Stage‐specific regulation of Gremlin1 on the differentiation and expansion of human urinary induced pluripotent stem cells into endothelial progenitors

Abstract Human urinary induced pluripotent stem cells (hUiPSCs) produced from exfoliated renal epithelial cells present in urine may provide a non‐invasive source of endothelial progenitors for the treatment of ischaemic diseases. However, their differentiation efficiency is unsatisfactory and the underlying mechanism of differentiation is still unknown. Gremlin1 (GREM1) is an important gene involved in cell differentiation. Therefore, we tried to elucidate the roles of GREM1 during the differentiation and expansion of endothelial progenitors. HUiPSCs were induced into endothelial progenitors by three stages. After differentiation, GREM1 was obviously increased in hUiPSC‐induced endothelial progenitors (hUiPSC‐EPs). RNA interference (RNAi) was used to silence GREM1 expression in three stages, respectively. We demonstrated a stage‐specific effect of GREM1 in decreasing hUiPSC‐EP differentiation in the mesoderm induction stage (Stage 1), while increasing differentiation in the endothelial progenitors' induction stage (Stage 2) and expansion stage (Stage 3). Exogenous addition of GREM1 recombinant protein in the endothelial progenitors' expansion stage (Stage 3) promoted the expansion of hUiPSC‐EPs although the activation of VEGFR2/Akt or VEGFR2/p42/44MAPK pathway. Our study provided a new non‐invasive source for endothelial progenitors, demonstrated critical roles of GREM1 in hUiPSC‐EP and afforded a novel strategy to improve stem cell‐based therapy for the ischaemic diseases.


| INTRODUC TI ON
Accumulating evidence suggest that bone marrow-derived circulating endothelial progenitor cells (EPCs) contribute to vascular repair. Recently, EPC transplantation has become an experimental therapy for ischaemic disease. 1 Several studies showed that EPC transplantation provides benefit for myocardial infarction 2 and limb ischaemia. 3,4 However, the amount of endogenous EPCs is limited.
In bone marrow or peripheral blood, the proportion of EPCs is only 0.01%. This number was even lower in cardiovascular patients. 5 Human pluripotent stem cells (hPSCs) are ideal candidates for cell-based regenerative repair, for they can self-renew indefinitely and potentially differentiate into any cell type. 5,6 HPSC-induced endothelial progenitors (hPSC-EPs) may provide the means for vascularization of tissue-engineered constructs and can serve as models to study vascular development and disease. 7 Lian et al have reported a rapid and efficient method for the production of hPSC-EPs and identifies Wnt/β-catenin signalling as a primary regulator for generating vascular cells from hPSCs. 8 This is a simple and efficient method for the conversion of hPSCs to CD34 + CD31+ endothelial progenitors.
Appropriate temporal activation of regulators of Wnt signalling was sufficient to drive multiple hPSC to differentiate to CD34 + CD31+ endothelial progenitors. In another article, Bao et al also used chemically defined albumin-free differentiation to induce human pluripotent stem cells to endothelial progenitor cells. 9 Human urinary induced pluripotent stem cells (hUiPSCs), which are induced from exfoliated renal epithelial cells present in urine, provided a non-invasive source for generating hPSCs. hUiPSCs also showed excellent differentiation potential and thus represent a good choice for producing pluripotent cells from normal individuals or patients with genetic diseases. 10 In this study, established hUiPSCs were used to acquire hUiPSC-induced endothelial progenitors (hUiPSC-EPs) from non-invasive source by Lian's method. Furthermore, we tried to study on the critical factors during the differentiation, improve the efficiency and clarify the mechanisms. Gremlin1 (GREM1) is a pro-angiogenic protein belonging to the cystine-knot superfamily that includes transforming growth factor-β proteins (TGF-β) and the angiogenic vascular endothelial growth factors (VEGFs). 11 GREM1 antagonizes bone morphogenetic proteins (BMPs) 2, 4 and 7, thereby preventing these ligands from interacting with their receptors. 12 GREM1 could bind to vascular endothelial growth factor receptor 2 (VEGFR2) and promote angiogenesis. 10 Our previous study showed that overexpression of GREM1 in human mesenchymal stem cells (hMSCs) has greater therapeutic effects against ischaemia compared with wild-type hMSCs by enhancing the survival of hMSCs and endothelial cells (ECs). 13 Other studies reported that GREM1 accelerates DMSO-induced cardiomyogenesis through inhibition of the BMP-signalling pathway. 14 However, the effect of GREM1 in the differentiation of endothelial progenitors is currently unknown.
In this study, we tried to clarify the effects of GREM1 during hUiP-SC-EP differentiation and expansion. We acquired hUiPSC-EPs in three specific stages, Stage 1 (Day 0-2, mesoderm induction), Stage 2 (Day 2-5, endothelial progenitors' induction) and Stage 3 (Day 5-8, endothelial progenitors' expansion). To determine the critical role of GREM1 during hUiPSC-EP differentiation and expansion, GREM1 gene was down-regulated by RNA interfering or GREM1 recombinant protein was added in the three stages, respectively. Our study may increase the differentiation efficiency and function of hUiPSC-EPs and provide a novel strategy to enhance stem cell-based therapy for ischaemic diseases.

