Production of erythriod cells from human embryonic stem cells by fetal liver cell extract treatment
© Liu et al; licensee BioMed Central Ltd. 2010
Received: 1 January 2010
Accepted: 10 August 2010
Published: 10 August 2010
We recently developed a new method to induce human stem cells (hESCs) differentiation into hematopoietic progenitors by cell extract treatment. Here, we report an efficient strategy to generate erythroid progenitors from hESCs using cell extract from human fetal liver tissue (hFLT) with cytokines. Human embryoid bodies (hEBs) obtained of human H1 hESCs were treated with cell extract from hFLT and co-cultured with human fetal liver stromal cells (hFLSCs) feeder to induce hematopoietic cells. After the 11 days of treatment, hEBs were isolated and transplanted into liquid medium with hematopoietic cytokines for erythroid differentiation. Characteristics of the erythroid cells were analyzed by flow cytometry, Wright-Giemsa staining, real-time RT-PCR and related functional assays.
The erythroid cells produced from hEBs could differentiate into enucleated cells and expressed globins in a time-dependent manner. They expressed not only embryonic globins but also the adult-globin with the maturation of the erythroid cells. In addition, our data showed that the hEBs-derived erythroid cells were able to act as oxygen carriers, indicating that hESCs could generate functional mature erythroid cells.
Cell extract exposure with the addition of cytokines resulted in robust erythroid -like differentiation of hEBs and these hEBs-derived erythroid cells possessed functions similar to mature red blood cells.
Red blood cells (RBCs) have been utilized as the treatment for severe blood loss and hematopoiesis study; but their clinic application has been constrained by limited quantities and compatibility issues. The availability of hESCs offers a great opportunity to produce large quantities of erythroid cells in vitro for transfusion, and to provide additional knowledge to the field of erythropoiesis. Previous studies have generated primitive erythroid cells from hESCs by embryoid body formation and stromal cell co-culturing [1–7]. However, the risk of mouse-related diseases and the low differentiation efficiency of hESCs are major limitations of the clinical application of this study.
Recently, we have established a method to produce relatively large number of human hematopoietic cells from hESCs, via a human-derived induction system, by using hFLSCs feeder cells and cell extract of hFLT. Use of this culture method enabled the production of 32.73% CD34+ from treated hEBs after 11 days of culture. More importantly, hEBs-induced hematopoietic cells predominantly yielded erythroid precursors when seeded on methylcellulose . Based on the above results, we isolated the 11- day hEBs from the co-culture system and transplanted them into liquid medium for a 16-day extending culture. During the 16-day culture, cytokines are used to first promote the proliferation and subsequently used for the maturation of erythroid precursors. This culture method enabled the production of about 5 × 106 fully differentiated erythroid cells from about 5 × 104 hEBs. The erythroid cells morphologically resembled fetal liver-derived erythroblasts, they mainly expressed embryonic hemoglobin and could be enucleated. Our results show that induction of hESCs into mature erythroid cells in vitro is possible by treatment with cytokine-supplemented cell extract.
The effects of hFLT cell extract treatment on hEBs
The capacity of erythroid-like development of hEBs
In a previous study, we found that cell extract treatment could influence differentiation of hEBs but only hFLT cell extract treatment could improve hematopoietic differentiation of hEBs . This experiment provided an opportunity to conduct a large-scale investigation of hESCs-derived erythropoiesis after hFLT cell extract treatment. Firstly, we treated hEBs with hFLT cell extract as described previously . Then the treated hEBs were co-cultured on the hFLSCs feeder in hEBs differentiation medium for the hematopoietic differentiation, and the untreated hEBs were culture in the same condition as a control.
