Infantile Hemangioma Originates From A Dysregulated But Not Fully Transformed Multipotent Stem Cell

Infantile hemangioma (IH) is the most common tumor of infancy. Its cellular origin and biological signals for uncontrolled growth are poorly understood, and specific pharmacological treatment is unavailable. To understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage derivatives. For this purpose we performed a high-throughput (HT) phenotypic and gene expression analysis of HemSCs, and analyzed HemSC-derived tumorspheres. We found that IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis and associated signaling pathways. These results show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to “rewire” or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers.

cells (such as medulloblastoma, glioblastoma, prostate, and colon cancer) 17 , and is expressed in the human embryo during the early stages of vascular development (4-week embryo) 19 . The normal human vascular compartment consists of multiple stem and progenitor cells 20 . In embryonic blood vessels, stem and progenitor cells contribute to endothelial cells, pericytes, and hemogenic endothelium; in adult blood vessels, stem and progenitor cells, found in an organ-specific vascular niche, contribute to pericyte, endothelial cell, and mesenchymal lineage-specific cells 20,21 . The possible vascular lineage models for normal endothelial cells include the hemangioblast, the hemogenic endothelium and the mesoderm-derived angioblast models. Thus, the normal vascular compartment consists of multiple stem and progenitor cells including adventitial, endothelial, hemangioblast, hemogenic and pericyte progenitor cells, mesenchymal stem/progenitor cell, and vascular stem cell.
At all stages of development, IH are heterogeneous (comprising endothelial cells, pericytes, myeloid cells, fibroblasts, and mast cells) and eventually involute into fibrofatty tissue (comprised of fat, fibroblasts and connective tissue) that replaces the vascular tissue 6 . This heterogeneity may result from multiple stem cells heterogeneously dysregulated at varying stages of development, and/or from a multipotent stem cell arrested in development. In proliferating-phase IH, the HemSC was identified as a rare CD133+ subset comprising approximately 1% of the tumor cell population, with vasculogenic potential (de novo vessel formation), clonogenic ability to self-renew, multipotential ability to give rise to several cellular lineages with distinct morphologies and gene expression profiles, and potential to regenerate GLUT1+ tumors containing the HemSC and the differentiated derivatives 6,10,14,16 . A rare subset (0.1-2%) of IH cells that express endothelial cell markers (such as platelet endothelial cell adhesion molecule-1 [PECAM-1/CD31], vascular endothelial-cadherin [VE-cadherin/CD144], CD34, and KDR/VEGFR-2/CD309 co-express the CD133 stem cell marker 6 . The identification of this subset suggests the existence of intermediate phenotypes (transitioning progenitor populations of interest) and stem cell heterogeneity.
We hypothesized that the HemSC is a vascular stem/progenitor cell whose proliferation is dysregulated, but not a fully transformed cell, which orchestrates IH pathophysiology through multiple signaling and regulatory networks. Therefore, to understand the process of hemangioma-genesis we characterized the progenitor hemangioma-derived stem cell (HemSC) and its lineage and non-lineage specific derivatives (Fig. 1). The results of our comprehensive phenotypic and gene expression analysis and the characterization of the tumorigenic potential of HemSCs in vitro show that IH derives from a dysregulated stem cell that remains in an immature, arrested stage of development.

Results
Molecular Signature. HemSCs have the potential to self-renew and give rise to differentiated cell types (derivatives) in vivo and in vitro 5,6,10,14,16,[22][23][24][25] . To analyse HemSC gene expression and identify specific molecular signatures, CD133 expressing cells were isolated from specimens of IH immediately after surgical excision as described in Materials and Methods. Consistent with previous reports, the HemSC was identified by flow cytometry as a rare CD133+ subset representing approximately 1% of the heterogeneous population. These cells were grown in vitro and subjected to gene expression analysis by qPCR for genes that encode the surface markers of endothelial, fibroblast, and mast cells -key cellular components of IH. At passages 3 and 7 in culture, the expression profile of these cells was heterogeneous and no longer pure, recapitulating the heterogeneity of the original tissue (data not shown).
