Molecular and Phenotypic Characterization of CD133 and SSEA4 Enriched Very Small Embryonic-Like Stem Cells in Human Cord Blood

ORIGINAL ARTICLE Molecular and phenotypic characterization of CD133 and SSEA4 enriched very small embryonic-like stem cells in human cord blood

A Shaikh1, P Nagvenkar1, P Pethe1, I Hinduja2 and D Bhartiya1

Very small embryonic-like stem cells (VSELs) are immature primitive cells residing in adult and fetal tissues. This study describes enrichment strategy and molecular and phenotypic characterization of human cord blood VSELs. Flow cytometry analysis revealed that a majority of VSELs (LIN−/CD45−/CD34+) were present in the red blood cell (RBC) pellet after Ficoll-Hypaque centrifugation in contrast to the hematopoietic stem cells (LIN−/CD45+/CD34+) in the interphase layer. Thus, after lyses of RBCs, VSELs were enriched using CD133 and SSEA4 antibodies. These enriched cells were small in size (4–6 μm), spherical, exhibited telomerase activity and expressed pluripotent stem cell (OCT4A, OCT4, SSEA4, NANOG, SOX2, REX1), primordial germ cell (STELLA, FRAGILIS) as well as primitive hematopoietic (CD133, CD34) markers at protein and transcript levels. Heterogeneity was noted among VSELs based on subtle differences in expression of various markers studied. DNA analysis and cell cycle studies revealed that a majority of enriched VSELs were diploid, non-apoptotic and in G0/G1 phase, reﬂecting their quiescent state. VSELs also survived 5-ﬂuorouracil treatment in vitro and treated cells entered into cell cycle. This study provides further support for the existence of pluripotent, diploid and relatively quiescent VSELs in cord blood and suggests further exploration of the subpopulations among them.

Leukemia (2015) 29, 1909–1917; doi:10.1038/leu.2015.100

INTRODUCTION selection and FACS.20 Aldefluor staining combined with the use of It was hypothesized that adult tissues may contain embryonic CD45 and Glycophorin A as lineage marker was also used to enrich − − + 24 remnants that are ‘lost’ during developmental organogenesis VSELs as CD45 /GlyA /Aldh cells. However, these reports that lie dormant and may give rise to some malignancies.1 neither provided comprehensive cellular characterization after The existence of such embryonic-like cells in mouse and human magnetic sorting nor have enriched VSELs using pluripotent stem tissues was demonstrated initially by Ratajczak and colleagues2 cell markers like OCT4 or SSEA4. Thus, certain questions regarding and were termed as very small embryonic-like stem cells (VSELs). properties of cord blood VSELs still remain unanswered. Attempts They are a population of developmentally primitive cells persisting were made to isolate VSELs using CXCR4 and negative selection in adult tissues.3 They become mobilized into peripheral blood for LIN-1 and CD45. However, these studies reported absence of during stress situations and tissue injury,4–7 and are enriched as pluripotency in VSELs.22,23 This inconsistency in data has led LIN–CD45–SCA1+ cell fraction in mice2 and as LIN–CD45–CD133+ to opposing views on the presence and functionality of cell fraction in humans.8 They express pluripotent markers and VSELs.22,23,25,26 have high nucleocytoplasmic ratio and undifferentiated open Unlike pluripotent embryonic stem (ES) and induced pluripo- chromatin.8,9 In vivo studies have demonstrated that VSELs exhibit tent stem (iPS) cells that divide rapidly in vitro and form teratoma characteristics of long‐term repopulating hematopoietic stem cells in mice,27,28 VSELs express pluripotent markers but are relatively (HSCs),10,11 are at the top of the hierarchy in the mesenchymal quiescent, do not divide when cultured feeder free and do not lineage12 and may differentiate into organ‐specific cells.13–16 It has form teratoma.2 Quiescence in murine VSELs has been attributed also been proposed that VSELs may initiate tumor growth,17 and to their unique genomic imprinting pattern,9 but the quiescent recently our group has proposed a role of VSELs in ovarian18 and nature of cord blood VSELs in terms of cell cycle status has not yet pancreatic19 cancers. been examined. Resistance of quiescent murine bone marrow However, there is still a lack of consensus on the isolation VSELs to radiotherapy10 and testicular and ovarian VSELs to protocols for VSELs and only a handful of studies are available chemotherapy29,30 has also been documented. However, similar describing them in cord blood.8,11,20–23 Various features of VSELs, effect of radio- or chemotherapy on human VSELs remains to be such as their small size, pattern of expression markers or a elucidated. combination of both, have been used to isolate them from cord This study aimed to enrich cord blood VSELs using cell surface blood. Initially, fluorescence-activated cell sorting (FACS) was markers CD133 and SSEA4 and further characterize them using employed to enrich VSELs among a population of CD34+/LIN−/ various approaches. The cell cycle status and the in vitro effect of 5- CD45− cells.8 This was a time-consuming protocol and not easily Fluorouracil (5-FU, a nucleotide analog that gets incorporated into adapted for clinical use. The method was then improved by using DNA and causes death of cycling cells whereas quiescent stem cells a combination of red blood cell (RBC) lysis, CD133 immunomagnetic are protected31,32) on cord blood VSELs has also been studied.

