scholarly journals Mature endothelium and neurons are simultaneously derived from embryonic stem cells by 2D in vitro culture system

2011 ◽  
Vol 15 (10) ◽  
pp. 2200-2215 ◽  
Author(s):  
Alessio Noghero ◽  
Marco Arese ◽  
Federico Bussolino ◽  
Anna Gualandris
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Embryonic Stem ◽  
In Vitro Culture System

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

Abstract We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

Long-Term Ex Vivo Maintenance and Expansion of Transplantable Human Hematopoietic Stem Cells

Blood ◽  
1999 ◽  
Vol 94 (5) ◽  
pp. 1623-1636 ◽  
Author(s):  
Chu-Chih Shih ◽  
Mickey C.-T. Hu ◽  
Jun Hu ◽  
Jeffrey Medeiros ◽  
Stephen J. Forman
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
In Vitro Culture System

We have developed a stromal-based in vitro culture system that facilitates ex vivo expansion of transplantable CD34+thy-1+ cells using long-term hematopoietic reconstitution in severe combined immunodeficient-human (SCID-hu) mice as an in vivo assay for transplantable human hematopoietic stem cells (HSCs). The addition of leukemia inhibitory factor (LIF) to purified CD34+ thy-1+ cells on AC6.21 stroma, a murine bone marrow–derived stromal cell line, caused expansion of cells with CD34+ thy-1+ phenotype. Addition of other cytokines, including interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor, and stem cell factor, to LIF in the cultures caused a 150-fold expansion of cells retaining the CD34+ thy-1+ phenotype. The ex vivo–expanded CD34+ thy-1+ cells gave rise to multilineage differentiation, including myeloid, T, and B cells, when transplanted into SCID-hu mice. Both murine LIF (cannot bind to human LIF receptor) and human LIF caused expansion of human CD34+ thy-1+ cells in vitro, suggesting action through the murine stroma. Furthermore, another human HSC candidate, CD34+ CD38− cells, shows a similar pattern of proliferative response. This suggests thatex vivo expansion of transplantable human stem cells under this in vitro culture system is a general phenomenon and not just specific for CD34+ thy-1+ cells.

scholarly journals Gelatin Electrospun Mat as a Potential Co-culture System for In Vitro Production of Sperm Cells from Embryonic Stem Cells

2020 ◽  
Vol 6 (10) ◽  
pp. 5823-5832
Author(s):  
Mina Vardiani ◽  
Marefat Ghaffari Novin ◽  
Morteza Koruji ◽  
Hamid Nazarian ◽  
Ellen Goossens ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Sperm Cells ◽  
In Vitro Production ◽  
Vitro Production

395 OVINE SPERMATOGONIA ISOLATION AND IN VITRO GROWTH ASSAY

2010 ◽  
Vol 22 (1) ◽  
pp. 354
Author(s):  
A. Qi ◽  
T. Wuliji ◽  
Y. Zhang
Keyword(s):  
Stem Cells ◽  
In Vitro Culture ◽  
Culture System ◽  
Culture Medium ◽  
Single Cells ◽  
Testicular Tissue ◽  
Basic Medium ◽  
Seminiferous Tubules ◽  
In Vitro Culture System