HUVEC control 29,30 . At the single-cell level, HemSCs showed variability in gene expression. For example, they showed homogeneous gene expression of the NOTCH, TIMP, EGF, and GAPDH genes; in contrast, Endoglin (ENG/CD105) gene expression was highly heterogeneous, ranging from high expression levels to no expression. These data were confirmed at the single-cell level by comparison of phenotypic expression (flow cytometry and bioimaging) of HemSC-specific cell surface markers (Fig. 2c). Our microarray data include normal human stem and progenitor cell subsets that can serve as a reference as human transcriptional controls for biomarker analysis, drug-targeting pathways, and for future studies. They provide a reference for global gene expression in different cell types, developmental stages, diseases, and experimental conditions such as regenerative medicine and reprogrammed human induced pluripotent [hiPS] subsets (Fig. 4). Of the sample biomarker gene set analyzed, SLC2A1 (GLUT1, clinical marker of IH), NT5E (CD73), MME (CD10), and CD44 were over-expressed compared to the normal progenitor cell controls (HUVEC, bone marrow SC, cord blood SC, and mesenchymal SC). In contrast, ILR1 and SLC7A5 (CD98) were also expressed in normal progenitor cell controls (mesenchymal SC and mesenchymal SC/cord blood SC, respectively). SLC2A1 was over-expressed in the glioblastoma cancer stem cell (CSC) control as well. The gene expression of mTOR (mammalian target of rapamycin), a drug-target, showed high variability (Supp. Fig. 2a). However, Protein Kinase B (AKT1), a kinase upstream of mTOR, was consistently over-expressed. The PPAR/Adipocytokine signaling pathway microarray analysis showed significant over-expression of transcription factors (PPARD, PPARG, KLF10, RXRB) and the clinical biomarker of IH, GLUT1 (SLC2A1) (Supp. Fig. 2b).
TGF-β Signaling -Endoglin (CD105) Gene Signature. Because the single-cell analysis of HemSCs showed high variability in the phenotypic (CD105) and genotypic (ENG) expression of endoglin we performed a further molecular analysis of the IH endoglin subset (Hem ENG+ ). The global gene expression profile of Hem ENG+ (100-200 Hem ENG+ sorted cells) was analyzed by microarray and qPCR (data shown as CD105 + subset control in Fig. 4b-g). The Hem ENG+ progenitor sub-population was compared with glioblastoma CSC, mesenchymal SC, bone marrow SC, cord blood SC, and HUVEC cells, which were FACS-enriched and sorted for purity before gene expression analysis. Prior to performing the gene expression studies, Hem ENG+ were FACS-enriched and sorted for CD105+ subset. The results showed that the endoglin gene was expressed at high levels in the Hem ENG+ sub-population. Molecular characterization of the bulk subset of Hem ENG+ showed a higher expression of Figure 2. Gene expression analysis of angiogenesis signaling. Agilent whole genome oligo microarrays (onecolor -Cy3 labeling) and 96-well qPCR arrays (SABiosciences) were used to examine expression profiles of genes in multiple signaling pathways involved in vasculogenesis/angiogenesis, normal and tumor development (a) qPCR analysis. 96-well qPCR arrays (SABiosciences) were used to examine expression profiles of genes in sorted cell populations. Fold change/regulation was calculated using delta delta C t method. The values were compared to fold change gene expression analysis using one-way analysis of variance (ANOVA) of samples (Partek Genomics Suite software) (Supp. Fig. 1). The scatter plot compares the normalized expression of significant genes on the array between HemSC and HUVEC (control) to exhibit large gene expression changes. The central line indicates unchanged gene expression (selected fold regulation threshold boundary set at 2). Data points in the upper left and lower right sections meet the selected fold regulation threshold. (b) Microarray and qPCR analysis of gene expression in HemSC relative to HUVEC. Microarray gene expression analysis was performed using one-way analysis of variance (ANOVA) of samples to compare the normalized expression of significant genes (in the angiogenesis signaling gene panel) between HemSC GLUT1+ and HUVEC (control). The gene list (Table 1) was created with specified criteria: 1) size of change defined as a fold change; and 2) significance of change defined as p-value with False Discovery Rate (FDR). qPCR analysis served as confirmation ( VEZF1 and a lower expression of PROM1/SALL4, suggesting endothelial differentiation with the involvement of TGF-β signaling. The PTGS1 (prostaglandin-endoperoxide synthase 1/COX1) gene was also over-expressed in the Hem ENG+ sub-population. PTGS1 gene expression is regulated by Kruppel-like factors, zinc finger transcription factors (such as KLF10) that also act as key transcriptional regulators of TGF-β signalling 31,32 . Specifically, KLF10 targets the KDR promoter 31,32 . PTGS1 gene expression is also induced by estrogen. Angiopoietin-like 4 (ANGPTL4/FIAF), a direct glucocorticoid receptor target, was exclusively expressed at high levels in the Hem ENG+ sub-population.  Cell Surface Marker Analysis. The analysis of surface markers can provide useful information for the development of diagnostic tools and indicate novel targets for antibody-based therapy of IH. Therefore, we performed a HT screening of primary IH-derived cells using a stem cell-specific panel of 240 monoclonal antibodies to surface antigens. The results were validated by flow cytometry, microarray (Table 3; Figs 3 and 4) and qPCR analysis of three primary IH cell cultures, using HUVEC, glioblastoma CSC, bone marrow SC, cord blood SC, and mesenchymal SC as controls.
