Expression of the 49 human ATP binding cassette (ABC) genes in pluripotent embryonic stem cells and in early- and late-stage multipotent mesenchymal stem cells

Abstract

The 49-member human ATP binding cassette (ABC) gene family encodes 44 membrane transporters for lipids, ions, peptides or xenobiotics, four translation factors without transport activity, as they lack transmembrane domains, and one pseudogene. To understand the roles of ABC genes in pluripotency and multipotency, we performed a sensitive qRT-PCR analysis of their expression in embryonic stem cells (hESCs), bone marrow-derived mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We confirm that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs. We observed that 44 ABCs were significantly expressed in hESCs, 37 in hES-MSCs and 35 in hMSCs. These variations are mainly due to plasma membrane transporters with low but significant gene expression: 18 are expressed in hESCs compared with 16 in hES-MSCs and 8 in hMSCs, suggesting important roles in pluripotency. Several of these ABCs shared similar substrates but differ regarding gene regulation. ABCA13 and ABCB4, similarly to ABCB1, could be new markers to select primitive hMSCs with specific plasma membrane transporter^low phenotypes. ABC proteins performing basal intracellular functions, including translation factors and mitochondrial heme transporters, showed the highest constant gene expression among the three populations. Peptide transporters in the endoplasmic reticulum, Golgi and lysosome were well expressed in hESCs and slightly upregulated in hMSCs, which play important roles during the development of stem cell niches in bone marrow or meningeal tissue. These results will be useful to study specific cell cycle regulation of pluripotent stem cells or ABC dysregulation in complex pathologies, such as cancers or neurological disorders.

Keywords: :

• embryonic stem cells
• gene expression
• human ATP binding cassette
• mesenchymal stem cells
• pluripotency
• stem cell niche

Introduction

There are 49 ATP binding cassette (ABC) genes in humans; these are part of a very large super-family represented in all living cells. Most ABC genes encode transmembrane transporters composed of two nucleotide binding domains (NBDs) and two transmembrane domains (TMDs), both of which are necessary for their activity.^1,2 ABCD, ABCG and some ABCB genes encode half-transporters composed of only one TMD and one NBD; they must homo- or hetero-dimerize to be biologically functional. ABC functions include the active uptake or efflux of important lipids, peptides, ions or xenobiotics across the plasma membrane (PM) or intracellular membranes of the endoplasmic reticulum, Golgi, peroxisome, mitochondria and lysosome.^2-5 By presenting specific lipids on the outer plasma membrane, some ABCs, especially of the ABCA subfamily, are also implicated in signaling apoptosis.^6,7 Interestingly, ABCE and ABCF subfamily proteins are truncated and lack a TMD; therefore, they have no transport function but play important roles in mRNA translation.^8-10 Functional connections exist between ABC expression and the pluripotency of embryonic stem cells, multipotency in early adult stem cells and cancer stem cells. Indeed, the expression of some ABC genes is under the control of pluripotency stem cell transcription factors, such as Oct-4 ^11,12 and SALL4.¹³ ABCB1 and ABCG2, which promote multidrug resistance (MDR) activity, are markers for a “side population” (SP) of pluripotent, multipotent or cancerous stem cells.^14-16 We and others previously demonstrated the enrichment by cell sorting of multipotent adult stem cells, represented by a growth factor receptor^low subpopulation.^17-22 We demonstrated that receptor^low stem cells respond to low cytokine concentrations for long-term self-renewal.^22,23 Consequently, the differential expression of ABC genes could provide further possibilities for this approach to be utilized to enrich and analyze normal primitive or cancerous stem cells.