1Stem Cell Biology Department, National Institute for Research in Reproductive Health, Mumbai, India and 2Jaslok Hospital & Research Centre, Mumbai, India. Correspondence: Professor D Bhartiya, Stem Cell Biology Department, National Institute for Research in Reproductive Health, JM Street, Parel, Mumbai 400012, India. E-mail: [email protected] or [email protected] Received 2 February 2015; revised 19 March 2015; accepted 7 April 2015; accepted article preview online 17 April 2015; advance online publication, 22 May 2015 Characterization of cord blood VSELs A Shaikh et al 1910 MATERIALS AND METHODS were captured by laser scanning confocal microscope (Carl Zeiss, The study was approved by the ethics committees of National Institute Oberkochen, Germany) using × 63 objective. for Research in Reproductive Health and Jaslok Hospital. Cord blood samples were collected after obtaining written informed consent from Quantitative real-time reverse transcription-PCR the participants with planned C-section deliveries and were processed Expression of pluripotency (OCT4A, OCT4, NANOG, SOX2, REX1 and TERT)as within 24 h. well as cell surface (CD133 and CXCR4) markers was studied by quantitative real-time reverse transcription-PCR using the CFX96 real-time PCR system Flow cytometry analysis of human cord blood (Bio-Rad Laboratories, Hercules, CA, USA). Details of primers, amplification Human cord blood was subjected to Ficoll-Hypaque centrifugation (details conditions and calculations for relative fold change are described in fi in Supplementary section) and the cells from both interphase layer and Supplementary section, Supplementary Table S2. Human fetal broblasts from RBC pellet were processed further to study HSCs (LIN−/CD45+/CD34+) were used as a calibrator to compare differences in marker expression − − + fl between RBC pellet and interphase cells, whereas unsorted cord blood cells and VSELs (LIN /CD45 /CD34 )by ow cytometry. The cells were stained + + for antibodies against lineage markers (LIN-1), CD45 and CD34 (details in were used as calibrator for studying CD133 and SSEA4 enriched cells. Melt fi Supplementary Table S1) for 60 min on ice. After washing, they were run curve analysis was performed at the end of every run to con rm the fi on BD FACS Aria (BD Biosciences, San Jose, CA, USA) and analyzed by using homogeneity of the PCR products that were also electrophoresed to con rm BD FACS Diva software. The method used for analysis of flow cytometry product size using 2% agarose gel (Bangalore Genei; data not shown). data is described in the Supplementary section. Cell cycle analysis Immunomagnetic enrichment of CD133- and SSEA4-positive cells For cell cycle and ploidy analysis, CD133+ and SSEA4+ enriched cells and The cells collected from the RBC pellet were subjected to immunomag- human embryonic stem (hES) cells were suspended in 0.5 ml of cold μ netic enrichment for CD133+ and SSEA4+ cells using EasySep APC and PE hypotonic solution containing 0.1% sodium citrate, 0.3 l/ml NP-40 (Sigma), μ Positive Selection Kits (Stem Cell Technologies, Vancouver, BC, Canada) 2 mg/ml RNaseA (Sigma) and 50 g/ml propidium iodide (Sigma). The tubes respectively. Briefly1×108 cord blood cells were stained with CD133-APC were placed in dark at 4 °C. Flow cytometry analysis was performed after a antibody (Miltenyi Biotech GmbH, Bergisch Gladbach, Germany) for 15 min minimum 20 min of hypotonic treatment. Fluorescent events were acquired fl at room temperature (RT), followed by incubation with APC selection on a BD FACS Aria ow cytometer and analyzed using with BD FACS DIVA cocktail for 15 min at RT. The cell suspension was then incubated with software and FCS Express 4 software. magnetic nanoparticles for 10 min at RT and placed on a magnetic particle concentrator. Similar protocol was followed for purification of SSEA4+ cells Evaluation of telomerase activity with the use of SSEA4 PE (BD Biosciences) and PE selection cocktail. Further + + Telomerase activity was determined by performing TRAP (Telomerase characterization studies were done on CD133 and SSEA4 enriched cells. Repeat Amplification Protocol) assay on CD133+ and SSEA4+ enriched cells, Cells in both positive and negative fractions were counted, assessed for hES cells (positive control) and human fetal fibroblasts. Cell lysates from viability and purity, used for immunophenotyping studies, making cell various sources (1–2×106) were used to measure telomerase activity using smears and for protein and RNA extraction (details in Supplementary TRAPeze RT kit (Millipore, Temecula, CA, USA) by following the fl section). Cells enriched for CD133 were also subjected to uorescent in situ manufacturer’s protocol (details given in Supplementary section). hybridization to rule out aneuploidy for chromosomes 13, 18, 21, X and Y (details in Supplementary section). Effect of 5-FU treatment on cord blood cells in vitro Total nucleated cells were obtained from cord blood by RBC lysis followed Immunophenotyping studies on CD133 and SSEA4 enriched cells by centrifugation at 1000 g for 10 min. Total nucleated cells were treated The CD133 and SSEA4 enriched cells were immunophenotyped for LIN-1, with 5-FU (Biochem, Mumbai, India, 25 μg/ml) prepared in Dulbecco’s CD45, CD34, SSEA4, CXCR4, CD133, OCT4A, STELLA and CXCR4 (antibody modified Eagle’s medium/F12 containing 10% fetal bovine serum details in Supplementary Table S1). Cell membranes were permeabilized (Invitrogen) for 24 h at 37 °C. Later, the cells were washed twice with using a 0.3% Triton X-100 solution (Sigma) in phosphate-buffered saline PBS and analyzed for percentage of CD133+ and CD34+ VSELs (LIN−/CD45−) (PBS) for OCT4 and STELLA. Briefly, the cells were incubated in FcR block and HSCs (LIN−/CD45+) as well as for cell cycle status by flow cytometry. for 20 min at 4 °C (Miltenyi Biotech) and then subjected to immunolabel- Cells were also studied for expression of pluripotent markers at both ing with directly labeled monoclonal anti-human antibodies (LIN-1, CD45, protein and transcript levels. CD34, SSEA4, CXCR4, CD133 and CXCR4) by incubating for 60 min at 4 °C. Indirect labeling was performed by incubating with the antibodies (OCT4A, STELLA and CXCR4) for 60min at 4°C followed by secondary Statistical analysis antibody labeling for 30 min at RT using Alexafluor goat anti-mouse IgG Arithmetic means and s.d. values of our flow cytometry data were 568 or IgG 488 (Molecular Probes, Invitrogen, Carlsbad, CA, USA). calculated using Graph Pad prism 6 (GraphPad, San Diego, CA, USA) Following washing with PBS, events were acquired on a BD FACS Aria software. Data were analyzed using Student’s t-test for unpaired samples. flow cytometer and analyzed using with BD FACS DIVA software and FCS Statistical significance was defined as Po0.05 and error bars in graphs Express 4 software (De Novo software). represent ± s.e.m.

