A role for intracellular calcium downstream of G-protein signaling in undifferentiated human embryonic stem cell culture

AbstractMultiple signalling pathways maintain human embryonic stem cells (hESC) in an undifferentiated state. Here we sought to define the significance of G protein signal transduction in the preservation of this state distinct from other cellular processes. Continuous treatment with drugs targeting Gαs-, Gα-i/o- and Gα-q/11-subunit signalling mediators were assessed in independent hESC lines after 7days to discern effects on normalised alkaline phosphatase positive colony frequency vs total cell content. This identified PLCβ, intracellular free calcium and CAMKII kinase activity downstream of Gα-q/11 as of particular importance to the former. To confirm the significance of this finding we generated an agonist-responsive hESC line transgenic for a Gα-q/11 subunit-coupled receptor and demonstrated that an undifferentiated state could be promoted in the presence of an agonist without exogenously supplied bFGF and that this correlated with elevated intracellular calcium. Similarly, treatment of unmodified hESCs with a range of intracellular free calcium-modulating drugs in biologically defined mTESR culture system lacking exogenous bFGF promoted an hESC phenotype after 1week of continuous culture as defined by co-expression of OCT4 and NANOG. At least one of these drugs, lysophosphatidic acid significantly elevates phosphorylation of calmodulin and STAT3 in this culture system (p<0.05). These findings substantiate a role for G-protein and calcium signalling in undifferentiated hESC culture

Thermoresponsive hydrogel maintains the mouse embryonic stem cell "naïve" pluripotency phenotype

A chemically defined hydrogel HG21, which allows enzyme-free passaging, is a substitute for gelatin allowing standardised and inexpensive mESC culture.</p

Infância, exclusão social e educação como utopia realizável

A infância, como construção social, tem sofrido, no decurso da 2ª modernidade, processos de reinstitucionalização que, em larga medida, põem em causa as representações e imagens das crianças, dominantes nos últimos 200 anos. A análise da (re)construção das identidades sociais e das subjectividades infantis constitui, desse modo, uma tarefa teórica da mais exigente actualidade. O que, entretanto, aqui se assinala é que este processo de reinstitucionalização da infância, apesar da construção de consensos globais sobre os direitos das crianças, tem vindo a aumentar os factores e as condições de exclusão das gerações mais jovens face aos direitos sociais e da cidadania. Neste artigo inventariam-se alguns dos principais indicadores de exclusão, considerando diversos espaços estruturais, e assinalam-se alguns dos pontos de ruptura por onde pode passar a construção de uma educação escolar centrada na afirmação activa dos direitos das crianças.Along the second modernity, childhood, as a social construction, has undergone reinstatement processes that question the representations and images of children that have prevailed over last two hundred years. An analysis of the (re) construction of the social identities and childhood subjectivity thus constitute a theoretical task of the most contemporary exigence. Nevertheless, even though a global consensus about children’s rights has been constructed, the increase of factors and conditions that exclude the youngest generations from social rights and citizenship, induced by this reinstatement process of childhood, is here emphasized. This paper lists some of the main indicators of exclusion, considering different structural spaces, and pinpoints some turning points through which the construction of a school education centered on the active affirmation of the children’s rights could be attained.CIFPEC/CIEC - Centro de Investigação em Estudos da Criança, UM (UI 644 e 317 da FCT

Hyaluronan in mesenchymal stromal cell lineage differentiation from human pluripotent stem cells:application in serum free culture

BACKGROUND: Hyaluronan (HA) is an extracellular glycosaminoglycan polysaccharide with widespread roles throughout development and in healthy and neoplastic tissues. In pluripotent stem cell culture it can support both stem cell renewal and differentiation. However, responses to HA in culture are influenced by interaction with a range of cognate factors and receptors including components of blood serum supplements, which alter results. These may contribute to variation in cell batch production yield and phenotype as well as heighten the risks of adventitious pathogen transmission in the course of cell processing for therapeutic applications. MAIN: Here we characterise differentiation of a human embryo/pluripotent stem cell derived Mesenchymal Stromal Cell (hESC/PSC-MSC)-like cell population by culture on a planar surface coated with HA in serum-free media qualified for cell production for therapy. Resulting cells met minimum criteria of the International Society for Cellular Therapy for identification as MSC by expression of. CD90, CD73, CD105, and lack of expression for CD34, CD45, CD14 and HLA-II. They were positive for other MSC associated markers (i.e.CD166, CD56, CD44, HLA 1-A) whilst negative for others (e.g. CD271, CD71, CD146). In vitro co-culture assessment of MSC associated functionality confirmed support of growth of hematopoietic progenitors and inhibition of mitogen activated proliferation of lymphocytes from umbilical cord and adult peripheral blood mononuclear cells, respectively. Co-culture with immortalized THP-1 monocyte derived macrophages (Mɸ) concurrently stimulated with lipopolysaccharide as a pro-inflammatory stimulus, resulted in a dose dependent increase in pro-inflammatory IL6 but negligible effect on TNFα. To further investigate these functionalities, a bulk cell RNA sequence comparison with adult human bone marrow derived MSC and hESC substantiated a distinctive genetic signature more proximate to the former.CONCLUSION: Cultivation of human pluripotent stem cells on a planar substrate of HA in serum-free culture media systems is sufficient to yield a distinctive developmental mesenchymal stromal cell lineage with potential to modify the function of haematopoietic lineages in therapeutic applications.</p

Derivation of the human embryonic stem cell line RCe006-A (RC-2)

AbstractThe human embryonic stem cell line RCe006-A (RC-2) was derived from a frozen and thawed blastocyst voluntarily donated as surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line exhibits expression of expected pluripotency markers and in vitro differentiation potential to three germinal lineage representative cell populations. It has a male trisomy 12 karyotype (47XY, +12). Microsatellite DNA marker identity and HLA and blood group typing data are available

Derivation of the human embryonic stem cell line RCe007-A (RC-3)

The human embryonic stem cell line RCe007-A (RC-3) was derived from a blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and HLA and blood group typing data is available

Derivation of the human embryonic stem cell line RCe014-A (RC-10)

AbstractThe human embryonic stem cell line RCe012-A (RC-8) was derived from a frozen and thawed day 5 embryo cultivated to the blastocyst stage. The embryo was voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data is available

Derivation of the human embryonic stem cell line RCe009-A (RC-5)

The human embryonic stem cell line RCe009-A (RC-5) was derived from a frozen and thawed Day 2 embryo voluntarily donated as unsuitable and surplus to requirement for fertility treatment following informed consent under licence from the UK Human Fertilisation and Embryology Authority. RCe009-A carries the common DF508 mutation on the cystic fibrosis trans-membrane regulator gene associated with the disease cystic fibrosis. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XX female karyotype and microsatellite PCR identity, HLA and blood group typing data are available

Derivation of the human embryonic stem cell line RCe010-A (RC-6)

AbstractThe human embryonic stem cell line RCe010-A (RC-6) was derived from a frozen and thawed blastocyst voluntarily donated as unsuitable and surplus to fertility requirements following ethics committee approved informed consent under licence from the UK Human Fertilisation and Embryology Authority. The cell line shows normal pluripotency marker expression and differentiation to the three germ layers in vitro. It has a normal 46XY male karyotype and microsatellite PCR identity, HLA and blood group typing data are available