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

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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The in vitro production and characterization of neutrophils from embryonic stem cells

Blood ◽

10.1182/blood-2003-04-1030 ◽

2004 ◽

Vol 103 (3) ◽

pp. 852-859 ◽

Cited By ~ 58

Author(s):

Jonathan G. Lieber ◽

Saiphone Webb ◽

Benjamin T. Suratt ◽

Scott K. Young ◽

Gary L. Johnson ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Es Cells ◽

Specific Antigen ◽

Embryonic Stem ◽

Specific Marker ◽

Mouse Bone Marrow ◽

Functional Evaluation ◽

In Vitro Production

AbstractAn embryonic stem (ES) cell/OP9 coculture system for the effective production of functional neutrophils is described. A 3-step differentiation strategy was developed that uses liquid culture, enabling reliable and abundant production of neutrophils at high purity without the need of sorting for isolation of mature neutrophils. Use of the OP9 stromal cell line significantly enhances the number, percentage, and duration of differentiated neutrophils produced from embryonic stem cells. Effective and sustained differentiation of ES cells into neutrophils provides a useful model system for studying neutrophil differentiation and function and the factors that regulate them. Morphologic and functional evaluation of these ES-derived neutrophils indicates that large numbers of mature neutrophils can be produced from pluripotent ES cells in vitro. Specifically, their morphology, ability to produce superoxides, flux calcium, undergo chemotaxis in response to macrophage inflammatory protein 2 (MIP-2), stain for the granulocyte-specific marker–specific chloroacetate esterase, and contain the neutrophil-specific markers Gr-1 and the mouse neutrophil-specific antigen indicates that they are comparable with purified mouse bone marrow neutrophils. They also express gelatinase and lactoferrin granule proteins. During the differentiation of these ES-derived neutrophils, regional areas of neutrophil production can be identified that have been designated as neutrophil generating regions (NGRs).

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Establishment of a feeder and serum-free culture system for human embryonic stem cells

Zygote ◽

10.1017/s0967199419000625 ◽

2020 ◽

Vol 28 (3) ◽

pp. 175-182

Author(s):

LiYun Wang ◽

RuiNa Zhang ◽

RongHua Ma ◽

GongXue Jia ◽

ShengYan Jian ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Culture System ◽

Embryonic Stem ◽

Hesc Line ◽

Self Renewal ◽

Serum Free ◽

Genes Encoding

SummaryStem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

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Magnetic Field-Magnetic Nanoparticle Culture System Used to Grow In Vitro Murine Embryonic Stem Cells

Journal of Nanoscience and Nanotechnology ◽

10.1166/jnn.2011.3089 ◽

2011 ◽

Vol 11 (1) ◽

pp. 36-44 ◽

Cited By ~ 3

Author(s):

Erika Regina Leal de Freitas ◽

Paula Roberta Otaviano Soares ◽

Rachel de Paula Santos ◽

Regiane Lopes dos Santos ◽

Elaine Paulucio Porfírio ◽

...

Keyword(s):

Magnetic Field ◽

Stem Cells ◽

Embryonic Stem Cells ◽

Culture System ◽

Magnetic Nanoparticle ◽

Embryonic Stem ◽

Murine Embryonic Stem Cells

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Differentiation of embryonic stem cells into a putative hair cell-progenitor cells via co-culture with HEI-OC1 cells

Scientific Reports ◽

10.1038/s41598-021-93049-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Nathaniel T. Carpena ◽

So-Young Chang ◽

Celine D. G. Abueva ◽

Jae Yun Jung ◽

Min Young Lee

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Hair Cell ◽

Culture System ◽

Embryonic Stem ◽

Organ Of Corti ◽

Cellular Interactions ◽

Mouse Escs ◽

Auditory Research

AbstractSeveral studies have shown how different cell lines can influence the differentiation of stem cells through co-culture systems. The House Ear Institute-Organ of Corti 1 (HEI-OC1) is considered an important cell line for in vitro auditory research. However, it is unknown if HEI-OC1 cells can promote the differentiation of embryonic stem cells (ESCs). In this study, we investigated whether co-culture of ESCs with HEI-OC1 cells promotes differentiation. To this end, we developed a co-culture system of mouse ESCs with HEI-OC1 cells. Dissociated or embryonic bodies (EBs) of ESCs were introduced to a conditioned and inactivated confluent layer of HEI-OC1 cells for 14 days. The dissociated ESCs coalesced into an EB-like form that was smaller than the co-cultured EBs. Contact co-culture generated cells expressing several otic progenitor markers as well as hair cell specific markers. ESCs and EBs were also cultured in non-contact setup but using conditioned medium from HEI-OC1 cells, indicating that soluble factors alone could have a similar effect. The ESCs did not form into aggregates but were still Myo7a-positive, while the EBs degenerated. However, in the fully differentiated EBs, evidence to prove mature differentiation of inner ear hair cell was still rudimentary. Nevertheless, these results suggest that cellular interactions between ESCs and HEI-OC1 cells may both stimulate ESC differentiation.

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