Immunofluorescence studies RESULTS The expression of nuclear (OCT4A, STELLA) and cell surface (CD45, CD34, Analysis of cord blood VSELs (CD34+Lin−CD45−) CD133, FRAGILIS) antigens along with cytoplasmic ACTIN was studied on cell smears. The fixed smears were washed with wash buffer (PBS VSELs (4–6 μm) are smaller in size than most blood cells and are lost 20 containing 0.3% bovine serum albumin and 0.1 mM EDTA), permeabilized at various steps during processing. In contrast to HSCs, gradient by 0.3% Triton X-100 for 10 min, washed in PBS, preblocked with 1% centrifugation depletes the initial number of VSELs by ∼ 50%.20 We bovine serum albumin and 5% normal goat serum (Bangalore Genei, have demonstrated presence of VSELs in the RBC fraction after Bangalore, India) and subsequently incubated with antibodies (antibody Ficoll-Hypaque centrifugation21 that was later also reported by details in Supplementary Table S1) overnight at 4 °C. Permeabilization other studies.22,23 Flow cytometry results of this study (Figure 1a with Triton X-100 was omitted for cell surface staining. After washing, − −/ + fl and Table 1) reveal that percentage of VSELs (LIN /CD45 CD34 )is cells were incubated with secondary antibody (1:500 dilution) Alexa uor higher in the RBC pellet (0.073 ± 0.008%) compared with interphase goat anti-mouse IgG 568 (OCT4 and STELLA) and Alexafluor donkey anti- − + + rabbit IgG 568 (FRAGILIS and CXCR4) for 2 h at RT. This step was omitted layer (0.03 ± 0.009%), whereas HSCs (LIN /CD45 /CD34 )aremore for directly labeled CD34, CD45, CD133 and SSEA4 antibodies. Negative in interphase layer (0.156 ± 0.027%) compared with that in RBC controls were incubated in the blocking solution with the omission of pellet (0.005 ± 0.003%). Using size-based beads as reference, size of + primary antibody. Counterstaining was done using 4,6-diamidino-2- VSELs were found to be between 4 and 6 μm and CD45 HSCs were phenylindole (Molecular Probes) or propidium iodide (Sigma). All images 46 μm (Supplementary Figure S1A) in size. Hematoxylin and eosin