Spermatogonia, as adult stem cells from the male reproductive system, are attracting strong interest from those studying male reproductive gamete preservation and developing new approaches in transgenic animals. Protein gene product 9.5 (PGP9.5) is a marker of sheep spermatogonia, which has been validated by J. R. Rodriguez-Sosa et al. (2006).The objective of this study was to develop an in vitro culture system for spermatogonial growth. Twenty 2- to 3-month-old rams were randomly selected at a local slaughter house for testicular tissue collection. Two-step enzymatic digestion methods were used for spermatogonia isolation from seminiferous tubules. In brief, mechanically isolated seminiferous tubules from testicular tissue were incubated in 1:1 1 mg mL-1 collagenase and hyaluronidase with 5 μg mL-1 DNase I for 20 min at 37°C. Most of the surrounding interstitial cells will fall off from seminiferous tubules by slightly pipetting. Seminiferous tubules were from cell suspensions after natural sedimentation in PBS and then were digested in 0.25% trypsin + 0.04% EDTAfor 5 to 7 min at 37°C to disassociate tubules into single cells. Data were analyzed with ANOVA procedures. Means of 5 specimens were presented. Of the total isolated cells, 19.7 ± 5.3% were identified as PGP9.5+cells, and 23.8 ± 3.6% were identified as c-kit+ cells. C-kit, the transmembrane tyrosine kinase receptor for stem cell factor, has been identified, which is expressed and functional in differentiating A1-A4 spermatogonia but not in spermatogonial stem cells (OhtaH2000). For in vitro culture of spermatogonia, DMEM supplemented with 1X ITS (insulin, transferrin, selenium), 100 μM β-mercaptoethano, 6 mM L-Glutamine, and 1X nonessential amino acids were used as basic culture medium. We have found that in primary spermatogonia culture, cells cultured together with testis somatic cells (sertoli cells) in basic medium supplied with 2.5% fetal bovine serum (FBS) were much more efficient than culturing with a supplement of 10% FBS. In primary culture, growing round-shaped cell colonies were visible from Day 5 in basic culture medium. In subculture, colonies were enzymically digested into smaller pieces from Day 8 to 10 and then placed onto mouse embryonic fibroblasts feeder layer in 2.5% FBS basic medium with the addition of 100 ng mL-1 glial cell-derived neurotropic factor, 10 ng mL-1 leukemia inhibitory factor, and 10 ng mL-1 basic fibroblast growth factor. PGP9.5+ spermatogonia cell colonies maintained their normal round shape until 4 to 5 passages. However, in subsequent passages, the colonies became flattened and cells gradually lost their interconnection and the growth pattern present in the early passages. Also in subsequent passages, cells began expressing more C-kit than PGP9.5. In conclusion, PGP9.5+ spermatogonia were successfully isolated from 2- to 3-month-old ram testis, and PGP 9.5 cell colonies were maintained and proliferated in the in vitro culture system up to 2 months.

391 A COMPARATIVE GENE EXPRESSION ANALYSIS BETWEEN SHEEP EARLY EMBRYONIC DEVELOPMENT AND PUTATIVE EMBRYONIC STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 352
Author(s):  
B. M. Murray ◽  
S. Schmoelzl ◽  
N. M. Andronicos ◽  
J. R. Hill ◽  
P. J. Verma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
In Vitro Culture ◽  
Culture Media ◽  
Embryonic Stem ◽  
Oct4 Expression ◽  
Pluripotency Markers

