Colloidal Self-Assembled Patterns Maintain the Pluripotency and Promote the Hemopoietic Potential of Human Embryonic Stem Cells

The generation of blood cells in a significant amount for clinical uses is still challenging. Human pluripotent stem cells-derived hemopoietic cells (hPSC-HCs) are a promising cell source to generate blood cells. Previously, it has been shown that the attached substrates are crucial in the maintenance or differentiation of hPSCs. In this study, a new family of artificial extracellular matrix (ECM) called colloidal self-assembled patterns (cSAPs: #1–#5) was used for the expansion of mouse and human PSCs. The optimized cSAP (i.e., #4 and #5) was selected for subsequent hemopoietic differentiation of human embryonic stem cells (hESCs). Results showed that the hematopoietic potential of hESCs was enhanced approx 3–4 folds on cSAP #5 compared to the flat control. The cell population of hematopoietic progenitors (i.e., CD34+CD43+ cells) and erythroid progenitors (i.e., CD71+GPA+ cells) were enhanced 4 folds at day 8 and 3 folds at day 14. RNA sequencing analysis of cSAP-derived hESCs showed that there were 300 genes up-regulated and 627 genes down-regulated compared to the flat control. The enriched signaling pathways, including up-regulation (i.e., Toll-like receptor, HIF-1a, and Notch) or down-regulation (i.e., FAs, MAPK, JAK/STAT, and TGF-β) were classic in the maintenance of hESC phenotype Real time PCR confirmed that the expression of focal adhesion (PTK2, VCL, and CXCL14) and MAPK signaling (CAV1) related genes was down-regulated 2-3 folds compared to the flat control. Altogether, cSAP enhances the pluripotency and the hematopoietic potential of hESCs that subsequently generates more blood-like cells. This study reveals the potential of cSAPs on the expansion and early-stage blood cell lineage differentiation of hPSCs.


INTRODUCTION
Substrate for anchor-dependent cells is crucial for self-renew and lineage commitment, including human embryonic stem cells (hESCs) (Murphy et al., 2014). In the last decades, substrates with different nanostructures such as nanogrooves and nanopillars have been applied to manipulate cell fate. Nanostructured surfaces can be further modified chemically using coatings or grafting technology to enhance biofunctionality. However, these surfaces are still far from an extracellular matrix (ECM) like substrate. ECM mimetic surface presenting hierarchical structures and multiple chemistries are rare to be found (Wang et al., 2016a).
Recently, a new family of substrates composed of various colloidal particles with different sizes and materials named selfassembled patterns (cSAPs) was developed in our group (Wang et al., 2015a). The particles can be pre-or post-modified, ultimately providing a complex surface of the cSAPs (Diba et al., 2019). Surface topography, roughness, hydrophilicity, chemistry, and even stiffness can be fine-tuned. The behaviors of human stem cells and adult cells have been investigated on the cSAPs (Wang et al., 2016b;Cui et al., 2019). Cell reprogramming of human fibroblasts into human induced pluripotent stem cells (hiPSCs) has also been studied on these new substrates (Wang et al., 2016c). cSAPs have shown the potential to control cell adhesion and subsequently the fate decision of cells.
Generation of blood cells in vitro with a significant amount for clinical uses is still challenging. Human pluripotent stem cells (hPSCs), including hESCs and hiPSCs, are promising sources to generate blood-like cells. The hematopoietic potential of hPSCs has a significant application in the cure of blood-related diseases such as Thalassemia (Papapetrou, 2017) or hemophilia (Wang et al., 2013). Several co-culture differentiation systems, such as the OP9 stromal cells and the aorta-gonad-mesonephros-derived stromal cells (AGM-S3) (Ledran et al., 2008), have been established for blood-like cell generation. These systems use a nature-inspired microenvironment to stimulate definitive hematopoiesis in vitro. These systems are useful to identify the function of critical genes in normal or abnormal hematopoiesis. For example, the AGM-S3 co-culture system could be used to check the detailed cellular and molecular mechanism of hematopoiesis influenced by the critical gene (Chen et al., 2017;Zeng et al., 2020). It also has potential utility in screening for compounds that promote human hematopoiesis, which is possible to set up a high throughput screening system for compound function screening using the AGM-S3 co-culture system (Chang et al., 2019). However, the current approach in vitro blood cell generation is far from satisfying clinical needs in terms of quality and number (Moreno-Gimeno et al., 2010). Therefore, the improvement of the efficiency of hematopoietic differentiation of hPSC-HCs is essential.
In this proof-of-concept study, human and mouse PSCs' morphology and growth were screened on five cSAPs. The selected cSAPs were used to expand and stimulate hESCs used for the downstream hematopoietic differentiation into HCs and blood-like cells. The outcomes imply that the expansion of hESCs (C) Surface wettability of the substrates was analyzed using water contact angle test (degrees). Five spots on each surface were analyzed (n 5). Error bar STDEV.
Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 771773 2 is a crucial step prior to the hematopoietic differentiation using the stromal cell co-culture system, and the cSAP is a valuable tool for in vitro blood cell production.