The most highly expressed markers (defined as greater than 10 -AF647+ %Parent) included Alkaline Phosphatase (AP), CD10, CD13, CD44, CD46, CD47, CD59, CD73, CD105, and CD147 (Tables 4 and 5; Fig. 5). The biologic classification of IH proposed by Mulliken and Glowacki identified "the endothelium in hemangiomas as characteristic of differentiation: Weibel-Palade bodies, alkaline phosphatase, and factor VIII production" 2 . Alkaline phosphatase (AP) is regarded as a benchmark pluripotent stem cell marker 33 . CD44 is a key marker of CSC 34 , along with CD133. CD44 controls normal development by influencing cell growth, survival, and differentiation 35,36 . In IH, CD44 was highly expressed in both the heterogeneous and HemSC populations. CD105 (Endoglin/ENG), a co-receptor for transforming growth factor-beta (TGF-β ), is involved in angiogenesis, particularly tumor angiogenesis 37 . CD13 (metalloprotease; aminopeptidase N; ANPEP/APN) is also an important regulator of angiogenesis and is overexpressed in tumor cells 36 . Within the tumor microenvironment, ANPEP expression is induced by vascular endothelial growth factor (VEGF), and high levels of CD13 have been associated with tumor progression in breast, ovarian, and prostate cancers 38 . CD10 (CALLA; Neprilysin; membrane metalloendopeptidase) is expressed in the tumor microenvironment, where it promotes endothelial cell growth and angiogenesis by mobilizing fibroblast growth factor-2 (FGF-2) and activating AKT1 signaling, a cell proliferation and prosurvival pathway 39,40 . HemSC-Derived Tumorsphere and Derivative Formation. HemSCs are characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis, and associated signaling pathways. In vitro tumorsphere formation assays were performed to investigate the tumorigenic and differentiation potential of the HemSC-derived tumorspheres (Figs 6-9). The results confirmed the HemSC as a rare CD133+ subset comprising approximately 1% of the tumor cell population, with an ability to self-renew, multipotential ability to give rise to several cellular lineages with distinct morphologies and gene expression profiles, and a potential to regenerate GLUT1+ tumorspheres containing the HemSC and the differentiated derivatives (pericytes).
To regenerate tumorspheres in vitro and analyze their differentiation potential HemSC-derived tumorspheres were first grown as non-adherent cultures in ultra low cluster plates and then transferred onto an adherent matrix (Fig. 6). The latter condition afforded the generation of differentiated derivatives that attached to, and spread on the culture substrate. HUVEC were used as a negative control and glioblastoma CSC as a positive control. In culture, glioblastoma CSC formed tumorspheres, whereas HUVEC formed no tumorspheres under the same experimental conditions (Fig. 6a). Bioimaging of HemSC-derived tumorsphere formation in non-adherent ultra low cluster plates showed no derivative formation besides the tumorspheres. However, after transferring to an adherent matrix they showed de novo formation of differentiated derivatives that attached to and spread on the culture substrate (Fig. 6b). Pericytes (HemPericytes) are abundant in IH proliferating-phase tissue and represent a key cellular component 6,41 . Platelet-derived growth factor receptor-beta (PDGFR-β ) is a pericyte surface marker [42][43][44][45] . Therefore, differentiated derivatives from the HemSC tumorspheres were characterized by immunostaining for PDGFR-β . A subset of adherent cells expressed PDGFR-β whereas the tumorspheres did not (Fig. 7). HT 21-h live imaging analysis of 13 distinct HemSC-derived tumorspheres showed de novo formation of pericyte derivatives from undifferentiated HemSCs, and a dynamic interaction between the HemSC -derived tumorsphere and its derivative population (with active movement and engagement of the derivative population) (Video 1-3). This finding showed that the de novo formation of pericyte-like cells originated from a heterogeneous population of stem cells in the tumorspheres, and that the HemSC is a multipotent stem cell arrested at an early stage of development (incompletely differentiated) with an ability to differentiate into several cellular lineages (lineage and non-lineage specific differentiation).
The HemSC-derived tumorspheres were characterized by flow cytometry and immunohistochemistry (Fig. 8). Flow cytometry analysis of pooled tumorspheres showed a heterogeneous population, similar to the population derived from monolayer culture of primary IH cells. HemSC was identified as a rare CD133+ subset comprising approximately 1% of the heterogeneous population, a frequency similar to that of HemSCs directly isolated from IH specimens. The immunohistochemical characterization of the HemSC-derived tumorspheres showed expression of GLUT1, the diagnostic marker of IH, as well as high levels of CD44, a well-established tumor stem cell marker. CD44 is highly expressed in IH cells, as shown by our genotypic and phenotypic analyses, and can be detected by immunohistochemistry in various normal and neoplastic tissues such as lymph nodes, melanoma, tumors of the testis. However, we did not find CD44 immunoreactivity in placenta (Fig. 9). Thus, the HemSC-derived tumorspheres recapitulated in vitro the gene expression pattern of IH.

