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

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 the matrix elasticity on the function of differentiated cardiomyocytes, whether it affects the induction of cardiomyocytes from pluripotent stem cells is poorly understood. Here, we examined the role of matrix rigidity on the 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 an 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 the 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

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

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

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 (CD166(low/neg)CD146(low/neg)CD73(+)CD44(low)BMPR1B(+)) 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 CD166(low/neg)BMPR1B(+) 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.Fil: Wu, Ling. University of California at Los Angeles; Estados UnidosFil: Bluguermann, Carolina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of California at Los Angeles; Estados UnidosFil: Kyupelyan, Levon. University of California at Los Angeles; Estados UnidosFil: Latour, Brooke. University of California at Los Angeles; Estados UnidosFil: Gonzalez, Stephanie. University of California at Los Angeles; Estados UnidosFil: Shah, Saumya. University of California at Los Angeles; Estados UnidosFil: Galic, Zoran. University of California at Los Angeles; Estados UnidosFil: Ge, Sundi. University of California at Los Angeles; Estados UnidosFil: Zhu, Yuhua. University of California at Los Angeles; Estados UnidosFil: Petrigliano, Frank A.. University of California at Los Angeles; Estados UnidosFil: Nsair, Ali. University of California at Los Angeles; Estados UnidosFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Li, Xinmin. University of California at Los Angeles; Estados UnidosFil: Lyons, Karen M.. University of California at Los Angeles; Estados UnidosFil: Crooks, Gay M.. University of California at Los Angeles; Estados UnidosFil: McAllister, David R.. University of California at Los Angeles; Estados UnidosFil: Van Handel, Ben. Novogenix Laboratories; Estados UnidosFil: Adams, John S.. University of California at Los Angeles; Estados UnidosFil: Evseenko, Denis. University of California at Los Angeles; Estados Unido

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

ABSTRACT: 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-␣ (TNF-␣) or interleukin (IL)-1␤ 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-␣ 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