The effects of hFLT cell extract treatment on gene expression
Production of red blood cells from hEBs
To monitor the differentiation of hEB cells into erythroid cells, CD71 (transferrin receptor) and glycophorin A (GLA) antigens were examined by flow cytometry assay throughout the culture. At the beginning of the liquid culture, CD71+ cells were detected at a low lever and GLA+ cells could not be detected at all (data not shown). CD71+ cells increased rapidly from day 1 to day 8, and reached 89.63% of their peak level at day 8 and subsequently decreased. About 34% of the cells were CD71+cells at the day 16. In contrast, however, GLA+ cells expanded significantly from day 8 to day 16. GLA+ cells could not reach 30% at the day 8, but peaked 51.82% at the end of culture (Figure 4B, C)
Analysis of hEBs-derived erythroid cells
To further observe the expression pattern of globins, we analyzed hEBs-derived erythroid cells at different days by western blotting. We found ζ, ε, α and γ globins proteins were detected by day 4 and β-globin protein could be found at day 16 (Figure 6B), which confirmed that erythroid cells generated from hEBs possessed the capacity to express the adult globin at the end of culture. These data indicated that globins expression of erythroid cells generated from hEBs might begin to switch from the embryonic to the adult type at day 16.
Functional analysis of hEBs-derived erythroid cells
To assess the function of the hEBs-derived erythroid cells, we measured oxygen dissociation of hEBs-derived erythroid cells and compared them with human cord blood (hCB) and human adult peripheral blood. As shown in Figure 6C, hEBs -derived erythroid cells displayed an oxygen dissociation curve similar to that of hCB, but shifted to the left as compared to the curve of human adult peripheral blood.
In this study, we first established a system to induce the production of a large number of erythroid cells from hESCs utilizing four standard procedures. In the first and second steps, the hEBs were treated with the cell extract from 14-weeks fetal liver and co-cultured with hFLSCs feeders. Cell extract treatment is a novel trans-differentiation strategy that can convert a somatic cell type into another type [11–15]. We recently reported a new method to promote the differentiation of human stem cells toward hematopoietic lineages by the treatment with cell extract of hFLT . The fetal liver is a very important organ of human hematopoiesis, and can generate not only transplantable hematopoietic stem cells, but also enucleated RBCs. We therefore speculated that hFLT cell extract treatment may be advantageous for erythroid line development as well. In the current study, we isolated hEBs from co-cultures after treatment with hFLT cell extract and examined their capacity for the erythroid development at different period of time. At same time the untreated hEBs were isolated from co-cultures as a control. We found that the treated hEBs in the co-culture system could express the erythroid associated genes and give rise to erythroid lineage colonies in semisolid medium, whereas the untreated hEBs did not express erythroid associated genes and mainly yielded CFU-GM with the same experimental conditions. Thus, our data provided strong evidence which shows that treated hEBs-derived hematopoiesis mainly generated erythroid cells.
Furthermore, we isolated the treated hEBs from co-cultures and induced them into erythroid cells in two -phases. At the beginning of the culture, addition of BMP4, Flt-3l, SCF, and EPO resulted in a rapid increase in cell numbers and an accumulation of differentiated cells. CD71 was used as a marker for early erythroid cells and GLA used as a marker for mature erythroid cells. During the first stage of liquid culture, we observed an obvious increase of CD71+ cells and a slight increase of GLA+ cells, which implied the generation and expansion of early erythroid cells from hEBs. From day 9 to day 16, stimulated by IGF-1, SCF and a high dose of EPO, most of the cells had characteristics of mature erythroid cells. During this stage we observed a peak in the percentage of GLA+ cells, but the percentage of CD71+ cells decreased at the end of culture.