Figure 1. Characterization of cells in the interphase layer and RBC pellet obtained after Ficoll-Hypaque centrifugation of cord blood. (a) Flow cytometric analysis of cells from interphase and RBC pellet (after RBC lysis) for evaluating number of VSELs (CD34+/CD45−/LIN−) and HSCs (CD34+/CD45+/LIN−). Events ranging from 3 μm are included in gate after comparison with different size beads having diameters of 1, 2, 4, 6, 10 and 15 μm. Cord blood-derived interphase and RBC pellet cells are shown on FSC vs SSC dot plot and gated (P1). Cells in P1 are analyzed for Lineage markers (LIN-1) expression. The LIN-1-negative cells were further analyzed for coexpression of CD45 and CD34. Note the percentage of VSELs is higher in RBC pellet, whereas the HSCs are more in interphase layer. Representative data of six independent experiments are shown. (b) Immunofluorescence staining of cells present in RBC pellet shows that they are positive for OCT4A, SSEA4, CD133, CXCR4, STELLA, FRAGILIS and ACTN but are negative for CD45. All images were taken under × 63 objective (Carl Zeiss, bar 10 μm). (c) Comparison of embryonic stem cell markers (OCT4A, OCT4, NANOG, SOX2, REX1 and TERT) and hematopoietic marker CD133 in interphase and RBC pellet by quantitative real-time reverse transcription-PCR. All the transcripts levels were higher in RBC pellet compared with interphase layer. The data represent fold difference obtained with respect to human fetal fibroblasts set as 1. **Po0.01 represents statistical significance between interphase layer and RBC pellet.

Table 1. Molecular and phenotypic differences between cells in the interphase layer and RBC pellet collected after Ficoll-Hypaque centrifugation of cord blood

Cells in RBC pellet Interphase cells

Morphology Small cells with high N/C ratio Larger cells with distinct cytoplasm Immunolocalization OCT4A+STELLA+CD133+FRAGILIS+CD34+SSEA4+CD45− Most cells are CD45+, few cells are CD34+and CD133+ Gene expression Higher expression of Very low expression of pluripotency and VSEL markers pluripotency transcripts (OCT4, OCT4A, SSEA4, NANOG, SOX2, REX1) VSEL transcripts (CD133, CXCR4) LIN−CD45−CD34+ VSELs 0.073 ± 0.008% 0.031 ± 0.009% LIN−CD45+CD34+ HSCs 0.005 ± 0.003% 0.156 ± 0.026% Abbreviations: HSC, hematopoietic stem cell; N/C, nucleus to cytoplasmic; RBC, red blood cell; VSEL, very small embryonic-like stem cell.

Figure 2. Characterization of CD133+ and SSEA4+ enriched cell fraction. (a) Phenotypic characterization of CD133 positive fraction by flow cytometry for pluripotent and hematopoietic markers. All CD133+ cells showed no expression of lineage markers but a fraction of CD133+ cells expressed CD34, SSEA4, OCT4A CXCR4 and STELLA. M1 represents average percentage (mean ± s.d.) of the positive fraction in CD133+ cell population and negative fraction in LIN-1 staining (gray histogram represents positive population and empty histogram represents unstained population). (b) Phenotypic characterization of SSEA4+-positive fraction by flow cytometry for pluripotent and hematopoietic markers. All SSEA4+ cells showed no expression of lineage markers but a fraction of SSEA4+ cells expressed CD34, CD133, OCT4A, CXCR4, and STELLA. M1 represents average percentage (mean ± s.d.) of the positively stained cells in SSEA4+ cell population and negative fraction in LIN-1 staining (gray histogram represents positive population and empty histogram represents unstained population). (c) Immunofluorescence staining on CD133+ cells show positive expression for CD133, CD34, SSEA4, STELLA, OCT4A and FRAGILIS. All images were taken under × 63 objective (Carl Zeiss, bar 10 μm).

