signaling. Decreased placental BCRP expression may result in reduced viability and hence functional deficit, contributing to the fetal growth restriction phenotype.—Evseenko, D. A., Murthi, P., Paxton, J. W., Reid, G., Emerald, B. S., Mohankumar, K. M., Lobie, P. E., Brennecke, S. P., Kalionis, B. Keelan, J. A. The ABC transporter BCRP/ ABCG2 is a placental survival factor, and its expression is reduced in idiopathic human fetal growth restriction. FASEB J. 21, 3592–3605 (2007)

Active transport across the human placenta: impact on drug efficacy and toxicity

The human placenta expresses a large number of transport proteins. The ATP-binding cassette (ABC) family of active efflux pumps, predominantly localised to the maternal-facing syncytial membrane of placental microvilli, comprise the major placental drug efflux transporters. A variety of other transporters are also expressed in the placenta that can facilitate xenobiotic transfer in both the maternal and fetal directions. Many drugs administered in pregnancy are ABC transporter substrates, and many are either teratogenic or fetotoxic. The invitro, invivo and clinical evidence reviewed in this article argues that active efflux of drugs by placental transporters helps to maintain its barrier function, reducing the incidence of adverse fetal effects. ABC transporter polymorphisms may explain the wide variability observed in fetal drug concentrations, incidence of teratogenesis or drug failure in pregnancies exposed to therapeutic agents. Although our understanding of the molecular mechanics and dynamics of placental drug transfer is advancing, much work is needed to fully appreciate the significance of placental drug transporters in the face of increasing drug administration in pregnancy.

Rigid microenvironments promote cardiac differentiation of mouse and human embryonic stem cells

Abstract While adult heart muscle is the least regenerative of tissues, embryonic cardiomyocytes are proliferative, with embryonic stem (ES) cells providing an endless reservoir. In addition to secreted factors and cell–cell interactions, the extracellular microenvironment has been shown to play an important role in stem cell lineage specification, and understanding how scaffold elasticity influences cardiac differentiation is crucial to cardiac tissue engineering. Though previous studies have analyzed the role of matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examine the role of matrix rigidity on cardiac differentiation using mouse and human ES cells. Culture on polydimethylsiloxane (PDMS) substrates of varied monomer-to-crosslinker ratios revealed that rigid extracellular matrices promote a higher yield of de novo cardiomyocytes from undifferentiated ES cells. Using a genetically modified ES system that allows us to purify differentiated cardiomyocytes by drug selection, we demonstrate that rigid environments induce higher cardiac troponin T expression, beating rate of foci, and expression ratio of adult α- to fetal β- myosin heavy chain in a purified cardiac population. M-mode and mechanical interferometry image analyses demonstrate that these ES-derived cardiomyocytes display functional maturity and synchronization of beating when co-cultured with neonatal cardiomyocytes harvested from a developing embryo. Together, these data identify matrix stiffness as an independent factor that instructs not only the maturation of already differentiated cardiomyocytes but also the induction and proliferation of cardiomyocytes from undifferentiated progenitors. Manipulation of the stiffness will help direct the production of functional cardiomyocytes en masse from stem cells for regenerative medicine purposes.

Human Developmental Chondrogenesis as a Basis for Engineering Chondrocytes from Pluripotent Stem Cells

Summary Joint injury and osteoarthritis affect millions of people worldwide, but attempts to generate articular cartilage using adult stem/progenitor cells have been unsuccessful. We hypothesized that recapitulation of the human developmental chondrogenic program using pluripotent stem cells (PSCs) may represent a superior approach for cartilage restoration. Using laser-capture microdissection followed by microarray analysis, we first defined a surface phenotype (CD166low/negCD146low/negCD73+CD44lowBMPR1B+) distinguishing the earliest cartilage committed cells (prechondrocytes) at 5–6 weeks of development. Functional studies confirmed these cells are chondrocyte progenitors. From 12 weeks, only the superficial layers of articular cartilage were enriched in cells with this progenitor phenotype. Isolation of cells with a similar immunophenotype from differentiating human PSCs revealed a population of CD166low/negBMPR1B+ putative cartilage-committed progenitors. Taken as a whole, these data define a developmental approach for the generation of highly purified functional human chondrocytes from PSCs that could enable substantial progress in cartilage tissue engineering.

Characterization and therapeutic potential of induced pluripotent stem cell-derived cardiovascular progenitor cells.