To investigate mechanisms underlying determination in stem cells, we previously developed and characterized an experimental system involving three cell types: pluripotent embryonic stem cells (hESCs), multipotent mesenchymal stem cells (hMSCs) and hESC-derived hMSCs (hES-MSCs). We and others selected hES-MSCs and demonstrated that they represent a more primitive population than hMSCs.^24-28 hMSCs play a major role in constituting and controlling various stem cell niches in bone marrow²⁹ or the meningeal barrier;³⁰ in this context, it is important to note that ABC transporters control the trafficking of peptides, including pleiotropic cytokines such as interleukin-1β, that could affect stem cell self-renewal, determination or apoptosis.^31,32

In this study, we used a highly sensitive qRT-PCR technique to measure and compare the expressions of all 49 human ABC genes in hESCs, hES-MSCs and hMSCs. We distinguish ABC genes displaying consistent high expression in the three populations, and those showing significant expression differences, and which may play roles during the transition from hESCs through hES-MSCs to hMSCs. These observations will be useful to better study the increasing number of pathologies in which ABC genes are mutated or their functions dysregulated.^33-35

Results

Expression levels of all 49 ABC genes in hESCs, hES-MSCs and hMSCs were determined by qRT-PCR analysis. A visual overview of these data are provided in a heat-map (Fig. 1A), which presents ABCs according to gene expression levels, together with known substrates, functions, subcellular localizations and associated diseases. Figure 1B summarizes the genes with high or medium (NCt < 29), low but significant (29 ≤ NCt < 35) and undetected (NCt ≥ 35) expression in each cell population. We observed that hESCs expressed a larger number (n = 44) of ABC genes compared with hES-MSCs (n = 37) and late hMSCs (n = 35). This suggests that these genes may have roles in maintaining pluripotency or in early hMSC development. This also confirms our previous results that hES-MSCs represent an intermediate developmental stage between hESCs and hMSCs.²⁴ Due to the high sensitivity of the qRT-PCR technique, we can observe in Figure 1B that the genes encoding ABC plasma membrane transporters are clearly better represented in hESCs (n = 20) than in the late-hMSC population (n = 8). This is also consistent with the fact that various receptor^low subpopulations have been widely studied to select more primitive stem cells.^17-22

Figure 1. Heat maps showing the expression levels of the 49 human ABC genes in hESCs, hES-MSCs and hMSCs. (A) Expression of all 49 human ABC genes in 14 samples of three stem cell types (hESCs, hES-MSCs and hMSCs). The 49 ABC genes are shown on the x-axis, ordered from high to low expression in each sub-family (ABCA to ABCG), according to the mean NCt values from the six hESC samples. The 14 samples are ordered on the y-axis with biological replicates (“a” and “b”). The color code ranges from high (NCt = 20, light green) through medium (black), to low but significant (NCt = 34.9, red) or undetectable (NCt ≥ 35, orange) gene expression. For each ABC, the structure, subcellular localization, function and substrates or association with human diseases^3-5,12,33,34 are represented by symbols, as detailed in the figure. (B) Overviews of mean ABC gene expression in the three stem cell types. ABC genes are ordered from high to low expression levels according to mean NCt values. Gene expression levels are sub-divided into categories as follows: high or medium (NCt < 29), low but significant (29 ≤ NCt < 35), and undetectable (NCt ≥ 35). We can observe that the ABCs with high gene expression are those representing intracellular basal functions. Virtually all ABCs with low but significant expression are plasma membrane transporters whose expression levels vary between the three stem cell populations. Seven of these genes (marked with asterisks) are expressed exclusively in pluripotent or primitive multipotent stem cells.

Figure 1B and radial plots in Figure 2 show that ABC gene expression in hESCs covered a wide range: 24 ABC genes were well expressed (NCt < 29); 20 had a low but significant expression (29 ≤ NCt < 35), and only five were not expressed at detectable levels (NCt ≥ 35). A quantitative and statistical comparison of the expression of all ABC genes in hESCs compared with hES-MSCs and hMSCs allowed the identification of genes without (Table 1) or with significant variation (Fig. 3).

