Human pluripotent stem cells as a model of trophoblast differentiation in both normal development and disease

Trophoblast is the primary epithelial cell type in the placenta, a transient organ required for proper fetal growth and development. Different trophoblast subtypes are responsible for gas/nutrient exchange (syncytiotrophoblasts, STBs) and invasion and maternal vascular remodeling (extravillous trophoblasts, EVTs). Studies of early human placental development are severely hampered by the lack of a representative trophoblast stem cell (TSC) model with the capacity for self-renewal and the ability to differentiate into both STBs and EVTs. Primary cytotrophoblasts (CTBs) isolated from early-gestation (6–8 wk) human placentas are bipotential, a phenotype that is lost with increasing gestational age. We have identified a CDX2+/p63+ CTB subpopulation in the early postimplantation human placenta that is significantly reduced later in gestation. We describe a reproducible protocol, using defined medium containing bone morphogenetic protein 4 by which human pluripotent stem cells (hPSCs) can be differentiated into CDX2+/p63+ CTB stem-like cells. These cells can be replated and further differentiated into STB- and EVT-like cells, based on marker expression, hormone secretion, and invasive ability. As in primary CTBs, differentiation of hPSC-derived CTBs in low oxygen leads to reduced human chorionic gonadotropin secretion and STB-associated gene expression, instead promoting differentiation into HLA-G+ EVTs in an hypoxia-inducible, factor-dependent manner. To validate further the utility of hPSC-derived CTBs, we demonstrated that differentiation of trisomy 21 (T21) hPSCs recapitulates the delayed CTB maturation and blunted STB differentiation seen in T21 placentae. Collectively, our data suggest that hPSCs are a valuable model of human placental development, enabling us to recapitulate processes that result in both normal and diseased pregnancies.

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Human Pluripotent Stem Cells Differentiated in Fully Defined Medium Generate Hematopoietic CD34+ and CD34− Progenitors with Distinct Characteristics

PLoS ONE ◽

10.1371/journal.pone.0014733 ◽

2011 ◽

Vol 6 (2) ◽

pp. e14733 ◽

Cited By ~ 19

Author(s):

Laurie Chicha ◽

Anis Feki ◽

Alessandro Boni ◽

Olivier Irion ◽

Outi Hovatta ◽

...

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Defined Medium ◽

Human Pluripotent Stem Cells

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Generation of Retinoic Acid-Dependent Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Blood ◽

10.1182/blood-2020-142468 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 35-35

Author(s):

Stephanie A Luff ◽

J Philip Creamer ◽

Carissa Dege ◽

Rebecca Scarfò ◽

Samantha Morris ◽

...

Keyword(s):