staining showed that VSELs in the RBCs pellet were small, spherical transcription-PCR (Figure 1c). Cells in the RBC pellet had cells with high nucleocytoplasmic ratio and thin rim of cytoplasm significantly higher mRNA transcript levels for OCT4A, OCT4, (Supplementary Figure S1B). NANOG, SOX2, REX1 and other cell surface markers like CD133 and Immunofluorescence studies (Figure 1b) showed positive CXCR4 compared with cells in the interphase layer. OCT4 encodes expression for pluripotency markers (nuclear OCT4A and cell for two major spliced variants, OCT4A and OCT4B, derived by surface SSEA4), primordial germ cell markers (STELLA and alternative splicing.34,35 OCT4A is localized to the nucleus and is an FRAGILIS) and early hematopoietic markers (CD133 and CD34). important marker to demonstrate pluripotent state of stem cells, Figure 1b also shows the absence of CD45, a pan-hematopoietic whereas cytoplasmic OCT4B is localized in cytoplasm and has no marker in few cells and dual staining of SSEA4 with actin reported biological function.36 The results of this study showed (PHALLOIDIN). These results confirmed the presence of pluripo- that the ratio of OCT4A to total OCT4 is almost one, suggesting tent stem cells with high nucleocytoplasmic ratio and thin rim of that RBC pellet contains majority of VSELs with nuclear OCT4A and cytoplasm in the RBC pellet. Expression of primordial germ cell not progenitors. The increased number of OCT4A-expressing cells markers was similar to earlier reports on murine BM-VSELs33 and could be attributed to the fetal nature of the cord blood and suggests that cord blood VSELs have a possible link to epiblast- justifies that cord blood may be a good source of VSELs. derived primordial germ cells. Table 1 summarizes the results mentioned above and describes The gene expression profile of cells from interphase layer and the key differences in cells present in the interphase layer and in RBC pellet was compared using quantitative real-time reverse RBC pellet after Ficoll-Hypaque centrifugation of cord blood.

Figure 3. Immunofluorescence staining, quantitative real-time reverse transcription-PCR and analysis of DNA content of CD133-enriched cells. (a) Dual immunofluorescence staining shows that CD133+ cells co-express OCT4 with SSEA4 and FRAGILIS with ACTIN and are also negative for CD45. All images were taken under × 63 objective (Carl Zeiss, bar 10 μm). (b) Expression of pluripotent (OCT4A, OCT4, NANOG, SOX2, REX1 and TERT) and hematopoietic (CD133, CXCR4) transcripts in CD133+ cells. The data represent average (mean ± s.e.m.) fold difference with respect to unsorted cells and is a mean of three experiments. *Po0.05 represents statistical significance between positive and negative fractions. (c) Ploidy and cell cycle analysis of CD133+ and SSEA4+ enriched cells and embryonic stem cells by flow cytometry using propidium iodide (PI). More than 90% of sorted positive fractions were diploid (as seen by dot plot of PE-A vs PE-W). The diploid cells were then analyzed + + on multicycle plot. A majority of CD133 and SSEA4 enriched diploid cells were in G0/G1 phase of cell cycle. The multicycle plot of diploid embryonic stem cells (positive control) shows all three phases of cell cycle (G0/G1, S and G2), with a majority of cells in the proliferative S phase. (d) Telomerase activity of CD133+ and SSEA4+ enriched cells is 2.5-fold higher than human fetal fibroblasts (HFFs). ES cells express maximum activity. *Po0.05 represents statistical significance when compared with HFF. **Po0.01 represents statistical significance when compared with HFF.

Because of their very small size, pluripotent VSELs have been CD34) and nuclear (OCT4A, STELLA) markers and subjected to unknowingly discarded as debris during isolation protocols and immunophenotyping studies. Results show that both CD133+ and have been aptly described as ‘missed pearls’.37 SSEA4+ expressed HSC (CD34), primordial germ cell (STELLA) and pluripotent (OCT4A) markers (Figures 2a and b). More than 95% of + + Enrichment and characterization of CD133 and SSEA4 enriched CD133 and SSEA4 enriched cells were negative for lineage VSELs marker (LIN-1) suggestive of their primitive nature as LIN-1 Immunomagnetic separation of cord blood cells demonstrated negative implies that they do not express mature blood cell 57% and 52% purity for CD133-APC and SSEA4-PE selection, markers (CD4, CD2, CD3, CD14, CD16, CD19 and CD56). It was also + + respectively (Supplementary Figure S2A). In both the magnetic observed that although CD133 and SSEA4 cells co-expressed separations, the negative fractions were devoid of positive cells CD34, there was a fraction of positive cells that were negative for + − (Supplementary Figure S2B). More than 90% cells were viable CD34 (Figures 2a and b). This CD133 /CD34 fraction is after magnetic sorting as analyzed by propidium iodide staining hypothesized to be responsible for the difference in in vivo + 11,38 + (data not shown). biological effects of CD133 cells. More than 60% of CD133 Both the positive and negative fractions obtained after and SSEA4+ cells expressed CXCR4 (Figures 2a and b). High immunomagnetic enrichment of CD133 and SSEA4 cells were expression level of CXCR4 correlates with circulatory nature of stained with various cell surface (LIN-1, CD133, SSEA4, CXCR4, cord blood.39 Intriguingly, the percentage of CD133+ cells