Figure 2. Expression of the 49 human ABC genes in hESCs. ABC genes are classified into subgroups as follows: (A) ABCA; (B) ABCB; (C) ABCC; (D) ABC-D, -E, -F and -G. Radial plots are sub-divided in areas as follows: high (NCt < 29, white), low but significant (29 ≤ NCt < 35, light-gray) and undetectable (NCt ≥ 35, gray) gene expression. Arrows and their directions indicate significant gene expression variations during development of hESCs to hES-MSCs and/or hMSCs. Information relating to structure, disease associations, subcellular localizations (bold) and substrates and specific functions (italics) derived from references^3-5,12,33,34 are detailed in the figure.

Table 1. ABC genes displaying similar expression in hESCs, hES-MSCs and hMSCs

Gene	NCt Means (± SD)	Localization	Structure	Substrates and specific functions
High expression (NCt < 26)
ABCE1	23.3 (0.5)	Cytopl	No TMD	mRNA translation
ABCF1	24.9 (0.6)	Cytopl	No TMD	mRNA translation
ABCF2	23.5 (0.6)	Cytopl	No TMD	mRNA translation
ABCF3	24.2 (0.6)	Cytopl	No TMD	mRNA translation
ABCB6	24.7 (0.6)	Mito	Half-Tr	Heme transport
ABCB7	24.7 (0.9)	Mito	Half-Tr	Heme transport
ABCB10	25.1 (0.6)	Mito	Half-Tr	Heme transport
ABCB8	25.1 (0.7)	Mito	Half-Tr	MDR
ABCD3	24.8 (0.7)	Pero	Half-Tr	Peroxysome biogenesis
ABCC1	24.5 (0.6)	PM	Full-Tr	Anion, lipid (GlcCer, SM, PC, S1P, LTC4), peptides (GSH, UGT), steroids and MDR
ABCC10	25.6 (0.8)	PM	Full-Tr	E(2)17betaG transport and MDR
Medium expression (26 ≤ NCt < 29)
ABCD1	26.7 (1.1)	Pero	Half-Tr	VLCFA transport
ABCG2	27.3 (1.2)	PM	Half-Tr	Transport of PC and PS, MDR and SP
ABCA1	26.2 (0.9)	PM	Full-Tr	Transport of Chol, PS, SM, PC
ABCC4	26.0 (0.9)	PM	Full-Tr	Anion transport and MDR
ABCC5	27.9 (0.7)	PM	Full-Tr	Anion transport and MDR
ABCA5	28.1 (0.9)	PM	Full-Tr	Unknown
Low expression (29 ≤ NCt < 35)
ABCC2	31.9 (1.6)	PM	Full-Tr	Anion transport and MDR
ABCA4	32.1 (1.8)	PM	Full-Tr	Transport of N-ret-PE
ABCA10	34.1 (0.8)	PM	Full-Tr	Chol transport
ABCB11	34.0 (1.2)	PM	Full-Tr	Biliary salt transport
Undetectable expression (NCt ≥ 35)
ABCB5	35 (0)	PM	Full-Tr	MDR
ABCC11	35 (0)	PM	Full-Tr	Transport of E(2)17betaG and steroids
ABCC12	35 (0)	PM	Full-Tr	Unknown
ABCG5	35 (0)	PM	Half-Tr	Sterol transport
ABCG8	35 (0)	PM	Half-Tr	Sterol transport

The ABC genes are categorized in four expression ranges according to the mean (± SD) NCt value of all samples. Localization, structure, substrates and specific function are as described in Figure 2. Half-Tr, Half-Transporter; Full-Tr, Full-Transporter.

Figure 3. Twenty-three ABC genes are significantly up- or downregulated during the development of hESCs toward hES-MSCs and hMSCs. Compared with the hESC reference group, relative quantification and standard deviation values for hES-MSCs (white) and hMSCs (hatched) were calculated as previously described^24,76 with the five reference genes indicated in Materials and Methods. Statistical significance (p < 0.05) is indicated by asterisks. For hESCs, mean NCt (± SD) values are shown below the x-axis. ABC genes are categorized into four sets, based on subcellular localizations and substrates: (A) intracellular transporters; (B) plasma membrane lipid transporters; (C) plasma membrane ion transporters; (D) ABCs with unknown function or substrate. Bold downward-pointing arrows (↓) indicate the abolition of gene expression (defined as NCt ≥ 35) in hES-MSCs or hMSCs. Abbreviations for substrates and specific functions (italics), and subcellular localizations (bold), are as described in Figure 2.

