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In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor’s Production

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Embryonic Stem Cell Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1341))

Abstract

Embryonic stem cells (ESCs) differentiation via embryoid body (EB) formation is an established method that generates the three germ layers. However, EB differentiation poses several problems including formation of heterogeneous cell populations. Herein, we described a differentiation protocol on enhancing mesoderm derivation from murine ESCs (mESCs) using conditioned medium (CM) from HepG2 cells. We used this technique to direct hematopoiesis by generating “embryoid-like” colonies (ELCs) from murine (m) ESCs without following standard formation of EBs. Our CM-mESCs group yielded an almost fivefold increase in ELC formation (p ≤ 0.05) and higher expression of mesoderm genes;-Brachyury-T, Goosecoid, and Flk-1 compared with control mESCs group. Hematopoietic colony formation from CM-mESCs was also enhanced by twofold at days 7 and 14 with earlier colony commitment compared to control mESCs (p ≤ 0.05). This early clonogenic capacity was confirmed morphologically by the presence of nucleated erythrocytes and macrophages as early as day 7 in culture using standard 14-day colony-forming assay. Early expression of hematopoietic primitive (ζ-globin) and definitive (β-globin) erythroid genes and proteins was also observed by day 7 in the CM-treated culture. These data indicate that hematopoietic cells more quickly differentiate from CM-treated, compared with those using standard EB approaches, and provide an efficient bioprocess platform for erythroid-specific differentiation of ESCs.

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Notes

  1. 1.

    *Keller et al. 1993 has reported that ESCs efficiently undergo differentiation in vitro to mesoderm and hematopoietic cells that this in vitro system recapitulates day 6.5 to 7.5 of mouse hematopoietic development. Embryonic stem cells differentiated as embryoid bodies (EBs) develop erythroid precursors by day 4 of differentiations, and by day 6, more than 85% of EBs contain such cells. The number of the EPO responsive precursors increased by day 8 of differentiation, them remained constant, and finally began to decline by day by day 12. Therefore, we hypothesized that day 5-8 are the period of mesoderm differentation in EB to recapitulate embryonic development based on the previous study reported.

    Reference ammended: 3(a). Irion S, Clarke RL, Luche H, Kim I, Morrison SJ, Fehling HJ, Keller GM (2010) Temporal specification of blood progenitors from mouse embryonic stem cells and induced pluripotent stem cells. Development 137(17):2829–2839. doi:10.1242/dev.0421193 (b). Keller GM, Kennedy M, Papayannopoulou T, Wiles MV (1993) Hemataopoietic commitment during embryonic stem cell differentiation in culture. Mol and Cell Biol: 473–486.

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Authors and Affiliations

  1. Biological Systems Engineering Laboratory, Department of Chemical Engineering and Chemical Technology, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK

    Iliana Fauzi, Nicki Panoskaltsis & Athanasios Mantalaris

  2. Department of Hematology, Imperial College London, Northwick Park & St. Mark’s campus, London, HA1 3UJ, UK

    Nicki Panoskaltsis

Authors
  1. Iliana Fauzi
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  2. Nicki Panoskaltsis
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  3. Athanasios Mantalaris
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Correspondence to Athanasios Mantalaris .

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Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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Fauzi, I., Panoskaltsis, N., Mantalaris, A. (2015). In Vitro Differentiation of Embryonic Stem Cells into Hematopoietic Lineage: Towards Erythroid Progenitor’s Production. In: Turksen, K. (eds) Embryonic Stem Cell Protocols. Methods in Molecular Biology, vol 1341. Humana Press, New York, NY. https://doi.org/10.1007/7651_2015_218

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  • DOI: https://doi.org/10.1007/7651_2015_218

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2953-5

  • Online ISBN: 978-1-4939-2954-2

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