CD34 timecourse

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Human H1 embyronic stem cells differentiated to CD34+ hematopoietic stem cells

Time course ID: human_H1
Sample provider:  Lesley Forrester and Richard Axton

Introduction

Embryonic stem cells (ESCs) are pluripotent, so they can differentiate into any cell type in the adult body. The direction of differentiation is determined by the combination of growth factors and cytokines to which the cells are exposed and this can be manipulated in experimentally to drive differentiation towards a specific cell type.

The ability to replace red blood cell loss using blood transfusion has been a major advance in reducing mortality due to accident or surgical procedures. However, blood for transfusion is always in short supply and techniques that would lead to a renewable source of red blood cells are urgently needed to alleviate this problem[1, 2].

In the body, all the different cell types of the blood (eg red blood cells, white blood cells and platelets) arise from multi-potent haematopoietic stem cell that are present in the adult bone marrow. During embryonic development these haematopoietic stem cells arise from the mesoderm germ layer via a complex interplay of transcription factors, cytokines and other regulators and our embryonic stem cell differentiation protocol is designed to recapitulate this process in culture.


Samples

The protocol used in these experiments was designed to generate red blood cells from human embryonic and induced pluripotent stem cells (Figure 1).

CD34 Fig1.png

Figure 1: Differentiation protocol for the production of red blood cells from human ESC and iPSCs.

The first step in the protocol involves the production of 3-dimensional embryoid bodies (EBs) either by culturing single cell suspensions in hanging drops or by culturing in mechanically cut squares in low adherent plates. EBs are dissociated after 3 days and cells are cultured in suspension for a further 6/7 days in conditions designed to generate haematopoietic progenitor cells (HPC) that can be monitored by the expression of CD34 and CD43 antigens on their cell surface and by the production of colony forming units in methylcellulose (Stem Cell Technology). The yield of CD34+/CD43+ cells varies between experiments but can be in the region of 60% of the total cell population. Subsequent culturing of these cells in appropriate cytokine cocktails results in the production of >90% erythroblasts by day 17 with the production of mature erythrocytes by 24 days.

Culture procedure used in Fantom analysis

In the experiments used for CAGE analysis, H1 hESCs were maintained on CellStart and Stem Pro (Invitrogen) plus bFGF and confluent cultures were passaged using EZYtool (Invitrogen). ESCs were dissociated to single cells and embryoid body (EB) formation was initiated in hanging drops (600 cells in 10μl) in Stemline II media in the presence of mesoderm inducing cytokines (including BMP4, Wnt3a, ActivinA) for 3 days. EBs were then disaggregated and cultured in suspension in the presence of haematopoietic cytokines (including VEGF, SCF, IGF2, TPO) for a further 7 days when cells were analysed by flow cytometry to assess the yield of cells showing CD34 and CD43 expression. This procedure is currently the subject of a patent application [3] which contains further details.

RNA preparation

RNA was harvested from three independent cultures of H1 cells (Day 0) Three independent differentiation experiments were set up and RNA was isolated from cells at day 3 and then at day 9. RNA was isolated using the RNA Bee protocol (Ambisco). Sample RNA was quantified using a Nanodrop spectrophotometer (Nanodrop, USA).

Quality control

Cells were analysed for expression of CD34 and CD43 at day 9 by flow cytometry.

Experiment  % CD34  %CD43  %CD34/43
1 51 27 17
2 53 44 27
3 53 40 26

Marker gene expression

Based on the extensive gene profiling of differentiating mouse ESCs[4, 5] a number of genes are predicted to increase or decrease during the differentiation protocol.

  • Pluripotency genes (eg Oct4, Nanog): expected to decrease during time course:
  • Mesoderm markers (eg Bry/T, Mixl1, Eomes): expected to increase at day 3:
  • Haematopoietic genes (eg CD34, CD43, Runx1 Scl/Tal, Lmo2): expected to be expressed at day 9/10

We noted that Oct4, Nanog and other pluripotency genes were downregulated in a comparable way to the other ESC time courses.  However we did not observe the expected rise in mesoderm marker genes. In particular the expression of CD34 and CD43 promoters was not detected, although there was significant expression of these proteins at day 9. It is possible that the timing of of the samples missed the peak of mesoderm specification.

We looked at the Mara data to see which genes were upregulated in our time course. The most interesting promoters and associated transcription factors from that analysis are:

  • TFEB, GTF-2I, Gata1, ZNF384, Evi1, Ebf1.

These are related to haematopoiesis and would be expected to increase as differentiation proceeds.

References

[1] Mountford, J., E. Olivier, and M. Turner, Prospects for the manufacture of red cells for transfusion. Br J Haematol, 2010. 149(1): p. 22-34.

[2] Mountford, J.C., et al., Red blood cells from pluripotent stem cells for use in transfusion. Regen Med, 2010. 5(3): p. 411-23.

[3] Mountford, J.C. and Olivier, E.N. Patent application PCT/GB2013/051917 Erythroid production. Initial filing 20 July 2013. Published 23 Jan 2014 as WO2014013255 A1. The Common Services Agency and the University of Glasgow.

[4] Schulz, H., et al., The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation. PLoS One, 2009. 4(9): p. e6804.

[5] Hescheler, J., et al., The FunGenES consortium: functional genomics in engineered embryonic stem cells. Stem Cell Rev, 2006. 2(1): p. 1-4.

[6] Patent application PCT/GB2013/051917 ‘Erythroid Production’ – Initial filing 20th July 2012, Published 23rd Jan 2014 as WO2014013255 A1. Mountford JC and Olivier EN. The Common Services Agency & the University of Glasgow.






















































































Beginning of non-public section

These cells were provided by collaborators Lesley Forrester and Richard Axton of the University of Edinburgh. The time course is Day 0, Day 3 and Day 9 of differentiation from embryoid bodies.

Details of the differentiation procedure to follow.

Zenbu configurations and status

Expression profiles

MARA based network results


Related samples

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References

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Quality control

https://fantom5-collaboration.gsc.riken.jp/webdav/home/arner/timecourse/time_course_main_paper_freeze_feb2013/qc_release_130226/human_H1/

ISMARA analysis results

All samples: http://ismara.unibas.ch/timecourses/ESC2HSC/ismara_report/index.html

Replicate averaged: http://ismara.unibas.ch/timecourses/CD34plus-avgd/averaged_report/index.html

For more information, see ISMARA.

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