A Novel Molecular and Functional Stemness Signature Assessing Human Cord Blood-Derived Endothelial Progenitor Cell Immaturity

<div><p>Endothelial Colony Forming Cells (ECFCs), a distinct population of Endothelial Progenitor Cells (EPCs) progeny, display phenotypic and functional characteristics of endothelial cells while retaining features of stem/progenitor cells. Cord blood-derived ECFCs (CB-ECFCs) have a high clonogenic and proliferative potentials and they can acquire different endothelial phenotypes, this requiring some plasticity. These properties provide angiogenic and vascular repair capabilities to CB-ECFCs for ischemic cell therapies. However, the degree of immaturity retained by EPCs is still confused and poorly defined. Consequently, to better characterize CB-ECFC stemness, we quantified their clonogenic potential and demonstrated that they were reprogrammed into induced pluripotent stem cells (iPSCs) more efficiently and rapidly than adult endothelial cells. Moreover, we analyzed the transcriptional profile of a broad gene panel known to be related to stem cells. We showed that, unlike mature endothelial cells, CB-ECFCs expressed genes involved in the maintenance of embryonic stem cell properties such as <i>DNMT3B</i>, <i>GDF3</i> or <i>SOX2</i>. Thus, these results provide further evidence and tools to appreciate EPC-derived cell stemness. Moreover this novel stem cell transcriptional signature of ECFCs could help better characterizing and ranging EPCs according to their immaturity profile.</p></div

Stemness genes expression array.

<p>Data are expressed as ΔCt. Transcript levels were normalized to GAPDH transcript level. Genes whose detection threshold is over 35 cycles are considered as not expressed and symbolized by “X”.</p

Endothelial and Stemness transcriptional signature of CB-ECFCs.

<p>(A) Transcript levels of endothelial markers. Quantitative RT-PCR analysis in ECFCs at passage P1 (n = 6) and adult HAECs at passage P3 (n = 4). Transcript levels were normalized to GAPDH transcript levels and relative to mean HAECs. (B) Transcript levels of stemness markers. Quantitative RT-PCR analysis in ECFCs at passage P1 (n = 6) and adult HAECs at passage P3 (n = 4). Transcript levels were normalized to GAPDH transcript levels and relative to mean hESCs (2 H9 samples at P45 and P52 and 1 H1 sample at P54). Error bars represent SEM (*p < 0.05).</p

Pluripotency of ECFC-derived iPSCs.

<p>(A) Immunostaining of iPSC-derived embryoid bodies revealed the expression of ectodermal (βIII tubulin, nestin), endodermal (AFP, HNF-3β), mesodermal (CD31, SMA) markers. Nuclei were stained with DAPI. Scale bars represent 100μm. (B) Immunostaining of teratomas induced in SCID mice after injection of 100,000 ECFC-derived iPSC1 containing derivatives of the 3 germ layers differentiation (βIII tubulin, AFP and SMA). Nuclei were stained with DAPI. Scale bars represent 100μm. (C) Histology of teratomas (hematoxylin/eosin staining) confirming differentiation into all 3 germ layers: mesoderm (1) endoderm (2) and ectoderm (3).</p

ECFC-derived iPSCs express typical hESC markers.

<p>(A) Immunofluorescence analysis of ECFC-derived iPSC1 line. Expression of stem cell markers: OCT3/4, SOX2, NANOG, PA, SSEA4, TRA-1-81. The cells were stained using the 488 and 546-conjugated Donkey Anti-Goat IgG or Goat Anti-Mouse IgG secondary antibody and the nuclei were counterstained with DAPI (blue). Scale bars represent 100 μm. (B) Quantitative RT-PCR analysis of the expression of endogenous (endo) and exogenous (exo) markers: <i>OCT3/4</i>, <i>SOX2</i>, <i>KLF4</i> and <i>C-MYC</i> in Ctrl ECFCs, transduced ECFCs (ECFC OKSM) and ECFC-derived iPSC1 at passage 4, 7 and 10. Transcript levels were normalized to GAPDH transcript levels and relative to mean hESCs (H9 samples at P45) as a calibrator.</p

Phenotypic and functional characterization of iPSC-derived endothelial cells.

<p>(A) Expression of specific endothelial markers (KDR, CD31 and CD144) by flow cytometry at passage P4. (B) CD31 and vWF immunofluorescent staining at passage 5. Scale bars represent 50μm. (C) Vascular-like network structures after 24h onto Matrigel^™ at passage P4. (D) Representative diacetylated low-density lipoprotein incorporation at passage P4. Scale bars represent 25μm. (E) TNF-α upregulated <i>ICAM-1</i> and <i>VCAM-1</i> at passage P5.</p

Morphology of ECFC-derived iPSCs and reprogramming efficiency of ECFCs and HAECs.

<p>(A) Cobblestone morphology of ECFCs before and 7 days after transduction, ECFC morphology changed and colonies were ALP positives. At passage 5 and 30, ECFC-derived iPSC1 showed typical characteristics of hESC colonies. (B-C) ECFC and HAEC reprogramming efficiency and rate. The reprogramming efficiency was estimated based on the number of ALP positive colonies. Error bars represent SEM (***p < 0.005).</p

EPDC’S phenotypical and functional characterization.

<p>(<b>A</b>) Primary colony’s phase contrast micrograph. (<b>B</b>) EPDC’s monolayer at passage 2. (<b>C</b>) Expression of specific endothelial markers (KDR, CD31/PECAM and CD144/VE-CAD) by flow cytometry. (<b>D</b>) CD31, (<b>E</b>) CD144 immunofluorescence staining. (<b>F</b>) Vascular-like network structures after 4 hours on Matrigel. (<b>G</b>) Characteristic diacetylated low-density lipoprotein incorporation.</p

Gene expression profile from HUAECs and EPDCs compared to HUVECs.

<p>(<b>A</b>) Sixty genes were analyzed using TaqMan® microfluidic cards on early passages HUAECs and EPDCs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084179#pone-0084179-g004" target="_blank">Figure 4</a> shows selection of 17 selected genes confirmed by Gene Expression Assays. Results were expressed as the ratio of expression between HUAECs vs HUVECs (light grey) and EPDCs vs HUVECs (dark grey). (<b>A</b>) Expression of venous (blue) and (<b>B</b>) arterial (red) markers (scale bars: SEM). (<b>C</b>) Table of numeric values. (*p<0.05, **p<0.001, ***p<0.0001) (qPCR expression analysis were performed at least 3 times, on independent cells batches and each time in triplicate).</p

Immunofluorescence analysis of arterio-venous markers in EPDCs under arterial and venous inductive conditions.

<p>(A) EPDCs were cultured under high or low VEGF concentrations. (B) High VEGF early passages EPDCs were cultured with 50 µM DAPT (red: arterial markers, bleu: venous markers).</p