Discussion
The data presented show that IH originates from a dysregulated but not fully transformed multipotent stem cell. IH is characterized by high expression of genes involved in vasculogenesis, angiogenesis, tumorigenesis, and associated signaling pathways. The identification of transcriptional regulator genes and analysis of gene expression at the single-cell level suggests the existence of intermediate phenotypes, unique subsets, and stem cell heterogeneity. Moreover, our observations point to an important role of the immune system in hemangioma-genesis, the strategies used in the proliferative phase of IH formation to evade immunosurveillance, and the potential benefits of immunotherapy. Our data also show that HemSC have the potential to regenerate GLUT1+ tumorspheres from which differentiated derivatives originate. The de novo formation of pericyte-like derivatives reflects the heterogeneity of a hemangioma-derived multipotential stem cell arrested at an early stage of development (incompletely differentiated) with the ability to differentiate into several cellular lineages (lineage-and non-lineage specific differentiation). Finally, we provide a comprehensive panel of potential biomarkers and drug-targets (which can be further explored for assay development), and propose novel potential therapeutic strategies for IH, identified by our comprehensive analysis.
Signature genes that differentiate HemSCs from the Hem-derivatives include over-expression of transcriptional regulators that encode zinc finger transcription factor proteins such as SALL4, tripartite motif (TRIM) proteins (RING type E3 ubiquitin ligases), and long non-coding RNAs (lncRNAs). Zinc finger genes, found in 3% of the human genome, encode proteins that serve as transcriptional regulators 46,47 . TRIM proteins regulate nuclear receptors, and have been associated with tumorigenic pathways and hormone (estrogen) responsive cancer cells 48 . Long non-coding RNAs (RNA transcripts with a regulatory function) are localized either in the nucleus or in the cytoplasm, where they are involved in transcriptional or post-transcriptional regulation 30,49,50 . For example, in HemSCs, the zinc finger transcription factor SALL4 is over-expressed. SALL4 is a stem cell factor normally restricted to human embryonic stem cells (hESCs) and somatic stem cells 16 . The zinc finger transcription factor vascular endothelial zinc finger 1 (VEZF1) is over-expressed in Hem-derivatives, HUVEC, and cord blood SCs. VEZF1 is normally expressed in endothelial cells during vascular development 51,52 . A rare subset of HemSCs that express the endothelial marker PECAM-1 co-express the transcription factors SALL4 and VEZF1. The identification of this subset further suggests the existence of intermediate phenotypes.
Gene expression is a dynamic process 53,54 . The variability observed at the single-cell level reflects true differences, as opposed to averaged measurements of a large number of cells such as those obtained by microarray and qPCR. Our microarray and qPCR analysis of Endoglin showed homogeneous expression that did not reflect the actual heterogeneity occurring at the single-cell level. Our qPCR/microarray analysis of bulk gene expression in 100-200 flow sorted HemSC showed that HemSCs differ from control HUVEC and are characterized by low expression of endoglin. However, the analysis of HemSC gene expression at the single-cell level (also validated by flow cytometry and bioimaging) clearly identified a heterogeneous expression pattern of the endoglin (ENG) gene, revealing a unique subset of cells (HemSC ENG+ ) and stem cell heterogeneity, with ENG expression levels varying from high to low/non-detectable. The identification of HemSC ENG+ and HemSC ENGprogenitor subsets suggests the existence of intermediate phenotypes (transitioning progenitor populations of interest); a hemangioma stem cell (hemangioma hemangioblast) dysregulated/arrested in development -differing from multiple stem cells (such as neural crest SC, bone marrow SC, cord blood SC, mesenchymal SC) heterogeneously dysregulated/ arrested at varying stages of development; and/or a stem cell marker that reflects tumor heterogeneity (the inactive normal stem cell vs. the active neoplastic stem cell involved in vascular pathogenesis [hemangioma-genesis]).