| Cell culture
Four pluripotent cell lines were used in this study. Two human urinary induced pluripotent stem cell (hUiPSC) lines (U1-hUiPSC, U5-hUiPSC) were kindly presented by Dr Guangjin Pan's group, who established these cell lines previously. 15 Two human embryonic stem cell (hESC) lines (H1-hESC, H9-hESC) were established in our laboratory as described previously. 16 Cells were maintained in mTeSR1 medium (STEMCELL Technologies) on matrigel-coated plates (BD Biosciences).

| Endothelial progenitors' differentiation
An established protocol was used for endothelial progenitors' differentiation of hUiPSC/hESCs without using VEGF. 8,9 Briefly, hU-iPSC/hESCs were cultured on Matrigel-coated six-well plates in mTeSR1 medium to 80%-90% confluence. The cells were dissociated into single cells with Accutase (Life Technologies). On Day 0, cells were treated with 6 μM CHIR99021 (Selleckchem) in DMEM/ F12 medium with 100 μg/ml ascorbic acid (A8960, Sigma). On Day 2 of differentiation, CHIR99021-containing medium was aspirated and DMEM/F12 medium with ascorbic acid was added and changed source for endothelial progenitors, demonstrated critical roles of GREM1 in hUiPSC-EP and afforded a novel strategy to improve stem cell-based therapy for the ischaemic diseases.

K E Y W O R D S
differentiation, endothelial progenitors, expansion, Gremlin1, human urinary induced pluripotent stem cells daily. On Day 5, hUiPSC/hESCs were successfully induced into endothelial progenitors. Cells were kept on expanding until Day 8.

| Flow cytometry (FACS)
Cells were dissociated into single cells with Accutase for 10 minutes at day 2, 5 or 8 in different experiments. The cell suspensions were incubated with fluorescent conjugated antibodies (Table S1) for 60 minutes at room temperature. Following incubation, the cells were re-suspended in PBS containing 2% FBS. For the apoptosis assay, cells were treated with 1 mM H 2 O 2 for 3 hours PI/ Annexin V were detected. A cell sorting analysis was performed with a FACS Caliber Flow Cytometer (BD Bioscience), and the data were analysed using the Cell Quest-Pro software (BD Bioscience).

| Tube formation assay
To assess the formation of capillary structures, 1 × 10 5 hUiPSC-EPs or hESC-EPs were suspended in 0.4 mL EGM-2 medium (Lonza) were plated into one well of 24-well tissue culture plate pre-coated with Matrigel (BD Bioscience). Tube formation was observed by light microscopy after 24 hour of incubation. Tube length was calculated and quantified by Image J software (the National Institute of Health).

| Western blot analysis
Cell lysates with equal total protein amounts were separated by SDS-PAGE gel. Proteins were transferred electrophoretically to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membranes were blocked in 5% milk in PBS-T (0.1% Tween 20) at room temperature for 1 hours The membranes were probed with primary antibody overnight (Table S3). The primary antibody was then identified by a horseradish peroxidase (HRP)-conjugated secondary antimouse or anti-rabbit antibody (1:1000; CST). Finally, the membranes were developed using an enhanced chemiluminescence advance detection kit (GE Healthcare) and exposed to x-ray films. The band density was analysed using Image J software (the National Institute of Health).