Stem Cells ◽

Retinoic Acid ◽

Pluripotent Stem Cells ◽

De Novo ◽

Conflicts Of Interest ◽

Human Pluripotent Stem Cells ◽

Human Embryos ◽

Dependent Manner ◽

Hematopoietic Stem

The generation of the hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a major goal for regenerative medicine. In the embryo, HSCs derive from a HOXA+ population known as hemogenic endothelium (HE) in a retinoic acid (RA)-dependent manner. Using hPSCs, we have previously identified a KDR+CD235a− mesodermal population that gives rise to a clonally multipotent HOXA+ definitive HE. However, this HE lacks HSC-like capacity in the absence of exogenous transgenes and is functionally unresponsive to RA treatment. Thus, the specification of an RA-dependent hematopoietic program from hPSCs has remained elusive. Through single cell RNA-seq (scRNA-seq) analyses, we identified that 2 distinct KDR+CD235a− populations exist prior to HE specification, distinguishable by CXCR4 expression. Interestingly, KDR+CD235a−CXCR4− mesoderm expressed CYP26A1, an RA degrading enzyme, and harbored definitive hematopoietic potential within hPSC differentiation cultures in the absence of RA signaling, indicating the HE specified from CXCR4− mesoderm as RA-independent (RAi). In sharp contrast, KDR+CD235a−CXCR4+ mesoderm exclusively expressed ALDH1A2, the key enzyme in the synthesis of RA, but lacked hematopoietic potential under the same culture conditions. However, the stage-specific application of RA signaling to CXCR4+ mesoderm resulted in the robust specification of CD34+HOXA+ HE with definitive erythroid, myeloid, and lymphoid hematopoietic potential, establishing this HE as RA-dependent (RAd). Furthermore, while RAi HE entirely failed to persist following murine hematopoietic xenografts, RAd HE transiently persisted within the peripheral blood and bone marrow of murine hosts. To assess whether these functionally distinct hPSC mesodermal progenitors are physiologically relevant to human embryonic development, we integrated scRNA-seq datasets from the hPSC mesodermal cultures and a gastrulating human embryo. These analyses revealed that in vivo, distinct KDR+CXCR4−CYP26A1+ and KDR+CXCR4+ALDH1A2+ populations can be found at the stage of emergent mesoderm, following patterning of nascent mesoderm. Additional comparison to later stage human embryos demonstrated that RAd HE has a more fetal-like HOXA expression pattern than RAi HE. Scoring of single fetal HE cells against hPSC-derived HE revealed that while some early fetal HE cells were similar to RAi HE, the late fetal HE cells, which are hypothesized to give rise to HSCs, were more similar to RAd HE. Lastly, as HSC-competent HE is expected to express arterial genes, we found a subset of late fetal HE with this phenotype that were exclusively similar to RAd HE. Collectively, these data represent the first ever characterization of RA-dependent hPSC-derived definitive hematopoiesis and its mesodermal progenitor. Additionally, we provide evidence for in vivo mesodermal and HE correlates for both RAi and RAd hematopoietic programs within human embryos. This novel insight into human hematopoietic development will serve as an important tool for modeling development and ultimately provide the basis for de novo specification of HSCs. Disclosures No relevant conflicts of interest to declare.

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Abstract 227: Cardiomyocytes from Human Pluripotent Stem Cells Predict Chemotherapy-Induced Cardiotoxicity

Circulation Research ◽

10.1161/res.111.suppl_1.a227 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Maxime Mioulane ◽

Gabor Foldes ◽

Sian Harding

Keyword(s):

Stem Cells ◽

Cell Death ◽

Pluripotent Stem Cells ◽

Fluorescent Dyes ◽

Embryonic Stem ◽

Chemotherapeutic Agents ◽

Human Pluripotent Stem Cells ◽

Toxicity Tests ◽

Dependent Manner ◽

Isolated Cells

The potential of human pluripotent stem cells-derived cardiomyocytes (hPSC-CM) to mimic human cardiac toxicity tests is largely unknown. Here we investigate the interaction between the chemotherapeutic agents Doxorubicin (DOX) and Lapatinib (LAP) and the protective effect of beta2 adrenergic signalling in hPSC-CM and murine myocytes. We hypothesized that death and survival pathways in hPSC-CM differ from those in animal-derived myocytes and thereby may predict better clinical outcome of chemotherapeutics. Human induced pluripotent or embryonic stem cell H7 cell lines were differentiated into cardiomyocytes (hiPSC-CM and hESC-CM, respectively), treated with drugs and stained with fluorescent dyes (Hoechst 33342, mitochondrial probes, effector caspases activation and impermeant nuclear dyes). Cell death profile was generated from high content screening and analysis (Cellomics). In isolated hPSC-CM, the anthracycline DOX induced apoptosis in a concentration-dependent manner (1-100µM), whereas LAP did not produce any toxicity. However, the combination of the two chemotherapeutic agents worsened the toxicity profile compared to DOX alone (p<0.001, n=12), consistent with clinical observations. In contrast, rat neonates were sensitive to LAP and showed a different cell death profile in DOX and drug combination experiments. Activation of apoptotic caspases in hESC-CM clusters was coincident with the dissipation of the mitochondrial membrane potential, while the potential was preserved in isolated cells during this phase. Beta2 adrenergic stimulation did not reverse DOX pro-apoptotic effect but modified toxicity-related nuclear events. In conclusion, we have shown that hPSC-CM reproduce the clinical adverse cardiac effects after treatment with anti-cancer drugs, making these cells a more relevant system for future drug development than animal-derived cells. However, our data suggest that cell death pathways involved may depend on culture condition for cardiomyocytes.

