In Vivo Generation of Bone Marrow from Embryonic Stem Cells in Interspecies Chimeras

Generation of bone marrow (BM) from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce all BM hematopoietic cell lineages from mouse ESCs in a rat. Based on FACS analysis and single-cell RNA sequencing, mouse ESCs differentiated into hematopoietic progenitor cells and multiple hematopoietic cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Transplantation of ESC-derived BM cells from mouse-rat chimeras rescued lethally-irradiated syngeneic mice and resulted in long-term contribution of donor cells to hematopoietic cell lineages. Altogether, a fully functional bone marrow was generated from mouse ESCs using rat embryos as “bioreactors”.

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230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab230 ◽

2007 ◽

Vol 19 (1) ◽

pp. 231

Author(s):

S. Wang ◽

X. Tang ◽

Y. Niu ◽

H. Chen ◽

T. Li ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

High Speed ◽

Es Cells ◽

Research Work ◽

Embryonic Stem ◽

Morphological Characteristics ◽

Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

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Transplantation of Retinal Progenitor Cells from Optic Cup-Like Structures Differentiated from Human Embryonic Stem Cells In Vitro and In Vivo Generation of Retinal Ganglion-Like Cells

Stem Cells and Development ◽

10.1089/scd.2018.0076 ◽

2019 ◽

Vol 28 (4) ◽

pp. 258-267 ◽

Cited By ~ 7

Author(s):

Song-Tao Wang ◽

Li-li Chen ◽

Peng Zhang ◽

Xiao-Bing Wang ◽

Yan Sun ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Retinal Ganglion ◽

Retinal Progenitor ◽

Optic Cup

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Evolutionarily Conserved Role of Nucleostemin: Controlling Proliferation of Stem/Progenitor Cells during Early Vertebrate Development

Molecular and Cellular Biology ◽

10.1128/mcb.01183-06 ◽

2006 ◽

Vol 26 (24) ◽

pp. 9291-9301 ◽

Cited By ~ 73

Author(s):

Chantal Beekman ◽

Massimo Nichane ◽

Sarah De Clercq ◽

Marion Maetens ◽

Thomas Floss ◽

...

Keyword(s):

Stem Cells ◽

Cell Proliferation ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Control Cell ◽

Embryonic Stem ◽

Physiological Role ◽

Specific Gene

ABSTRACT Nucleostemin (NS) is a putative GTPase expressed preferentially in the nucleoli of neuronal and embryonic stem cells and several cancer cell lines. Transfection and knockdown studies indicated that NS controls the proliferation of these cells by interacting with the p53 tumor suppressor protein and regulating its activity. To assess the physiological role of NS in vivo, we generated a mutant mouse line with a specific gene trap event that inactivates the NS allele. The corresponding NS −/− embryos died around embryonic day 4. Analyses of NS mutant blastocysts indicated that NS is not required to maintain pluripotency, nucleolar integrity, or survival of the embryonic stem cells. However, the homozygous mutant blastocysts failed to enter S phase even in the absence of functional p53. Haploid insufficiency of NS in mouse embryonic fibroblasts leads to decreased cell proliferation. NS also functions in early amphibian development to control cell proliferation of neural progenitor cells. Our results show that NS has a unique ability, derived from an ancestral function, to control the proliferation rate of stem/progenitor cells in vivo independently of p53.

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Vascular Progenitor Cells Isolated From Human Embryonic Stem Cells Give Rise to Endothelial and Smooth Muscle–Like Cells and Form Vascular Networks In Vivo

Circulation Research ◽

10.1161/circresaha.107.150201 ◽

2007 ◽

Vol 101 (3) ◽

pp. 286-294 ◽

Cited By ~ 187

Author(s):

Lino S. Ferreira ◽

Sharon Gerecht ◽

Hester F. Shieh ◽

Nicki Watson ◽

Maria A. Rupnick ◽

...