Colloidal Self-Assembled Patterns
Five substrates were selected from our library and fabricated according to our previous protocol (Wang et al., 2016b) ( Figure 1A). Briefly, cSAPs #1 and #2 was composed of SiO 2 with 5 μm diameter and polystyrene (PS) with 200 or 400 nm diameter, #3, #4, and #5 was composed of SiO 2 with 2 μm diameter and PS with 65 nm diameter, carboxy-PS (PSC) with 50 or 100 nm diameter, and the cSAPs were fabricated within the 24-well tissue culture plates (TCPS, Falcon). TCPS was the flat control group in this study.
Scanning electron microscopy (SEM, ZEISS SUPRA ® 55, Carl Zeiss, Germany) and water contact angle (WCA) was used to characterize the surface structures and wettability of these cSAPs using an automated contact angle measurement device (PT-705B, Precise Test, China) at room temperature. Other detailed characterizations have been done previously (Wang et al., 2016b).
All cells used in this study were passaged three times on cSAPs prepared in 24-well tissue culture plate or control surfaces coated by Matrigel or Gelatin for further research. The experimental group was named cSAP #X (X 1, 2, 3, 4, 5) and TCPS (as a flat control). Cell inoculatiion density of mESCs and miPSCs in experimental group was 2 × 10 4 cells/cm 2 . Cell density of hESCs and hiPSCs was adjusted according to the colony quantity. Cell culture in experimental group was same to the routinely cultured cells, mESCs and miPSCs was passaged at a ratio of 1:6, hESCs and hiPSCs was passaged at a ratio of 1:3. Medium was changed every day. Cell morphology was captured every day by an inverted microscope (Olympus-IX71).

Real-Time qPCR
Total RNA was extracted from cells with the MiniBEST universal RAN extraction Kit (Takara, Japan). cDNA was made with 1 mg of total RNA based on the manufacturer's protocol using the PrimeScript TM RT Master Mix (Takara, Japan), and subsequent real-time qPCR was carried out in triplicates, using the 2 × RealStar Green Mixture (GENE STAR, China) on Light Cycler 96 System cording to manufacturer's instructions (Roche, United State). Amplifications were performed using the following conditions: 95°Cfor 2 min, followed by 40 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 30 s. Gene expression was normalized to Gapdh, and then the TCPS control. The sequences of all primers used were listed in Supplementary Table S1.

Flow Cytometry
Flow cytometry experiments were performed according to a previous report (Chen et al., 2017). Briefly, the co-cultured cells at D8 or D14 were dissociated with 0.25% trypsin-EDTA solution. The cell suspension was obtained by filtration through a 70-μm nylon mesh. Before immunostaining, cells were blocked by rabbit serum at 4°C for 30 min. Cells were stained with the anti-CD34/CD43 antibodies (at D8 and D14), and anti-CD34/CD45 or anti-GPA/CD71 antibodies (at D14) at 4°C for 30 min (Supplementary Table S2). Flow cytometric analysis was performed using the FACS Canto II system (BD Biosciences). All FACS data were analyzed using FlowJo 10 software.

RNA Sequencing
H1 hESCs cultured on cSAPs and flat control (TCPS) for two passages (4 days/passage) were dissociated with 0.5 mM EDTA for RNAseq. Total RNA of each 1 × 10 6 cells was extracted by 1 ml TRIzol (Life Technologies) and purified according to the standard instruction. RNA Sequence analysis was performed by BGI Tech (Shenzhen, China). The analysis of gene function was performed with the multi-group data mining system of Dr. Tom (http:// report.bgi.com). Gene changes on cSAP #5 compare to TCPS over 2-fold were defined as significant.