Figure 4. Effect of 5-Fluorouracil (5-FU) treatment on cord blood cells. (a) Flow cytometric analysis of cells before (control (C)) and post 5-FU (treated (T)) for evaluating number of VSELs (CD45−/LIN−) and HSCs (CD45+/LIN−) coexpressing CD133. The cells in size range from 3 to 15 μm (C1, T1) and were analyzed for expression of lineage marker LIN. LIN−cells (C2, T2) were then analyzed for cells CD45+/CD133+ (HSCs; C4, T4) and CD45−/CD133+ (VSELs; C3, T3). Note there is no change in VSELs and HSCs numbers post treatment. Representative data of three independent experiments are shown. (b) Similar analysis as above on HSCs and VSELs coexpressing CD34 shows an increase in numbers post 5-FU treatment. Representative data of three independent experiments are shown. The VSELs and HSCs were back gated on FSC/SSC plot and results show that LIN−/CD45− VSELs are smaller in size compared with LIN−/CD45+ HSCs. (c) Dual immunoﬂuorescence staining shows coexpression OCT4 with CD133 and SSEA4 with CD133. Bar 20 μm. (d) Cell cycle analysis of untreated and 5-FU treated cord blood cells by ﬂow cytometry using propidium iodide (PI). Note that cord blood cells post treatment seem to enter S phase.

co-expressing SSEA4 and vice versa was very low (Figures 2a and Markers speciﬁc for pluripotent stem cells (OCT4A, SSEA4), b). Results suggest the existence of distinct heterogeneity among primordial germ cells (STELLA, FRAGILIS) and primitive hematopoietic subpopulations of VSELs. As purity of selection using CD133 or cells (CD133, CXCR4, CD34) were studied by immunoﬂuorescence SSEA4 was just more than 50%, negative fractions and con- staining on CD133+ and SSEA4+ enriched cells. Both CD133+ and taminant populations were also analyzed. The negative cell SSEA4+ enriched cells expressed nuclear OCT4A and STELLA fraction that comprised progenitors and committed blood cells and cell surface markers SSEA4, CD133, CXCR4 and CD34 showed only the expression of CD34 and CXCR4 (Supplementary (Figure 2c and Supplementary Figure S3). CD133+ enriched cells Figures S2C and D). The percentage of other target cells was very were negative for CD45, but co-expressed OCT4A and SSEA4. low when compared with their positive counterparts. In the They also co-localized ACTIN and FRAGILIS. Hematoxylin and eosin contaminant population (CD133− and SSEA4− cells in positive staining of CD133+ and SSEA4+ fractions (supplementary Figure fractions), the presence of target cell populations was found to be S4A) showed the presence of small, spherical cells with high negligible (Supplementary Figures S3A and B). nucleocytoplasmic ratio, whereas larger cells with multi-lobed