The endoglin gene encodes a transmembrane auxiliary receptor for TGF-β 55 . TGF-β signaling is important in normal vascular development (vasculogenesis, angiogenesis, and embryonic vascular assembly) and plays a role in tumor angiogenesis 32,55-61 . In early development, endoglin is required for hemangioblast specification, is a marker for an embryonic progenitor, the hemangioblast (in addition to the hemangioblast marker KDR/ VEGFR-2/CD309), and serves as an important regulator of hematopoietic and endothelial lineage commitment [56][57][58][59][62][63][64][65][66][67] . Endoglin expression levels are heterogeneous in different cell sub-populations. For instance, during yolk sac development, blood cells are characterized by low expression, whereas endothelial cells present high expression of this receptor 56 . In mouse embryonic stem cells-derived embryoid bodies endoglin marks the hemangioblast on day 3 of differentiation, and in the absence of endoglin (Eng −/− ) there is a significant reduction in hemangioblast frequency. Eng-null (Eng −/− ) mice die by embryonic day 10 due to abnormal vasculature development 56,68 . Within normal tissue, endoglin is highly expressed in active vascular endothelial cells during embryogenesis and in syncytiotrophoblasts of term placenta, and is expressed at low levels in resting endothelial cells 55 . In active, normal endothelial cells, endoglin expression is required for TGF-β receptor ALK1 (activin A receptor type II-like 1/ACVRL1) signaling, which promotes proliferation, migration, and angiogenesis 69 . ALK1 expression indirectly inhibits ALK5 (TGF-β receptor 1/TGFβ R1/ TGFR1). In resting (quiescent) endothelium, endoglin is expressed at low/non-detectable levels. Thus, TGF-β /ALK5 signaling inhibits cell proliferation and migration. In vascular pathology, endoglin expression is highly expressed and upregulated in tumor endothelium, placenta-derived cells (in pre-eclampsia), and endothelial cells in response to vascular injury 32,55 . Endoglin expression is regulated and stimulated by hypoxia and TGF-β signaling pathways 70 . Further single-cell characterization of the HemSC ENG+ (PROM1 High /ENG High ) and HemSC ENG− (PROM1 High /ENG Low ) sub-populations, and analysis of the endoglin zinc finger/promoter domain (SP/KLF) 32 can differentiate the hemangioma hemangioblast from the derivative population, the HemSC (dysregulated subset) from the other stem cells present in the vascular compartment, and the inactive normal stem cell vs. the active neoplastic stem cell.
Several observations point to an important role of the immune system in hemangioma-genesis. The initial stage of IH proliferation generally coincides with the onset of maturation of the immune system (at age 6-12 months), and a significant number of CD8+ T cells have been identified in IH 71,72 . During the transition from the proliferation to the involution phase of IH, the immune system can also play a role with variation in indoleamine 2,3-dioxygenase (IDO) expression levels 72 . Understanding the specific crosstalk between the host immune system and IH during these stages of tumor development is important; for example, to identify the strategies HemSCs use to evade immunosurveillance and the potential benefits of immunotherapy. We analyzed the hemangioma cell surface proteome by a comprehensive stem cell surface marker screening panel including monoclonal antibodies to molecules that modulate the immune response. Based on our results and data published in the literature, an interplay can be envisaged between the immune system and hemangioma-genesis. IH arises at the time of transition from immunotolerance to immunocompetence, which can enhance the growth of IH. The onset of immunocompetence at birth coincides with a transition to a higher number of adult immune stem cells than fetal immune stem cells [73][74][75][76][77] . Our analysis of the cell surface markers in the proliferative phase of IH formation reflects mechanisms to escape immunosurveillance. CD47, "a 'don't eat me' signal for phagocytic cells" is expressed on the Scientific RepoRts | 6:35811 | DOI: 10.1038/srep35811 surface of IH cells as well as on "all human solid tumor cells" 78 . In addition to the 'don't eat me' sirens, tumors use several alternative strategies to evade immunosurveillance and a variety of these can be identified in IH. The generation of adenosine by CD73, highly expressed by IH, suppresses the T cell response within the tumor microenvironment 79 . This immunosuppressive effect promotes tumorigenesis [79][80][81] . CD59 and CD46, also highly expressed in IH, prevent complement-mediated tumor cell lysis 82 . CD59 (Protectin) serves as a line of defense, acts against complement-mediated lysis by incorporating into a membrane attack complex (MAC), and is regarded as a powerful inhibitor of complement cytolysis. CD46, a complement regulatory protein, inhibits complement activation and serves to protect the host cell against attack 82 . Additional cell surface markers included CD10 (zinc-binding metalloendopeptidase and diagnostic pediatric lymphoma and leukemia cell surface marker) 36,39,40 , and CD13 (zinc-binding metalloprotease aminopeptidase, pericyte cell surface marker, and myeloid [normal and neoplastic] cell surface marker) 36,38 .