Intracellular ABC members.

ABCE1 and the ABCF subfamily members, which lack a TMD, do not act as transporters, but as important translation factors.^8-10 These genes showed the highest expression levels in this study, without significant differences between hESCs, hES-MSCs or hMSCs (Table 1 and Fig. S2).

ABCB6, ABCB7 and ABCB10, which encode mitochondrial membrane heme transporters,^36-38 and ABCB8, whose MDR function protects mitochondrial DNA,³⁹ remained highly and stably expressed during mesodermal development (Table 1 and Fig. S2). Three peptide transporters: ABCB2 and ABCB3 (endoplasmic reticulum)⁴⁰ and ABCB9 (lysosome),⁴¹ were well expressed (Fig. 2B) and slightly upregulated between hESCs and both mesenchymal populations (Fig. 3A). This is consistent with the provision by hMSCs of stem cell niches (see Fig. 3E) for pluripotent and multipotent stem cells.^29,30,42 ABC transporters in these stem cell niches could promote the traffic of peptides with pleiotropic effects on self-renewal or differentiation.^31,32

Figure 3E. (E) A typical Hematopoietic Stem Cell (HSC) niche. Shown is a small HSC with a high nucleo-cytoplasmic ratio and prominent nucleoli, embedded in an adipocyte full of lipid droplets. Strong plasma membrane interdigitation is observed between the adipocyte and the HSC, suggesting possible lipid transport.

The four half-transporters of the ABCD subfamily are located in the peroxisome, being involved in energy production.⁴³ ABCD1 and ABCD3, which are involved in neuropathologies,³³ were not modulated during mesenchymal development (Fig. 2D and Table 1), whereas ABCD2 and ABCD4 were downregulated (Figs. 2D and 3A).

Plasma membrane ABC transporters (PM-ABCs).

PM-ABCs with significant expression variation.

As shown in Figure 3, lipid transporters with overlapping substrates, and functions often showed different gene regulation. This is particularly true for ABC cholesterol transporters, which were highly upregulated in hMSCs for ABCA6 and ABCA9 (Fig. 3B), and slightly for ABCA2, whereas they were downregulated or even abolished for ABCG4, ABCA3, ABCA7, ABCB1 and ABCG1 (Fig. 3B). Similarly for ABCs with MDR functions, ABCA8 was highly upregulated, while ABCB1 expression was abolished in hMSCs (Fig. 3B). The overlapping nature of ABC substrates could partly explain why research on ABC inhibitors to study the functions of these genes or the suppression of drug resistance has proved so challenging.⁴⁴

Among ABC lipid transporter genes that were up- or downregulated, most of them displayed low but significant expression levels in hESCs. Only ABCA2 and ABCA7, which have been identified as genetic risk factors in Alzheimer disease,^34,45,46 and ABCA3, an important transporter mutated in neonatal surfactant deficiency disease,^33,34 were well expressed by hESCs.

Compared with hESCs, ABCB1 was stably expressed in hES-MSCs but undetected in hMSCs (Fig. 3B). ABCB1 could therefore be a useful membrane marker for the selection of more primitive hMSCs from bone marrow, similar to hES-MSCs. However, ABCB1 has already been described for its important MDR function involved in SP selection. This MDR function complicates cancer chemotherapeutic treatment but protects the brain by the blood-brain barrier. By exporting xenobiotics or small peptides, Parkinson or Alzheimer diseases could be prevented.³⁴ ABCB4 is a specific phosphatidylcholine transporter, and its defect causes intrahepatic cholestasis.^33,34 Similarly to ABCB1, ABCB4 could be a new membrane marker for the selection of primitive hMSCs with an ABCB4^low phenotype.