The optimization of culture media to support the isolation of embryonic stem cells relies on methods to monitor whether the pluripotent state of the cultured cells has been maintained. We developed a panel of gene expression assays that allowed us to correlate molecular measures of pluripotency or lineage differentiation with a developmental time course. By conducting quantitative PCR analysis of sheep embryos over Day 6.5 to 24 and sheep inner cell mass (ICM) cells cultured over 25 days, we tested whether culture media designed to inhibit differentiation are able to maintain sheep ICM cells in a pluripotent state. Briefly, embryos were collected from Merino ewes (n = 50, 3 years) at Day 6.5, 12, 16, 20, and 24 post-AI. Embryos were collected from the dissected uterine tracts of slaughtered ewes, excluding Day 6.5 blastocysts, which were surgically recovered from superovulated ewes. For the in vitro culture, Day 6.5 ICM cells were isolated by immunosurgery and cultured on mitomycin-C-treated mouse embryonic fibroblasts in an inhibitor-based medium (3i, based on Ying Q-L et al. 2008 Nature 453, 519-523). Real-time PCR assays for pluripotency (OCT4, SOX2, NANOG) and differentiation (ectodermal: FGF5, PAX6; endodermal: GATA4, GATA6, Somatostatin; mesodermal: BMP4, Connexin40) of sheep candidate genes were conducted on cDNA prepared from these samples and normalized against the reference genes RPL19 and RPS26. In in vivo embryos, pluripotency markers OCT4, SOX2, and NANOG all decreased between Day 6.5 and Day 20, although OCT4 expression spiked around Day 16. More interestingly, pluripotency expression decreased during in vitro culture, with NANOG expression completely lost by passage 2 at Day 11 and OCT4 expression at an equivalent Day 24 embryo basal level by Day 14. The endodermal markers GATA6 and GATA4 decreased between Day 6.5 and Day 12, respectively, although in vitro GATA4 was only expressed once at Day 7. In vivo FGF5 and both PAX6 and Somatostatin displayed a delayed onset, increasing expression from Day 16 and 20, respectively, whereas the ectodermal markers were already expressed by Day 7 in vitro. Both mesodermal markers Connexin40 and BMP4 presented minor fold changes in both data sets. In conclusion, this study has verified the primer sets and described a sheep in vivo embryo gene expression profile comprising both pluripotent and differentiation candidates. Furthermore, the decrease of pluripotency markers together with the appearance of differentiation markers during in vitro culture of ICM cells suggest that culturing ICM cells in 3i media is not sufficient to maintain a sheep-specific pluripotent population of cells. Therefore, future studies will be aimed at manipulating the current in vitro system to focus on maintaining pluripotent genes such as NANOG and OCT4 in culture.

Generation of Red Blood Cells From Human Embryonic Stem Cells (ES) with Increased Efficacy

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4334-4334
Author(s):  
Anand S Srivastava ◽  
Babak Esmaeli Azad ◽  
Rosalia De Necochea Campion ◽  
Ewa Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Red Blood Cells ◽  
Human Embryonic Stem Cells ◽  
Blood Cells ◽  
Culture System ◽  
Embryonic Stem ◽  
Employment Equity ◽  
Equity Ownership

Abstract Abstract 4334 It is estimated that for every unit of donated blood, two units are required in North America. The current rate of blood donation is stagnant while the need increases by 6–8% annually. In order to overcome this difficulty, we have developed an improved method to generate red blood cells from human embryonic stem cells (H9) with increased efficacy. In addition to xeno-free conditions and standard cytokine cocktail used for hematopoietic differentiation of human embryonic stem cells (Carrier et al. J Transl Med. 2009; Vol 7: 27), we have introduced a new method of improved growth and differentiation of human ES cells with hypoxia-induced mesenchymal stem cells, obtained from allogeneic adult bone marrow donors. This technique increased efficacy of red blood cell production by 5–25 fold. We have developed a bioscaffold–> microsphere-based culture system with highly porous surface allowing culturing of a very large number of embryonic stem cells per one culture condition. This culture system avoids shear forces and damage to the cells, and facilitates removal and recycling of the microspheres. The in vitro obtained human ES-derived red blood cells are enucleated and do not produce tumors (efficacy of enucleation is 65–95%). The laser-based system is utilized to eliminate nucleated cells from the culture. The problem with hES-derived red blood cells is that they are produced in small numbers and process is very costly. We are developing a 3-phase bioreactor with computerized programming, which will increase every step of the differentiation process and allow recycling of feeder cells and cytokines. In this system we will utilize iron-loaded microspheres coated with hypoxia-processed mesenchymal stem cells as a main culture unit. The in vitro generated human ES-derived red blood cells upscaled in a bioreactor will be used for the off-shelf production of red blood cells for clinical use. Disclosures: Srivastava: Giostar: Employment, Equity Ownership. Azad:Dnamicroarray, Inc.: Employment, Equity Ownership. Carrier:Giostar: Consultancy; Samaritan Pharmaceuticals: Consultancy; Entest Biomedical: Consultancy, Equity Ownership; America Stem Cells: Consultancy, Equity Ownership; Millenium: Speakers Bureau; NovaRx: Employment, Equity Ownership.

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