Background Cardiovascular progenitor cells (CPCs) have been identified within the developing mouse heart and differentiating pluripotent stem cells by intracellular transcription factors Nkx2.5 and Islet 1 (Isl1). Study of endogenous and induced pluripotent stem cell (iPSC)-derived CPCs has been limited due to the lack of specific cell surface markers to isolate them and conditions for their in vitro expansion that maintain their multipotency. Methodology/Principal Findings We sought to identify specific cell surface markers that label endogenous embryonic CPCs and validated these markers in iPSC-derived Isl1+/Nkx2.5+ CPCs. We developed conditions that allow propagation and characterization of endogenous and iPSC-derived Isl1+/Nkx2.5+ CPCs and protocols for their clonal expansion in vitro and transplantation in vivo. Transcriptome analysis of CPCs from differentiating mouse embryonic stem cells identified a panel of surface markers. Comparison of these markers as well as previously described surface markers revealed the combination of Flt1+/Flt4+ best identified and facilitated enrichment for Isl1+/Nkx2.5+ CPCs from embryonic hearts and differentiating iPSCs. Endogenous mouse and iPSC-derived Flt1+/Flt4+ CPCs differentiated into all three cardiovascular lineages in vitro. Flt1+/Flt4+ CPCs transplanted into left ventricles demonstrated robust engraftment and differentiation into mature cardiomyocytes (CMs). Conclusion/Significance The cell surface marker combination of Flt1 and Flt4 specifically identify and enrich for an endogenous and iPSC-derived Isl1+/Nkx2.5+ CPC with trilineage cardiovascular potential in vitro and robust ability for engraftment and differentiation into morphologically and electrophysiologically mature adult CMs in vivo post transplantation into adult hearts.

Visible-light-initiated hydrogels preserving cartilage extracellular signaling for inducing chondrogenesis of mesenchymal stem cells.

Hydrogels have a unique opportunity to regenerate damaged cartilage tissues by introducing mesenchymal stem cells (MSCs) in a highly swollen environment similar to articular cartilage. During cartilage development, collagen-cell interactions play an important role in mediating early mesenchymal condensation and chondrogenesis with transforming growth factor-β1 (TGF-β1) stimulation. Here, a hydrogel environment that can enhance cell-matrix interactions and chondrogenesis by stabilizing type-II collagen (Col II) and TGF-β1 into photopolymerizable (methacrylated) chitosan (MeGC) with simple entrapment and affinity binding is demonstrated. The MeGC hydrogel was designed to gel upon initiation by exposure to visible blue light in the presence of riboflavin, an aqueous initiator from natural vitamin. The incorporation of Col II into MeGC hydrogels increased cellular condensation and deposition of cartilaginous extracellular matrix by encapsulated chondrocytes. MeGC hydrogels containing Col II supported the release of TGF-β1 in a controlled manner over time in chondrogenic medium and the incorporated TGF-β1 further enhanced chondrogenesis of encapsulated chondrocytes and MSCs, especially synovial MSCs. Subcutaneous implantation of hydrogel cultures showed greatly improved neocartilage formation in constructs loaded with TGF-β1 compared with controls. These findings suggest that cartilage mimetic hydrogels have a high potential for cartilage repair.

Lysophosphatidic acid mediates myeloid differentiation within the human bone marrow microenvironment.

Lysophosphatidic acid (LPA) is a pleiotropic phospholipid present in the blood and certain tissues at high concentrations; its diverse effects are mediated through differential, tissue specific expression of LPA receptors. Our goal was to determine if LPA exerts lineage-specific effects during normal human hematopoiesis. In vitro stimulation of CD34+ human hematopoietic progenitors by LPA induced myeloid differentiation but had no effect on lymphoid differentiation. LPA receptors were expressed at significantly higher levels on Common Myeloid Progenitors (CMP) than either multipotent Hematopoietic Stem/Progenitor Cells (HSPC) or Common Lymphoid Progenitors (CLP) suggesting that LPA acts on committed myeloid progenitors. Functional studies demonstrated that LPA enhanced migration, induced cell proliferation and reduced apoptosis of isolated CMP, but had no effect on either HSPC or CLP. Analysis of adult and fetal human bone marrow sections showed that PPAP2A, (the enzyme which degrades LPA) was highly expressed in the osteoblastic niche but not in the perivascular regions, whereas Autotaxin (the enzyme that synthesizes LPA) was expressed in perivascular regions of the marrow. We propose that a gradient of LPA with the highest levels in peri-sinusoidal regions and lowest near the endosteal zone, regulates the localization, proliferation and differentiation of myeloid progenitors within the bone marrow marrow.