We studied tumorsphere formation by HemSCs to investigate their tumorigenic and differentiation potential. CD44 served as a highly expressed cell surface marker to validate the expression profile in the HemSC-derived tumorsphere and tissue array analysis. We used PDGFR-β , a highly specific pericyte marker 83 , to identify the HemPericytes among the cultured derivatives. Our analysis revealed abundant PDGFR-β + HemPericytes. In normal vascular development, endothelial cells and pericytes are derived independently with distinct lineages 20,21 although there are "intimate interactions between endothelial cells and pericytes" 44 . During angiogenesis, PDGFR-β + cells are attracted to the vascular endothelium and secrete vascular endothelial growth factor (VEGF) 43,44 . Pericytes control endothelial cell proliferation and differentiation via a paracrine and cell-cell contact mechanism, and take an active role in angiogenesis as a survival factor. Thus, HemSC-derived pericytes may serve as a survival factor for the proliferating IH. Importantly, the de novo formation of pericyte-like derivatives reflects the stem cell heterogeneity of HemSCs in the tumorsphere (including undifferentiated perivascular mesenchymal cells among other stem cells) and a hemangioma-derived multipotential stem cell arrested at an early stage of development (incompletely differentiated) with an ability to differentiate into several cellular lineages (lineageand non-lineage specific differentiation).
The results of our study provide insight into the cellular and molecular mechanisms underlying the complex clinical diversity in IH; however, the limited number of our patient samples is insufficient to assess the clinical variability for classification. We used cellular, molecular, and genetic techniques -in particular flow cytometry -for assay development of a comprehensive panel of potential biomarkers and drug-targets. Our specific panel (Table 6) can be used for future studies of large cohorts for biomarkers and therapeutic targets (existing or novel drugs); the construction and analysis of gene regulatory networks; the characterization of hemangioma-derived stem cell subsets by established methods of multiparameter analysis (e.g. the identification of multiple cell surface markers will afford polychromatic flow cytometry [PFC] 84 investigation to further characterize dysregulated HemSCs); the study of IH-derived stem cell subsets by novel techniques and applications (e.g. isoform sequencing platforms for novel genes/gene isoforms and single-cell transcripts); the dissection of IH heterogeneity (including different cell types) for clinical/pathological classification; and the development of novel precision medicine isogenic models, such as patient-derived human induced pluripotent stem cells for a precision medicine approach to the treatment of the individual patient. For practical applications, an antibody panel can be designed to identify expression of GLUT1 (diagnostic clinical marker for IH) and IH cell surface signature markers (such as CD10, CD13, CD44 and CD73) by immunohistochemistry, an established method routinely used for in vitro diagnostic assays and a useful adjunct to diagnostic histology.
Besides surgical excision, current pharmacological treatments for IH, corticosteroids or propranolol, are administered for several months and have adverse effects. Rapamycin, an mTOR inhibitor, has been shown to inhibit HemSC self-renewal and vascular differentiation potential in patient-derived hemangioma stem cells 85 . Our microarray data for HemSCs GLUT1 High showed high variability in mTOR gene expression but significant over-expression of AKT1 (one of the upstream kinases in adipocytokine signaling pathways). Our gene expression analysis show high levels of PPARγ , a "pro-adipogenic" and anti-inflammatory response gene. The expression of the placental glucose transporter (such as GLUT1) is regulated by glucocorticoids (adrenal steroids) and the nuclear receptor PPARγ (which interacts with estrogen to regulate and promote adipogenesis) 30,86 . Researchers have performed quantitative studies to investigate the role of "pro-adipogenic" genes (such as PPARγ ) in hemangioma-genesis, proliferation to involution, and their interaction with propranolol, a beta-blocker used in the treatment of IH 29 . Targeting the adipocytokine/PPAR signaling pathway affords an alternative therapeutic strategy to challenge the fibrofatty differentiation of IH, which can result in life-long, function-compromising effects including disfigurement. CD73 serves as a potential biomarker and drug target for IH, with the adenosine pathway serving a critical role in immune activation and inflammation 87 . To target the immunosuppressive effects that promote tumorigenesis [79][80][81] , immune response modifiers, such as humanized monoclonal antibody to CD73, can serve as a complementary treatment. For instance, a humanized monoclonal antibody to CD73 may inhibit IH growth by blocking CD73 and stimulating the activity of T cells.