To further understand the erythropoiesis of hESCs, we examined globins expression of erythroid cells from hEBs. According to our previous study, erythroid cells derived from hEBs only expressed embryonic globins but not adult globin . However, in this study we found that hEBs-derived erythroid cells expressed both the embryonic and adult globins at the end of culture. One possible explanation for this result relates to the complex process of mammalian erythropoiesis. In the primitive hematopoiesis wave, blood islands in the yolk sac transiently generate nucleated RBCs. In the later stage of development, the fetal liver is the primary site for production of transplantable hematopoietic stem cells and enucleated RBCs. Definitive hematopoiesis ultimately shifts to the bone marrow, the site for production of life-long adult-type hematopoiesis. Erythrocytes from different hematopoietic sites expressed different types of globins. Yolk sac-derived erythrocytes express only embryonic globins, and erythroblasts produced in fetal liver express embryonic globins and a small amount of adult globin. Erythroblasts from bone marrow primarily expressed adult globin. In our inducing system, hEBs were treated with hFLT cell extract and co-cultured with hFLSCs. It is possible that this condition mimics the environment of the fetal liver and results in similar globin expression of the hEBs-derived erythroid cells as with fetal liver-derived erythroid cells.
A critical issue for clinical application of hESCs is whether they can generate functionally mature progenies. In our system, enucleated RBCs could first be observed at day 8 of liquid culture. The number of enucleated RBCs increased rapidly from day 12 to 16. The erythroid cells produced from treated hEBs morphologically resembled RBCs from human fetal liver. Capability of carrying oxygen is an important function of RBCs. To determine if hEBs-derived erythroid cells possessed the same function as normal RBCs, we analyzed the function of hEBs-derived erythroid cells and found that the oxygen dissociation curve of hEBs-derived erythroid cells shifted to left compared to the curve of human adult blood. To our knowledge, the oxygen dissociation curve of hCB was shifted left when compared to that of human adult peripheral blood . Our results implied that hEBs-derived erythoid cells were also able to function as oxygen carriers and the oxygen dissociation pattern of these cells was more similar to fetal blood cells than adult blood cells. Thus RBCs derived from hEBs in our system may act as an alternative resource of RBCs from blood.
Hematopoietic differentiation of hESCs has been achieved by using a variety of experimental approaches[1, 2]. In addition, the successful derivation of RBCs from hCB has been achieved by many published assays[3, 4]. However, there are only a few published reports on erythroid differentiation from hESCs. Trans-differentiation of a somatic cell type into another cell type would be beneficial for producing replacement cells for potential therapeutic applications. In this study, we first reported that fetal liver cell extract-based treatment induced the differentiation of hESCs into RBCs safely and efficiently. Our method provides a useful tool for studying the molecular mechanisms of hematopoietic development, and will be valuable in the production of RBCs for transfusion.
hESC H1 cells were obtained from WiCell Research Institute, Inc. (Madison, WI, USA). To prevent cells from differentiating, cells were co-cultured with irradiated (20 cGy) mouse embryonic fibroblast (MEF) cells and maintained in knock-out Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% serum replacer (SR), 1% nonessential amino acids (NEAA), 1 mM L-glutamine (all from Invitrogen Corporation, Carlsbad, CA, USA), 0.1 mM β-mercaptoethanol (Sigma-Aldrich, USA) and 4 ng/ml human basic fibroblast growth factor (bFGF) (R&D Systems, USA).
The hFLSCs were isolated and cultured as previously described .
Preparation of cell extract
The hFLT cell extract was prepared, as described previously [18, 19], from 15-week human aborted fetal liver tissue obtained with informed consent. Briefly, the hFLT cell extract was prepared as follows: cells were washed in phosphate-buffered saline (PBS) and resuspended in cell lysis buffer. Cells were lysed with a dropping pipette and centrifuged at 15,000 × g for 15 minutes at 4°C. The supernatant was removed, filtered and stored at -80°C.
Inducing erythroid differentiation of hEBs
Step 1. Cell Extract treatment of hEBs
Undifferentiated hESCs were harvested at 80% confluence after collagenase IV digestion and transferred to 10-cm low cell-binding dishes to form hEBs in hEBs medium containing KO-DMEM supplemented with 20% FBS, 1% NEAA, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. The hEBs were then treated with hFLT extract. Briefly, the 2-day hEBs were treated with SLO in Ca2+-Mg2+-free Hanks' balanced salt solution (Gibco-BRL, Invitrogen) for 50 minutes at 37°C. About 100 μl of hFLT cell extract containing an XX ul or % ATP-generating system (1 mM ATP, 1 mM GTP, 1 mM NTP, 10 mM phosphocreatine, and 25 μg/ml creatine kinase, Sigma) were added to replace the SLO and incubated for 60 minutes at 37°C. To reseal plasma membranes CaCl2 (2 mM) was added to the hESCs culture medium, and cells were cultured overnight at 37°C.