Among the ABCC subfamily members, we observed two groups of ion transporters (ABCC8 and ABCC9, and ABCC3, ABCC6 and ABCC7) that underwent opposite changes in regulation from hESCs to early and late hMSCs (Fig. 3C). This again stresses the fact that different transporters for similar substrates might have quite different gene regulation. ABCC7 was not expressed during mesodermal development (Fig. 3C) but during endodermal development, where its mutation causes cystic fibrosis.^47,48

Stably-expressed PM-ABCs.

Some PM-ABCs maintained a stable gene expression during mesenchymal development (Table 1 and Fig. S2). Apart from ABCC1 and ABCC10, these ABC genes have a medium or low but significant expression (Table 1 and Fig. S2). They transport lipids (ABCA1, ABCA4, ABCA10, ABCC1 and ABCG2), anions (ABCC1, ABCC2, ABCC4 and ABCC5), xenobiotics (ABCC1, ABCC2, ABCC4, ABCC5, ABCC10 and ABCG2), bile salts (ABCB11) or have unknown substrates (ABCA5) (Table 1).^3-5 Four ABCs (ABCC1, ABCC2, ABCC4 and ABCC5) combine MDR and anion transport activities together.

ABCs with unknown function.

ABCA5 and ABCA13 are full transporters localized on the plasma membrane, but their substrates are as yet unknown. ABCA5 remained well and stably expressed in all three cell populations (Table 1 and Fig. S2).

Expression of ABCA13 was unchanged between hESCs and hES-MSCs but was abolished in hMSCs (Fig. 3D), making this gene a possible new candidate for the selection of primitive hMSCs, comparable to ABCB4. ABCA13 has been implicated in schizophrenia, bipolar disorder and depression,⁴⁹ but initial results need to be confirmed.⁵⁰

Expression of ABCC13, a pseudogene,⁵¹ was detected in hESCs, but abolished in both hES-MSCs and hMSCs (Fig. 3D). ABCC13 gene expression has been detected in fetal liver and primitive Hematopoietic Stem Cells (HSCs),^51,52 but its functions in hESCs or HSCs remain to be clarified.

Undetected ABCs.

Only five ABCs (ABCB5, ABCC11, ABCC12, ABCG5 and ABCG8) were not detected in hESCs, hES-MSCs and hMSCs (Table 1 and Figs. 1A, B and 2). They only appear during endoderm and ectoderm development.^53-55 They transport steroids (ABCC11) or sterols (ABCG5 and ABCG8), or have MDR (ABCB5) or unknown activities (ABCC12) (Table 1).

Discussion

This study has analyzed for the first time the expression of all 49 human ABC genes in self-renewing pluripotent hESCs and in early and late multipotent mesenchymal stem cell cultures. Notwithstanding post-transcriptional and posttranslational regulation, strikingly high concordances have been described between the levels of specific mRNAs and those of the corresponding proteins within the same cell population, both for stem cells⁵⁶ or adult differentiated tissues.^57,58

Previous studies that selected the most primitive multipotent stem cells (such as mesenchymal, epidermal or hematopoietic primitive stem cells), which rapidly lose pluripotent, symmetric and exponential self renewal, revealed that these cells express cytokine receptors at much lower levels than later asymmetrically and non-exponentially dividing multipotent stem cells.^22,59-61 PM-ABCs mediate the transport of various signaling molecules, such as peptides into and out of the cell, and modulate the lipid environment of membranes surrounding cytokine receptors, affecting their internalization and thus signaling function.^2,3,62 Consistent with the expression of cytokine receptors in early stem cells and the role of PM-ABCs in cytokine signaling, in the present study we observed that it was the pluripotent hESCs that displayed the largest number of low-expressed PM-ABC transporters (Fig. 1B). The requirement for a substantial proportion of these ABCs appears to be hESC-specific, as the expression of seven of the 18 “low-expressed” PM-ABC transporters was abolished during the mesenchymal development process. Among these genes, we identified ABCA13 and ABCB4 as well as ABCB1^14,63 as good candidates for new markers for the enrichment from hMSCs of more primitive stem cell populations, such as hES-MSCs. In contrast to many ABC plasma membrane transporters, intracellular ABCs that control basal functions such as translation or energy production display a much higher level of expression, which remains constant during the development from hESCs to hMSCs.