In conclusion, our data show that IH originates from a dysregulated but not fully transformed multipotent stem cell, which orchestrates IH pathophysiology through multiple signaling and regulatory networks. The potential biomarkers we identified can afford the development of diagnostic tools and precision-medicine therapies to "rewire" 88 or redirect cellular transitions at an early stage, such as signaling pathways or immune response modifiers. Our investigative approach can be used to characterize dysregulated progenitor cell subsets involved in other diseases and identify druggable targets for their treatment. For example, our microarray data include normal human stem and progenitor cell subsets. This information provides a valuable tool for researchers to evaluate biomarker analysis and drug-targeting pathways, as well as a reference for studies of global gene expression in different cell types, developmental stages, diseases (e.g. investigating progenitor cells in an inflammatory environment), and cell-based therapies such as for regenerative medicine.       [cord blood SC]; and mesenchymal CD133+ [mesenchymal SC]). The HemSC were isolated as follows. Specimens of IH were obtained during surgery and a pathologist confirmed the diagnoses. HemSCs were isolated from these specimens immediately after surgical excision as described by Khan et al. 14 . IH samples were minced with a scalpel and digested using collagenase (Roche) to obtain a single-cell suspension. Cells expressing CD133 were isolated using the magnetic microbead cell sorting system (Miltenyi) and seeded onto adherent fibronectin coated plates (BD Biosciences) (in EBM-2 medium supplemented with 20% fetal bovine serum [FBS] and penicillin/streptomycin [Lonza]). When 60-80% confluency was reached, the cells were passaged and seeded on adherent fibronectin/matrigel coated cell culture plates (BD Biosciences) and non-adherent ultra-low attachment cell culture plates (Corning) for HemSC-derived tumorsphere formation. Prior to gene expression experiments, early passage, pure HemSC -CD133 (Miltenyi) positive cells were isolated by fluorescence-activated cell sorting.  Tumorsphere Bioimaging. Using the Applied Precision Personal DV live-cell imaging system, the study design included 21-h imaging studies with live imaging of HemSC-Derived tumorsphere and derivative formation/interaction. Controlled studies included use of nuclear stain, cell surface markers, the specificity controls (Ms IgG2a, κ ), and beads tagged with the secondary for exact instrument intensity measurements.  were analyzed by HT flow cytometry using the BD -HT sampler instrument (LSRII HTS) and high-content bioimager, Cellomics ArrayScan HC (high-content and high-throughput) imaging microscope. Cell Surface Marker Screening of HemSCs -Early passage cells were analyzed and flow sorted using the BD Aria II (BD Biosciences) instrument, FACSDiva (BD Biosciences) and FlowJo software (FlowJo, LLC). Appropriate compensation studies were conducted. Using a HT approach, 500 CD133+ /CD31−cells (Miltenyi) were sorted directly into 96-well plates and introduced to 20 μ L/well of purified antibodies, conjugated with AlexaFluor 647 detection reagent (BD Biosciences), and fixed with CytoFix fixation buffer (BD Biosciences). The profiles of the human cell surface markers (high expression markers from heterogeneous cell population) were analyzed by HT flow cytometry using the BD LSRII HTS instrument.

Gene Expression Analysis
HT Gene Expression Analysis. Prior to gene expression analysis, early passage IH (H41, H48, H50, H52, H53) HemSCs (progenitor) and differentiated cells (derivatives), glioblastoma CSC, mesenchymal SC, bone marrow SC, cord blood SC, and HUVEC cells were FACS enriched and sorted for purity. The microarray data were confirmed by qPCR and single-cell gene expression for the highest expressing genes for precise measurement. In addition, HemSC gene expression data were confirmed by comparison of phenotypic expression of HemSC specific cell surface markers. The microarray data are available at the GEO database (http://www.ncbi.nlm.nih. gov) -GEO accession number GSE34989.
Microarray. The cells were flow sorted directly into Miltenyi SuperAmp lysis buffer for SuperAmp preparation kit, and RNA amplification was performed on a global PCR protocol. The mRNA was isolated using magnetic bead technology; 250 ng of amplified cDNA was used for gene expression analysis (~40,000 genes) using Agilent whole genome oligo microarrays (one-color -Cy3 labeling). Data analysis was performed with Gene Set Enrichment Analysis (GSEA) software (with standard gene set as permutation type, 1,000 permutations and log2 ratio of classes as metric for ranking genes) and Partek Genomics Suite software. With the Partek Genomics Suite software, data were normalized and gene expression analysis was performed using one-way Analysis of Variance (ANOVA) of samples. The gene list was created with specified criteria: 1) size of change defined as a fold change > 1.5 or fold change < -1.5; and 2) significance of change defined as p-value with False Discovery Rate (FDR) < 0.05 or p-value with False Discovery Rate (FDR) < 0.1. The gene expression intensity data images were generated with Partek Genomics Suite Software. qPCR. Quantitative real-time PCR was performed with an Applied Biosystems 7300 instrument using SYBR Green PCR Master Mix (Qiagen). 96-well PCR arrays (SABiosciences) were used to examine expression profiles of genes in sorted cell populations. Per manufacturer's instructions: per well (25.0 μ l final volume) = 12.5 μ l RT 2 SYBR Green qPCR Mastermix (#330520)/10.5 μ l ddH2O/1.0 μ l cDNA (up to 250 ng)/1.0 μ l (10 μ M) PCR primer pair assay; thermal profile protocol is specific for Applied Biosystems 7300 instrument: Stage 1: 95 °C, 10 min; Stage 2: 40 cycles of (95 °C, 15 sec; and 60 °C, 60 sec). For data analysis, fold regulation cut-off was set as 2 and the methods used for housekeeping genes (HKG)/internal controls included a list of genes selected from the entire 96 well plate including 5 HKG that had small changes in the threshold cycle (CT) values across all samples. C t values for these genes were then geometrically averaged and used for the Δ Δ CT calculations.