Step 2. hFLSCs feeder co-culture
The treated hEBs were cultured with hFLSCs feeder in hEBs differentiation medium for 11 days containing 80% IMDM (Invitrogen), 30% FBS (Invitrogen), 1% NEAA, 2 mM L-glutamine, 0.1 mM beta-mercaptoethanol. After some days of culture, cells were collected and analyzed to identify erythroid differentiation. Total RNA was isolated from hEBs at different days of co-culture using the following the manufacturer's protocol. We analyzed mRNA expression for markers of erythroid cells by RT-PCR. In addition, the EB cells at different days of co-culture were collected and seeded in MethoCult GF -H4434 semisolid medium (Stem Cell Technologies, USA) to determine their capacity for erythroid development.
Step 3. Erythroid differentiation of hEBs in liquid medium with cytokines
The hEBs that possessed the greatest capacity for erythroid development were dissociated into single-cell suspensions by collagenase IV treatment. Cells were induced into erythroid cells by two phases: from day 1 to day 8, cells were cultured in medium composed of IMDM supplemented with 30% FBS, BMP4 (20 ng/mL), SCF (100 ng/mL), EPO (2 U/mL), and Flt-3l(20 ng/mL); from day 9 to day 16, cells were cultured in medium composed of IMDM supplemented with 30%FBS, IGF-1 (20 ng/mL), SCF (100 ng/mL), EPO (4 U/mL). About 1 to 2 mL fresh medium was added to each plate every 2 to 3 days,.
Primer sequences for RT-PCR
Primer sequence (sense)
Primer sequence (antisense)
TCTGGCTACAA G AGGAGAAG
TGACA ACCCCAGGTCTTA GG
Clonogenic progenitor cell assay
Hematopoietic clonogenic assays were performed in 35-mm low adherent plastic dishes using MethoCult GF -H4434 semisolid medium (Stem Cell Technologies, USA) consisting of 1% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 50 ng/mL stem cell factor, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL IL-3, 20 ng/mL IL-6, 20 ng/mL granulocytecolony-stimulating factor (G-SF), and 3 units/mL erythropoietin(EPO). After culturing for 12-14 days, colonies were scored according to their cellular morphology.
Primer sequences for Real-time PCR
Primer sequence (sense)
Primer sequence (anti-sense)
The cell extract was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA). After blocking in TBST containing 5% dry-fat skim milk, the membrane was incubated with anti-globin antibodies (Santa Cruz Biotechnology), followed by HRP-labeled goat anti rabbit IgG. The protein bands were visualized by enhanced chemiluminescence (ECL; Pierce).
Flow cytometric analysis
The trypsinized individual cells were incubated with the following FITC-conjugated and PE-conjugated monoclonal antibodies: anti-human CD71, anti-human glycophorin A (B&D Biosciences, USA) for 30 minutes at 4°C. Cells were washed with PBS for three times, and analyzed by flow cytometeric analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA, USA).
Cells were dropped onto slides and fixed for 20 minutes in 4% paraformaldehyde and were stained with Wright-Giemsa reagents (Fisher Scientific) following manufacturer's instructions.
Functional Assays for Erythroid Cells
Results were expressed as means ± SEM. Statistical significance was determined using Student t- test. Results were considered significant at P < .05.
This work was supported by National High Technology Research and Development Program of China (No:2006AA02A107), the Major State Basic Research Program of China (No:2005CB522702), and The Project of Beijing Municipal Science & Technology Commission (No: Z0005190043331).
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