Keyword(s):

Stem Cells ◽

Smooth Muscle ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Vascular Networks ◽

Vascular Progenitor Cells

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Androgenetic Embryonic Stem Cells Form Neural Progenitor Cells In Vivo and In Vitro

Stem Cells ◽

10.1634/stemcells.2007-0877 ◽

2008 ◽

Vol 26 (6) ◽

pp. 1474-1483 ◽

Cited By ~ 14

Author(s):

Timo C. Dinger ◽

Sigrid Eckardt ◽

Soon Won Choi ◽

Guadelupe Camarero ◽

Satoshi Kurosaka ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Progenitor Cells ◽

Neural Progenitor Cells ◽

Embryonic Stem ◽

Neural Progenitor

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Improved Analysis of Hematopoietic Engraftment by Non-Invasive In Vivo Bioluminescent Imaging of Transplanted Human Embryonic Stem Cell-Derived Hematopoietic Cells.

Blood ◽

10.1182/blood.v106.11.1270.1270 ◽

2005 ◽

Vol 106 (11) ◽

pp. 1270-1270

Author(s):

Jonathan L. Linehan ◽

Xinghui Tian ◽

Julie K. Morris ◽

Dan S. Kaufman

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Es Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Scid Mice ◽

Cell Lineages ◽

Non Invasive ◽

Flow Cytometric

Abstract Animal transplantation models are essential to characterize the long-term in vivo engraftment capacity of putative hematopoietic stem cells derived from human embryonic stem cells (hESCs). We have previously demonstrated that hESCs can be routinely utilized to derive multiple hematopoietic cell lineages. Here, we use in vivo bioluminescence imaging (BLI) of stable luciferase (luc)-expressing hESCs to noninvasively monitor the dynamics of transplantation, engraftment, and growth of hESC-derived hematopoietic cells within individual animals over an extended time course. Luc expression under control of an EF1α promoter was introduced into the H1 hESC line using a self-inactivating lentiviral vector. Undifferentiated hESC colonies that stably expressed luciferase were established and selected, and the pluripotent capability of luc+ hESCs was first explored by teratoma formation. Undifferentiated luc+ human ES cells were intramuscularly injected into NOD/SCID mice. The dynamics of survival and growth of the hESCs was monitored by BLI using the IVIS Imaging System (Xenogen) at regular time points post-transplantation. There was a decrease of luminescent signal during the first 1–2 weeks. This was followed by a dramatic increase in luminescent signals after about 5 weeks, which correlated with teratoma size. Immunohistochemical analysis confirmed stable luc-expression in multiple differentiated cell types within the teratomas. We next used BLI to examine luc+ H1 hESCs that were induced to undergo hematopoietic differentiation by co-culture with S17 cells, to give rise to H1/S17 cells. Flow cytometric studies confirmed hematopoietic cells (CD34+, CD45+, CD31+, and c-kit+ cells) were derived from these differentiated luc+ hESCs, with 5–10% of H1/S17 cells being CD34+. Hematopoietic progenitors that gave rise to colonies of mature luc+ blood cells in a standard CFU assay were also observed from the H1/S17 cells. Luc-expression of differentiated hESCs was maintained at similar levels to those of the undifferentiated ES cells. To define the in vivo potential of luc+ hESC-derived hematopoietic cells, hESCs were allowed to differentiate on S17 cells for two weeks. SCID-repopulating cell studies were done by intravenous (iv) injection into sublethally irradiated NOD/SCID mice. After iv injection of 2–3 x106 unsorted luc+ H1/S17 cells, BLI showed the brightest signal in the lung at day 0 (within 2 hours), followed by a rapid decline in signal on the next day (day1). On day 8, most luc+ cells were detected in the abdomen and liver. Subsequently, after 6–12 weeks, multiple engraftment loci were identified in hematopoietic tissues. Flow cytometric analysis of bone marrow from these mice confirmed the presence of hESC-derived human CD45+ cells. Engraftment was also demonstrated after direct intra-bone marrow injection with as few as 60,000 CD34+ cells sorted from luc+ H1/S17 cells. Again, stable engraftment can be monitored by BLI for 8+ weeks. These results demonstrate that BLI has several important advantages as an effective non-invasive approach to track and quantitatively monitor in vivo engraftment of hematopoietic or other cell lineages derived from hESCs.

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