Immunofluorescence Staining
Cells were cultured in multi-well plates were fixed with 4% paraformaldehyde (PFA) for 30 min at 4°C, and washed three times with wash buffer (5% FBS in PBS). They were then incubated with membrane permeation reagent (PBS containing 0.3% Triton-100 and 5% FBS) at 4°C for 30 min, stained overnight at 4°C with anti-OCT4/SOX2/SSEA4 antibodies (mouse antihuman), washed three times with PBS containing 5% skim milk, and then incubated for 30 min at room temperature with secondary antibodies (FITC-conjugated secondary Ab, goat anti-mouse) (Supplementary Table S2). Nuclei were labeled with DAPI. After washing three times with PBS, the sample was imaged under a fluorescence microscope.

Statistical Analysis
All data are presented as means ± SD; statistical analyses were performed by GraphPad Prism 8 using the Student's t-test, oneway ANOVA with Dunnett post hoc test, and two-way ANOVA with Tukey's post hoc test. p < 0.05 was considered significant.

RESULTS
3.1 Colloidal Self-Assembled Patterns Characterization cSAPs were fabricated by mixing different particles together and depositing them on the tissue culture plates (TCPS); after evaporation, particles were distributed on the surface according to the principle of self-assembly ( Figure 1A). The surface topography of cSAPs was measured by SEM and showed that the large particles and small particles were orderly distributed on the surfaces ( Figure 1B). Due to the differences of the surface on chemestry and topography, surface wettability showed that water contact angle (WCA) of cSAP #1 and #2 (SiO 2 5 μm; PS 200 and 400 nm) was more hydrophobic (85.8 ± 3.5 and 96.8 ± 4.6 degree), than the cSAP #3, #4, and #5 (SiO2 2 μm; PS 65 nm, PSC 50 and 100 nm), and the WCA of cSAP #3, #4, and #5 was 31.8 ± 1.1, 32.3 ± 3.2, and 25.8 ± 2.4 degree, respectively ( Figure 1C).

Colloidal Self-Assembled Patterns Maintain Pluripotency of miPSCs, hiPSCs, and hESCs
The colony morphology of miPSCs on cSAPs was significantly different from that on the TCPS control ( Figure 2A). miPSCs colonies on cSAPs were more 3D-like, especially on the cSAP #1 and #2. The PSC colonies were dome-like morphology on the cSAP #4 and #5, neither 3D spheroids nor 2D-like colonies. miPSCs cultured on cSAPs without LIF (Leukemia Inhibitory Factor) had a higher percentage of Oct4-GFP positive cells after 7 days than TCPS without and even with LIF, indicating that cSAPs could maintain the pluripotency of PSCs Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 771773 5 ( Figure 2B and Supplementary Figure S1A). Percentage of Oct4-GFP positive cells was more than 90% on cSAPs, except the cSAP #3, which was similar to the cells cultured within LIF (i.e., ∼80%).
Gene expression analysis showed that pluripotent markers (i.e., Oct4, Nanog, and Sox2) of miPSCs were similar (i.e., Oct4 and Nanog, fold changes < 1.5) or higher (i.e., Sox2, fold changes > 1.5) on the cSAPs compared to the TCPS control without LIF ( Figure 2C). Gene expression of miPSCs was also analyzed under LIF conditions. The results showed that Sox2 expression was significantly higher on cSAPs than TCPS, while Oct4 and Nanog were similar between surfaces ( Figure 2D). The high expression of Sox2 may indicate neural stem and progenitor cells in some cSAPs groups (Ellis et al., 2004). The effects of cSAPs on miPSCs and mESCs were different because three genes of mESCs expressed similarity between surfaces (fold changes < 1.5, Figure 2C and Supplementary Figure S1B).
Gene expression of mesoderm markers of miPSCs was higher on cSAPs than the TCPS control without LIF (Supplementary Figure S1C). On average, the expression of mT, mSnail2, and mFoxa2 on cSAPs, except cSAP #5, was significantly higher than the TCPS control (fold changes > 2, Supplementary Figure S1C). hiPSCs and hESCs also showed different colony morphology compared with TCPS and slight differences between cSAPs, similar to miPSCs on cSAPs showing a more 3D-like colony morphology ( Figures 3A,B). For hESCs can not grow well without Matrigel, all surfaces used in culturing hESCs were precoated with Matrigel. FACS analysis (Figure 4 and Supplementary Figure S2) and the hematopoietic colony form assay ( Figure 5) showed that only H1 hESCs cultured on cSAP#4 and #5 had better hematopoietic differentiation efficiency than the TCPS control when co-cultured with AGM-S3, so only #4 and #5 were chosen for further analysis of hESCs pluripothency. Besides, immunostaining of pluripotent markers, i.e., OCT4, SOX2, and SSEA4, showed that passaged hESCs were in high pluripotency on cSAPs, i.e., cSAP #4 and cSAP #5, and TCPS ( Figure 3C).
According to RNAseq analysis, the genes regulated on cSAPs were enriched in mineral absorption, longevity regulating, Toll-like receptor signaling, HIF-1a signaling, Notch signaling that were up-regulated, and the focal adhesion, TGF-β signaling, PI3K-Akt signaling, and MAPK signaling that were down-regulated ( Figure 6B). According to the RNA-seq results we found that expression of CAV1 and CXCL14 was down-regulated ( Figure 6B), and was consistent with qPCR verification ( Figure 6C). CXCL14 is found concentrated in the focal adhesions, the down-regulated expression of CXCL14 (∼2.79 fold) on cSAP #5 resulted in lower expression of PTK2 (∼1.61 fold) and VCL (∼2.83 fold) ( Figure 6C). The dot-plot also showed focal adhesion was enriched in down-regulated DEGs ( Figure 6B). CAV1 can function as a scaffolding protein contribute to the activation of the MAP kinase pathway (Mineo et al., 1996;Engelman et al., 1998). In this study, the down-regulated MAPK pathway in Figure 6B may be the result of lower CAV1 (∼3.90 fold) expression on cSAP #5 ( Figure 6C). Lower expression of CAV1 can promote hematopoietic differentiation of hESCs when co-cultured with stromal cells under hematopoietic induction condition (Choi et al., 2012). Therefore, cSAPcultured hESCs having higher blood cell differentiation ability could be a consequence of down-regulation of focal adhesion and MAPK signaling ( Figure 6D).