It is well established that hMSCs play important roles in forming and maintaining stem cell niches,²⁹ for example in bone marrow (as illustrated in Fig. 3E). Our data on the differential expression of ABC genes between hESCs and hMSCs may help shed light on these mechanisms. Previous studies indicate that membrane lipid composition and receptor or cytokine intracellular trafficking play an important role,^3,64 but more in-depth functional studies are still needed to understand the mechanisms involved.

One adult tissue derived from mesenchymal cells is the meningeal barrier of the brain. The expression of ABC transporters in this tissue is vital to maintain the equilibrium of lipids, peptides and small molecules within this compartment, and thus for normal brain function.^34,65 Consistent with this, the dysregulation of several ABC genes is associated with various neurological disorders, including Parkinson and Alzheimer diseases.^34,45 As the etiologies of these conditions are likely multi-factorial in nature, an understanding of the roles of ABCs will require collaborative efforts between clinicians, geneticists and cell and molecular biologists.

Materials and Methods

Cell lines and culture conditions.

hES2 and hES3 cell lines were provided by ES Cell International (ESI) and H7 by the Wisconsin Institute. They were cultivated according to supplier’s instructions or as previously described in references 24, 66 and 67.

Two different hES-MSC lines were used. The first hES-MSC1 (P51RAC), obtained from hESC H1,⁶⁸ was generously provided by Dr. Emmanuel Olivier and Dr. Eric E. Bouhassira.²⁵ The second was derived from hES2 in our laboratory as previously described in reference 24. Human bone-marrow derived hMSCs were obtained, isolated and amplified²⁴ in accordance with international guidelines and French bioethics laws. All stem cells used for expression analysis were of a high quality, according to morphological assessment and pluripotency-marker expression.^24,66,67

A long-term human Dexter-type culture providing a Hematopoietic Stem Cell (HSC) niche, used for the image in Figure 3E, was generated as previously described in reference 69.

Gene expression analysis.

Preparation of stem cell mRNA, cDNA and quantitative real-time PCR (qRT-PCR) were performed as previously described in reference 24. Microfluidic TaqMan^® Human Endogenous Control Arrays (Applied Biosystems) and TaqMan^® ABC gene Arrays were used as previously described in reference 70 and 71. Five reference genes (RPLP0, POLR2A, IPO8, PPIA, GAPDH) were chosen according to their consensus stability values (Fig. S1 and Table S1),^72‑75 and their geometric mean expression levels applied to derive normalized cycle threshold (NCt) and 2^-NCt values for the expression of ABC genes, as previously described in reference 24. Relative quantifications and standard deviation (SD) of gene expression were calculated using the 2^-ΔΔCt method,^24,76 and statistical significance tests were performed as previously described in reference 24.

Acknowledgements

This work was supported by the Centre National de la Recherche Scientifique (CNRS)-UPR9045 and by the FP6 European Integrated Project “Genostem” (#503161). R.B. was supported by a CNRS Fellowship (509723), and I.P. was supported by a Fellowship from Prof. R. Frydman. We thank Dr. E. Olivier and Dr. E.E. Bouhassira for supplying the P51RAC (hES-MSC1) cell line. The work in this paper benefited from the facilities of the QPCR platform of IMAGIF (Centre de Recherche de Gif, www.imagif.cnrs.fr).

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Note

Supplemental material can be found at: www.landesbioscience.com/journals/cc/article/20023