Leukemia (2015) 1909 – 1917 © 2015 Macmillan Publishers Limited Characterization of cord blood VSELs A Shaikh et al 1915 nuclei were seen in the negative fractions. Higher transcript levels DISCUSSION of OCT4A, NANOG, SOX2, REX1, CD133 and CXCR4 were observed in It has been more than 15 years since human ES cell lines were first + + both CD133 and SSEA4 enriched VSELs compared with the reported.27 These cells have associated immunological rejection unsorted cord blood cells (Figure 3b and Supplementary Figure issues and to overcome this, human iPS cells were developed in S4B). 2007,28 and derivation of patient-specific human ES cell lines by somatic cell nuclear transfer was successfully reported in 2013.41 Ploidy and cell cycle analysis of CD133 and SSEA4 enriched VSELs In addition, sword of teratoma formation hangs on these Ploidy status of CD133+ and SSEA4+ enriched VSELs was studied pluripotent stem cells if and when they will be used for using propidium iodide-based flow cytometry. A majority of regenerative medicine. Current data suggest that ES/iPS cells give + + rise to their fetal counterparts and thus their ability to regenerate CD133 enriched (98.17%) and SSEA4 enriched (92.31%) cells 42 were found to be diploid in nature (Figure 3c). Normal ploidy adult organs needs further evaluation. Alternatively, pluripotent status was further confirmed by fluorescent in situ hybridization VSELs that exist in adult body tissues also require in-depth studies analysis for chromosomes 13, 18 and 21. Fluorescent in situ before their clinical potential may be exploited. VSELs were + recently reported to self-renew and increase in numbers after hybridization analysis of CD133 enriched VSELs demonstrated 43 absence of aneuploidy with two signals each for 13, 18 and 21. In treatment with pituitary gonadotropins in mouse bone marrow fl as well as after pregnant mare serum gonadotropin treatment to addition, uorescent in situ hybridization for X and Y chromo- 30 somes ruled out any X- and Y-related defects (Supplementary chemoablated mouse ovary. Using different strategies, VSELs have been shown to differentiate into cells of all three germ layers Figure S4D). Similar absence of aneuploidy was also observed in 16 44 45 SSEA4+ cells (data not shown). including skeletal structures, hepatic cells, pancreatic cells, neuronal lineage,46 lung epithelium,14 endothelial cells,47 testicu- Cell cycle analysis on diploid-enriched VSELs showed that a 29 30,13 majority of CD133+ enriched (96.79 ± 1.16%) and SSEA4+ enriched lar sperm and oocytes in ovary. The ability of VSELs to self- renew in vivo give rise to tissue-specific progenitors and (95.4 ± 0.83%) cells were in Go/G1 phase of cell cycle. Less than 5% of CD133+ and SSEA4+ enriched cells were in the S phase regenerate adult organs needs to be acknowledged even though (Figure 3c). In contrast to the enriched VSELs, higher numbers of they do not divide rapidly in vitro and fail to form teratoma like ES/ hES cells were in the S phase (48.63 ± 2.30%). Comparison iPS cells. The ease of isolating them from autologous source and of percentage of cells from the two enriched cell populations quiescent status may be an added advantage for regenerative and hES cells in various phases of cell cycle is shown in medicine. supplementary Figure S4C. Presence of majority of VSELs in the In this study, a majority of VSELs were found to settle along with the RBCs after Ficoll-Hypaque centrifugation and could be G0/G1 phase of cell cycle is suggestive of their quiescent nature similar to their murine counterparts.9 In addition, sub-G1 peak was enriched successfully using CD133 and SSEA4 cell surface markers. Thus, a unique strategy to enrich VSELs from cord blood is not observed during cell cycle analysis of enriched cells, thus + + marking the absence of apoptotic cells. described. The CD133 and SSEA4 cells were found to be phenotypically a primitive population, with most cells being TRAP assay was used to analyze telomerase activity levels in + + CD133+ and SSEA4+ enriched fractions, fibroblast cells and ES negative for CD45 and lineage markers. These CD133 and SSEA4 cells. The CD133+ and SSEA4+ sorted VSELs had almost 2.5 times cells also expressed pluripotent (OCT4A, SSEA4), primordial germ higher telomerase activity compared with fibroblast cells and 10 cell (STELLA, FRAGILIS) and primitive hematopoietic (CD133, CD34, times lower enzyme activity compared with ES cells (Figure 3d). CXCR4) markers. The pluripotent and primitive status of these cells was also validated by increased mRNA transcripts of genes regulating pluripotency and other markers (OCT4A, NANOG, SOX2, Effect of 5-FU treatment on cord blood cells in vitro REX1, CD133 and CXCR4) in positive fractions compared with the 5-FU treatment for 24 h (Figures 4a and b) resulted in a 1.5-fold negative fractions. All of the above results confirm that CD133- decrease of cells expressing lineage markers (52.90 ± 7.99 vs − and SSEA4-positive fractions were enriched for VSELs. 82.29 ± 1.61%). The treatment did not affect HSCs (LIN /CD45+; The immunophenotyping studies done to further char- − − 0.108 ± 0.026 vs 0.108 ± 0.051%) and VSELs (LIN /CD45 ; 0.034 ± acterize these enriched populations presented subtle hetero- 0.081 vs 0.035 ± 0.076%) that coexpressed CD133. However, CD34 geneity among VSELs in terms of marker expression. Among the coexpressing HSCs (0.150 ± 0.041 vs 0.336 ± 0.173%) and VSELs LIN−/CD45− cells, CD133+/CXCR4+ comprised a larger fraction (0.051 ± 0.009 vs 0.103 ± 0.017%) almost doubled compared with compared with CD133+/OCT4A+ and CD133+/STELLA+ cells, control (Figures 4a and b). The increase in CD34-positive cells whereas CD133+/SSEA4+ cells were rare and possibly represented possibly represents their activation for re-establishing homeostasis the most primitive VSELs in agreement with earlier report of Zuba- as suggested earlier.40 Cells that survived 5-FU treatment Surma et al.20 Our results suggest that SSEA4 could also be used expressed OCT4A, CD133 and SSEA4 (Figure 4c), suggesting their along with CD133 to isolate primitive VSELs from cord blood. pluripotent state. Quantitative real-time reverse transcription-PCR Takahashi et al.48 recently reported that LIN−CD34− cells expres- results showed that OCT4A and OCT4 transcripts were slightly sing CD133 may represent a more primitive cell population increased compared with untreated cells, but there was no compared with LIN−CD34+ cells that were thought to be most significant effect of 5-FU on CD133, SOX2 and NANOG transcripts primitive cells in the hematopoietic system.49 Similar carefully (Supplementary Figure S5A). In addition, compared with untreated designed studies to detect subpopulations among the stem cells 48 controls where majority of cells (498%) were in G0/G1 phase, an will help shed more light on the HSC hierarchy. Cells expressing increase in cells in the S phase was evident post 5-FU treatment LIN−/CD45−/CD34−/CD133+/SSEA4+ may possibly be the most (0.79 ± 0.74% control versus 6.38 ± 0.84% after 5-FU, Figure 4d and primitive and rare VSELs. The heterogeneity among morphologi- Supplementary Figure S5B). cally similar enriched VSELs was also demonstrated in murine To conclude, VSELs can be enriched using cell surface markers OCT4+ VSELs where microarray analysis from similarly isolated 20 CD133 and SSEA4 from the RBCs pellet obtained after Ficoll- murine OCT4+ VSELs revealed differences in expression of several Hypaque centrifugation of cord blood. VSELs are diploid, non- genes and VSELs were subclassified into ES-like, epiblast-like and apoptotic and quiescent and demonstrate higher expression of OCT4+ VSELs.50 pluripotent and other stem cell markers and telomerase activity. Existence of heterogeneity among VSELs may also be respon- VSELs survive 5-FU treatment and CD34+-expressing cells get sible for differences that are being observed in their chara- ‘activated’ as evident by their entry into the cell cycle. cteristics especially related to their stemness and self-renewal