DISCUSSION
Pluripotent stem cells (PSCs) can self-renew unlimitedly and differentiate into three germ layers in vitro. For example, under specific induction conditions, hPSCs can be differentiated sequentially to mesodermal cells, hematopoietic progenitor cells, and mature hematopoietic cells (Lim et al., 2013). In vitro hematopoietic differentiation facilitates a better understanding of hematopoiesis and embryonic development. Besides, the production of blood-like cells is significant in regenerative medicine.
Nowadays, three major in vitro differentiation systems have been established for the generation of hESC-derived HCs, including embryoid body (EB) culture, co-culture with feeder cells, and culture with extracellular matrix (ECM) proteincoated surfaces (Chen et al., 2014). Among these methods, the microenvironments for hESC expansion are critical to determining the subsequent HSC generation (Ma et al., 2008;Slukvin, 2013). ECM protein-coated surfaces, such as fibronectin, collagen IV, laminin, collagen I, entactin, and heparin-sulfate proteoglycan, have been developed to induce differentiation of mesodermal and hematopoietic lineages under more chemical defined conditions (Chen et al., 2011;Nakamura-Ishizu et al., 2012). However, few studies manipulated the adhesion status of hESCs prior to hematopoietic differentiation. Furthermore, previous studies heavily relied on biological modulators such as paracrine molecules and medium molecules, while the effects of biophysical stimulations during the process were rarely explored.
It has been demonstrated that the surface coating of cell culture substrate was able to modulate the fate of cells, including directional differentiation of mesenchymal and pluripotent stem cells (Wang et al., 2016a). Surface decoration with nanostructures and bioactive signals can reconstruct the stem cell niche's microenvironments, which provide biophysical cues to the attached cells (Dalby et al., 2007). A previous study demonstrated that reduction of focal adhesions of mESCs was able to be maintained the cells in an undifferentiated and pluripotent state, while stronger cell adhesion resulted in stem cell differentiation (Taleahmad et al., 2017). The current study employed that colony morphology and adhesive force of PSCs varied on different cSAPs and the TCPS control. Further analysis demonstrated that PSCs' pluripotent state was adhesion-and morphology-dependent, where more 3D-like colonies on cSAPs have higher pluripotency than the 2D culture. cSAPs can affect adhesion molecules and regulate the pluripotency of PSCs. This Frontiers in Cell and Developmental Biology | www.frontiersin.org November 2021 | Volume 9 | Article 771773 9 phenomenon is consistent with previous studies using different materials (Zujur et al., 2017).
Previous studies did show that physical cues on the substrate facilitate the generation of hESC-HCs. For example, graphene oxide (GO) had been reported to promote Endothelial-to-Hematopoietic Transition (EHT) and then the hESC-HC generation (Garcia-Alegria et al., 2016). In the current study, cSAPs don't have superior conductivity or biofunctionality but hierarchical micro-and nanostructure and dual chemistries. It has been demonstrated that these properties will alter protein adsorption from the medium, cell adhesion, cell migration, and ECM synthesis of adhered cells (Wang et al., 2011;Wang et al., 2012;Wang et al., 2015b). cSAPs can maintain the pluripotent status of PSCs and stimulate mesodermal commitment. The chemical and physical properties of different cSAPs can maintain the pluripotency of PSCs to a different extent. According to a previous report, PSCs in a naïve state have more robust self-renew capability and limited potential toward lineage differentiation (Lee et al., 2017). Besides, the naïve PSCs need capacitation for triggering multi-lineage differentiation (Rostovskaya et al., 2019). According to the colony morphology, cSAPs may reverse the cells back into a naïvelike state (e.g., cSAP #2) or a state of capacitation (e.g., cSAP #5). The result implies that cSAP-derived hESCs have a better hematopoietic potential due to optimal cell adhesion and colony formation.