Single-Cell Gene Expression.
To survey gene expression profiles by a HT approach, fluorescence-activated cell sorting was performed using BD Aria II, 100 cells per well and for single-cell samples 1 cell directly flow sorted into the wells of 96-well plates containing Platinum Taq reverse transcriptase, polymerase master mix (Invitrogen) and primers for each gene target (SABiosciences) as per Fluidigm manufacturers Protocol 41 instructions and as described in the Nature Methods workflow for single-cell profiling 27 . The 2-step process included STA (specific target amplification) reaction (RT-STA cycling conditions and an Exonuclease I treatment method to remove unincorporated primers). The RT-STA solution (9.0 μ l per well) consisted of: 5 μ l CellsDirect 2X Reaction Mix/0.2 μ l SuperScript III RT Platinum Taq Mix/1 μ l 10X Primer Mix (500 μ M)/2.8 μ l Nuclease Free H2O (Invitrogen). The thermal cycling profile protocol was as follows: Stage 1: 50 °C, 15 min; Stage 2: 95 °C, 2 min; Stage 3: 20 cycles of (95 °C, 15 sec; and 60 °C, 4 min). The Exo I treatment method (total volume 3.5 μ l [per 9 μ l RT-STA solution]) consisted of: 2.52 μ l Nuclease Free H2O, 0.36 μ l Exonuclease I Reaction Buffer (10X), and 0.72 μ l Exonuclease I (20units/μ l) (New England BioLabs). The thermal cycling protocol was as follows: Stage 1: 37 °C, 30 min; Stage 2: 80 °C, 15 min. The final concentration of STAreaction + Exonuclease I was diluted 5-fold in this experiment. The human cDNA library (Biochain) was treated with RTA-STA/ExoI treatment method, diluted 5-fold with additional serial dilutions (1:3). In the NTC, negative control condition, no cells were sorted at all but RT-STA was applied. 5 primer assays were used (SABiosciences) with GAPDH as housekeeping gene and 4 high expressing genes reflecting involvement of multiple pathways (EGF/ENG/NOTCH/TIMP1).

Tumorsphere Analysis
HemSC-derived Tumorspheres. The in vitro tumorsphere formation assay involved a three-dimensional (3D) culture system with two main approaches. The first approach for tumorsphere growth consisted in the growth of spheres as a suspension in non-adherent 6-well plates, and the second approach involved the culture of spheres on Matrigel-or fibronectin-coated plates (BD Biosciences). STEMPro hESC-human embryonic stem cell culture medium (Invitrogen) or EBM-2 medium supplemented with 20% FBS and penicillin/streptomycin (Lonza) was used.
In one week of culture in non-adhesive plates, the cells in suspension started to form tumorspheres that grew in suspension or loosely attached to the culture substrate. The tumorspheres were captured with a sterile 25-ml serological pipet for analysis (by cytospin or paraffin embedded plasma-thrombin clot formation as described). For in vitro differentiation analysis, the spheres were transferred on Matrigel-or fibronectin-coated plates (BD Biosciences).
For immunohistochemical analysis, the culture medium was gently removed from the culture dishes. Human plasma was added, followed by the addition of thrombin. The spheres remained embedded in the plasma-thrombin clot were paraffin-embedded. Sections of paraffin-embedded plasma-thrombin clots were stained with hematoxylin and eosin (H&E) and for validation studies, tumorsphere sections were stained with 1) GLUT1 antibody -diagnostic clinical marker for IH; and 2) CD44 highest expressing marker in heterogeneous cell surface marker screening panel. For independent validation, the NYU Immunohistochemistry (IHC) Core Facility processed the sections according to the manufacturers' protocols. The primary antibodies used for immunohistochemical analysis (provided by Dr. Luis Chiriboga, NYU IHC Core Facility) were GLUT1 (Ventana 760-4526) and CD44 (Dako M7082). The primary antibody was detected with biotinylated goat anti mouse IgG antibodies (provided in the detection kit from the respective manufacturers). Additional controls included the use of tissue microarray for IHC.
For tumorsphere HT bioimaging and differentiation analysis, the HemSC-derived tumorspheres and differentiated derivatives were stained for the pericyte cell surface marker PDGFR-β (purified antibody to CD140b [clone 28D4]) (BD Biosciences) and directly conjugated with AlexaFluor 647 goat anti-mouse Ig. Prior to live imaging, tumorspheres and derivatives were stained with cell-permeable nucleic acid stain -Hoechst 33342 (BD Pharmingen).