Abstract
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.
Highlights
Substrate for anchor-dependent cells is crucial for self-renew and lineage commitment, including human embryonic stem cells (Murphy et al, 2014)
3.1 Colloidal Self-Assembled Patterns Characterization colloidal self-assembled patterns (cSAPs) were fabricated by mixing different particles together and depositing them on the tissue culture plates (TCPS); after cSAPs Promote the Hemopoietic Potential of human embryonic stem cells (hESCs) evaporation, particles were distributed on the surface according to the principle of self-assembly (Figure 1A)
The Pluripotent stem cells (PSCs) colonies were dome-like morphology on the cSAP #4 and #5, neither 3D spheroids nor 2D-like colonies. mouse induced pluripotent stem cells (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 cSAPs Promote the Hemopoietic Potential of hESCs
Summary
Substrate for anchor-dependent cells is crucial for self-renew and lineage commitment, including human embryonic stem cells (hESCs) (Murphy et al, 2014). Nanostructured surfaces can be further modified chemically using coatings or grafting technology to enhance biofunctionality. These surfaces are still far from an extracellular matrix (ECM) like substrate. 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 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 been studied on these new substrates (Wang et al, 2016c). Cell reprogramming of human fibroblasts into human induced pluripotent stem cells (hiPSCs) has 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
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