Controlling the cellular status of hESCs during expansion can enhance the hemopoietic potential in the AGM-S3 co-culture system. cSAPs coating with Matrigel or proteins can be seen as an artificial ECM that modulates the adhesion of hESCs during expansion. cSAP derived hESCs can generate 2-4 folds of HCs compared to the TCPS control. According to RNAseq data, a flock of genes related to mineral absorption, such metallothionein 1 (MT1) family, MX1, and NR3C1, and the signaling pathways of Toll-like receptor, HIF-1a, Notch were up-regulated. The genes related to cell adhesion and signaling pathways of MAPK, Jak-STAT, and TGF-β were downregulated.
The changes of these genes and pathways play vital roles in hematopoiesis. For example, Notch signaling controls the hematopoiesis and inflammation process (Šućur et al., 2020). Toll-like receptors and HIF-1a signaling can influence the generation of hematopoietic stem and progenitor cells (Capitano, 2019;Wielockx et al., 2019). The JAK/STAT signaling pathway controls about 50 cytokine signals to orchestrate hematopoiesis (Staerk and Constantinescu, 2012;Morris et al., 2018). MAPK signaling is involved in the generation of hematopoietic stem and progenitor cells (HSPC), erythropoiesis, mylogenesis (Geest and Coffer, 2009). TGF-β signaling can influence the generation of HSCs (Blank and Karlsson, 2015). Some of these genes were not directly relevant to hematopoiesis, which mechanisms need further research to discover. For example, many transcript factors, such as FOS, EGR1, FOXG1, TF, and other types of genes, such as MT1 family (Metallothionein), DDX60 (Probable ATP-Dependent RNA Helicase), MX1(Interferon related GTPase), indicated a close couple to the promotion effects caused by culturing hESCs with cSAP. However, the mechanism of cSAP induced biological changes is unclear. The relationship between changing transcriptional profiles and promoting hematopoiesis needs to be elucidated using further analysis such as gene sequence and proteomics at a single cell level.

CONCLUSION
The hemopoietic potential of hESCs is critical in blood generation and related regenerative medicine. This study demonstrates that the adhesion and pluripotency of hESCs are crucial in subsequent hemopoietic differentiation. cSAP can be a new family of artificial ECMs (protein-or peptidemodified cSAPs) where the surface presents complex physical and chemical cues. cSAP modulate hESC adhesion and the ability to conduct hemopoietic differentiation. By merely manipulating hESCs using cSAPs, the number of hematopoietic progenitors and erythroid progenitors can be enhanced 3-4 folds. Altogether, cSAP could be the next-generation tool for hESC expansion and blood cell generation.

DATA AVAILABILITY STATEMENT
The RNAseq data presented in the study are deposited in the Array Express repository, accession number E-MTAB-11123.

AUTHOR CONTRIBUTIONS
P-YW and BC conceived and supervised this study. JLn, KL, and ME conducted material fabrication and characterization. JZ, WS, and DY conducted cell culture and data analysis. JH helped processing and uploading the RNAseq data. JLu, FM, and PK provided useful comments. JLn, BC, and P-YW wrote and revised the manuscript. All authors have approved the final version of the manuscript.