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Stepwise Signaling and Low Oxygen Promote Hematopoietic Progenitor Emergence From Human Pluripotent Stem Cells

Blood ◽

10.1182/blood.v118.21.1317.1317 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1317-1317 ◽

Cited By ~ 2

Author(s):

Hiroshi Endo ◽

Naoya Takayama ◽

Tomo Koike ◽

Hiromitsu Nakauchi ◽

Koji Eto

Keyword(s):

Stem Cells ◽

Growth Factor ◽

Pluripotent Stem Cells ◽

Blood Cells ◽

Early Phase ◽

Late Phase ◽

Human Pluripotent Stem Cells ◽

Low Oxygen ◽

Hematopoietic Progenitor ◽

Oxygen Conditions

Abstract Abstract 1317 Hematopoietic systems in mouse models have been well characterized based on the defined cell surface markers present on fetal and adult hematopoietic stem cells (HSCs) and their blood derivatives. In humans, by contrast, hematopoietic ontogeny and the subsequent hierarchy have not been determined. Human pluripotent stem cells (ES cells, ESCs and iPS cells, iPSCs) are embryo-type cells and a promising cell source for studying the ontogeny of blood cells within a differentiation system. We previously established an in vitro co-culture method using C3H10T1/2 mesenchymal stromal cells, whereby vascular endothelial growth factor (VEGF) promotes the appearance of CD34+ hematopoietic progenitor cells (HPCs) from human ESCs or iPSCs (Takayama et al., Blood, 2008; Takayama et al., J Exp Med, 2010). Here we demonstrate that the use of a combination of low oxygen and growth factors is suitable for cells at specific developmental stages that include CD56+CD326- mesodermal progenitors during the early phase (days 0–4), CD34+CD56+CD90+CD105+CD43-KDR- hemangioblasts during the mid-phase (days 5–7), and CD43+ hematopoietic and KDR+ endothelial cells during the late phase (days 8–10). During mid-phase, application of basic fibroblast growth factor (bFGF, 10 ng/ml) under 1% O2 significantly increased numbers of CD43+ cells by 5-fold, as compared to cells without bFGF under 21% O2. Administration of a MYC inhibitor (50 μM) to cells during the early-phase, and tumor growth factor beta (TGF-beta) receptor inhibitor (SB431542, 10 μM) during mid-phase, also stimulated generation of CD34+CD43+ HPCs (1.5-fold and 3-fold in increase respectively). Notably, although this protocol resulted in a prominent yield of mesodermal progenitors during the early phase and of HPCs during the late phase (which were 50% and 10% of all derivatives from human iPSCs, respectively), this signaling manipulation had the opposite effect at other stages. For example, bFGF or TGFbeta receptor inhibition significantly depressed HPCs during the early-phase. The signaling modulation and low oxygen conditions in our co-culture system did not require factors known to affect ex vivo human CD34+ HSC / HPC expansion from cord blood cells, which contain stem cell factor, thrombopoietin, FMS-like tyrosine kinase 3 ligand, erythropoietin, interleukin (IL)-3 and IL-6. Our novel culture protocol implicates new players in the stepwise development from a pluripotent state to blood cell generation. These players appear to be governed by circumstances resembling a developmental niche with lower oxygen conditions. Disclosures: No relevant conflicts of interest to declare.

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Controlled Growth and the Maintenance of Human Pluripotent Stem Cells by Cultivation with Defined Medium on Extracellular Matrix-Coated Micropatterned Dishes

PLoS ONE ◽

10.1371/journal.pone.0129855 ◽

2015 ◽

Vol 10 (6) ◽

pp. e0129855 ◽

Cited By ~ 9

Author(s):

Chiemi Takenaka ◽

Hiroshi Miyajima ◽

Yusuke Yoda ◽

Hideo Imazato ◽

Takako Yamamoto ◽

...