| Statistical analysis
All data are presented as the mean ± SEM obtained from at least three independent experiments. Comparisons between groups were

| Stage-specific expression of GREM1 during the differentiation and maintenance of hUiPSCs into endothelial progenitors
To explore a non-invasive source of endothelial progenitors, we in-  Figure 1C). QRT-PCR results showed hPSC markers OCT4, Nanog and SOX2 decreased after differentiation ( Figure 1D). On the contrary, EPC markers, CD34, CD31, CD144 and VEGFR2 increased ( Figure 1E). The expression of hPSC and EPC markers on each day showed similar results ( Figure S1). FACS analysis confirmed the success of EP differentiation, as indicated by CD34/CD31 and VEGFR2/CD144 double positive rates ( Figure 1F).
We found that the protein expression of endothelial progenitors, especially CD34 and VEGFR2, was significantly increased after differentiation ( Figure S2).
To clarify the mechanism of differentiation, GREM1 expression during the process was detected by WB and qRT-PCR. WB results revealed there was few GREM1 protein expression in hUiPSCs or hESCs, while high GREM1 expression was detected in endothelial progenitors (

| Knock-down of GREM1 during Stage 1 promoted the differentiation and expansion of hUiPSCs into endothelial progenitors
Although GREM1 mRNA expression was relatively low, it was knock-down in Stage 1 to clarify the effects during mesoderm induction stage. At Day 2, the expression of GREM1 mRNA could be detected (Ct value was around 27), although the protein level of GREM1 protein was too low to be detected. Therefore, we pro-
The proliferation and apoptosis of endothelial progenitors were also detected. Ki67 expression showed the positive cells were decreased from (43.43 ± 2.63)% to (17.33 ± 2.17)%, P < .05 ( Figure 5A,B). Cell cycle detected by FACS showed that cell ratio at G1 phase increased from (82.71 ± 1.44) % to (91.82 ± 0.64)% F I G U R E 2 GREM1 expression during hUiPSC/hESCs-EPs cell differentiation. A, GREM1 protein expression of hUiPSC-EPs was determined by WB. B, GREM1 protein expression of hESC-EPs was determined by WB. C, GREM1 mRNA expression of hUiPSC-EPs was detected by qPCR. D, GREM1 mRNA expression of hESC-EPs was detected by qPCR. E, RNA samples were collected at successive differentiation days for hUiPSC-EPs, and expression of GREM1 and related genes were tested by qPCR. F, GREM1 and related genes were tested in hESC-EPs by qPCR. The data represent mean ± SEM of three independent experiments. * P < .05

| Knock-down of GREM1 during Stage 3 inhibits the function and expansion of hUiPSCs into endothelial progenitors
The effects of GREM1 were then detected by using siGREM1 during

| Stage-specific addition of recombinant protein GREM1 influenced the differentiation and expansion of hUiPSCs into endothelial progenitors
To improve the differentiation and expansion of endothelial progenitors, we used recombinant protein GREM1 during different stages, respectively. We determined that the addition of GREM1

| Recombinant protein GREM1 during Stage 3 promoted endothelial progenitors' expansion and activated the downstream pathway
As addition of GREM1 during Stage 3 increased the differentiation efficiency, we detected the role of GREM1 during this maintenance of endothelial progenitors' stage. GREM1 was added during  Figure S4). To determine the mechanism through which GREM1 regulates endothelial progenitors' maintenance, we evaluate the canonical signal transduction pathway VEGFR2/Akt and VEGFR2/p42/44MAPK. We also found that the addition of recombinant protein GREM1 in endothelial progenitors' expansion stage (Stage 3) promoted the expansion of hUiPSC-EPs, which was accomplished through VEGFR2/ Akt and VEGFR2/p42/44MAPK pathway.

| D ISCUSS I ON
HiPSCs have been generated with varied efficiency from multiple tissues. Yet, acquiring donor cells is, in most instances, an invasive procedure that requires laborious isolation. 18

ACK N OWLED G EM ENTS
The authors would like to thank Dr Guangjin Pan's group for providing us with hUiPSCs used in this study. We thank Dr Phei Er Saw for revising the manuscript. We thank Prof. Te Liu for giving lots of good advice for the implementation of the project.

CO N FLI C T O F I NTE R E S T
All authors declared no conflicts of interest.

E TH I C A L A PPROVA L
All experimental research on animals followed internationally recognized guidelines, the Declaration of Helsinki, and all guidelines in China.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and/or analysed during the current study are available from the corresponding author on reasonable request.