© 2015 Macmillan Publishers Limited Leukemia (2015) 1909 – 1917 Characterization of cord blood VSELs A Shaikh et al 1916 potential. Kassmer and Krause51 have suggested that different ACKNOWLEDGEMENTS subpopulations of VSELs have different potentials, and it is likely We thank Dr Mukherjee, Gayatri Shinde, Sushma Khavale, Nivedita Dhavale, Dr Babu that only a subset of them are capable of giving rise to cells from Rao, Dr Tamhankar, Harshvardhan Gawade and Dr Khatkhatay for their help. We also the three germ layers on a single-cell level. The heterogeneity thank University Grants Commission, New Delhi, for support toward doctoral noted in the cord blood in this study may reflect different states of program of Ambreen Shaikh. Financial support for the study was provided by Indian differentiation of VSELs, with a possibility that LIN−/CD45−/CD34−/ Council of Medical Research, Government of India, New Delhi (Accession no. CD133+/SSEA4+ VSELs may give rise to LIN−/CD45−/CD34−/ RA/214/01-2015). SSEA4−/CD133+ cells that may further differentiate into LIN−/ − − + + CD45 /SSEA4 /CD133 /CD34 cells. Thus, further studies are REFERENCES required to understand heterogeneity in cord blood VSELs and 8 – the ability of subpopulations to undergo multilineage differentia- 1 Virchow R. Archive fuerpathologische (Editorial). Anat Physiol Klin Med 1855; :23 54. 22 fl 2 Kucia M, Reca R, Campbell FR, Zuba-Surma E, Majka M, Ratajczak J et al. tion. Danova-Alt et al. tested 22 markers by ow cytometry on A population of very small embryonic-like (VSEL) CXCR4 (+) SSEA-1(+) Oct-4+ whole cord blood and reported higher expression of CD31, CD84 stem cells identified in adult bone marrow. Leukemia 2006; 20:857–869. and CXCR4 and absence of ES cell markers. 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