Keyword(s):

Stem Cells ◽

Extracellular Matrix ◽

Pluripotent Stem Cells ◽

Defined Medium ◽

Human Pluripotent Stem Cells ◽

Controlled Growth

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Cryopreservation of Human Pluripotent Stem Cells in Defined Medium

Current Protocols in Stem Cell Biology ◽

10.1002/9780470151808.sc01c17s31 ◽

2014 ◽

Vol 31 (1) ◽

Cited By ~ 6

Author(s):

Weiwei Liu ◽

Guokai Chen

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Defined Medium ◽

Human Pluripotent Stem Cells

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Culturing of human pluripotent stem cells in a mesoderm biased state

10.21203/rs.3.pex-1100/v1 ◽

2021 ◽

Author(s):

Dylan Stavish ◽

Christopher Price

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Defined Medium ◽

Human Pluripotent Stem Cells ◽

Pluripotency Factors

Abstract Here we present a new culturing system, PRIMO Plus, to generate mesoderm biased human pluripotent stem cells by cross antagonism of pro-differentiation and pro-pluripotency factors in a fully defined medium.

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Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner

Cytotherapy ◽

10.1016/j.jcyt.2014.02.009 ◽

2014 ◽

Vol 16 (7) ◽

pp. 881-892 ◽

Cited By ~ 43

Author(s):

Katarzyna Drela ◽

Anna Sarnowska ◽

Patrycja Siedlecka ◽

Ilona Szablowska-Gadomska ◽

Miroslaw Wielgos ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Umbilical Cord ◽

Hypoxia Inducible Factor ◽

Oxygen Atmosphere ◽

Dependent Manner ◽

Low Oxygen ◽

Undifferentiated State

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Two distinct trophectoderm lineage stem cells from human pluripotent stem cells

10.1101/762542 ◽

2019 ◽

Cited By ~ 4

Author(s):

Adam Mischler ◽

Victoria Karakis ◽

Jessica Mahinthakumar ◽

Celeste Carberry ◽

Adriana San Miguel ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Fetal Tissue ◽

Human Pluripotent Stem Cells ◽

Placental Development ◽

Human Trophoblast ◽

Trophoblast Stem Cells ◽

Distinct Cell ◽

Additional Cell

SummaryTrophoblasts are the principal cell type of the placenta. The use of human trophoblast stem cells (hTSCs) as a model for studies of early placental development is hampered by limited genetic diversity of existing hTSC lines, and constraints on using human fetal tissue or embryos needed to generate additional cell lines. Here we report the derivation of two distinct stem cells of the trophectoderm lineage from human pluripotent stem cells. The first is a CDX2- stem cell equivalent to primary hTSCs – they both exhibit identical expression of key markers, are maintained in culture and differentiate under similar conditions, and share high transcriptome similarity. The second is a CDX2+ putative human trophectoderm stem cell (hTESC) with distinct cell culture requirements and differences in gene expression and differentiation relative to hTSCs. Derivation of hTSCs and hTESCs from pluripotent stem cells significantly enables construction of models for normal and pathological placental development.

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Cell cycle regulators control mesoderm specification in human pluripotent stem cells

10.1101/632307 ◽

2019 ◽

Cited By ~ 1

Author(s):

Loukia Yiangou ◽

Rodrigo A. Grandy ◽

Sanjay Sinha ◽

Ludovic Vallier

Keyword(s):

Cell Cycle ◽

Stem Cells ◽

Pluripotent Stem Cells ◽

Human Pluripotent Stem Cells ◽

Bmp Signaling ◽

Dependent Manner ◽

Cell Cycle Regulators ◽

Lateral Plate ◽

Mesoderm Specification ◽

Mesoderm Formation

ABSTRACTMesoderm is one of the three germ layers produced during gastrulation from which muscle, bones, kidneys and the cardiovascular system originate. Understanding the mechanisms controlling mesoderm specification could be essential for a diversity of applications, including the development of regenerative medicine therapies against diseases affecting these tissues. Here, we use human pluripotent stem cells (hPSCs) to investigate the role of cell cycle in mesoderm formation. For that, proteins controlling G1 and G2/M cell cycle phases were inhibited during differentiation of hPSCs into lateral plate, cardiac and presomitic mesoderm using small molecules or by conditional knock down. These loss of function experiments revealed that CDKs and pRb phosphorylation are necessary for efficient mesoderm formation in a context-dependent manner. Further investigations showed that inhibition of the G2/M regulator CDK1 decreases BMP signaling activity specifically during lateral plate mesoderm formation while reducing FGF/ERK1/2 activity in all mesoderm subtypes. Taken together, our findings reveal that cell cycle regulators direct mesoderm formation by controlling the activity of key developmental pathways.

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