High-resolution molecular validation of self-renewal and spontaneous differentiation in clinical-grade adipose-tissue derived human mesenchymal stem cells

Abstract

Improving the effectiveness of adipose-tissue derived human mesenchymal stromal/stem cells (AMSCs) for skeletal therapies requires a detailed characterization of mechanisms supporting cell proliferation and multi-potency. We investigated the molecular phenotype of AMSCs that were either actively proliferating in platelet lysate or in a basal non-proliferative state. Flow cytometry combined with high-throughput RNA sequencing (RNASeq) and RT-qPCR analyses validate that AMSCs express classic mesenchymal cell surface markers (e.g., CD44, CD73/NT5E, CD90/THY1, and CD105/ENG). Expression of CD90 is selectively elevated at confluence. Self-renewing AMSCs express a standard cell cycle program that successively mediates DNA replication, chromatin packaging, cyto-architectural enlargement, and mitotic division. Confluent AMSCs preferentially express genes involved in extracellular matrix (ECM) formation and cellular communication. For example, cell cycle-related biomarkers (e.g., cyclins E2 and B2, transcription factor E2F1) and histone-related genes (e.g., H4, HINFP, NPAT) are elevated in proliferating AMSCs, while ECM genes are strongly upregulated (>10-fold) in quiescent AMSCs. AMSCs also express pluripotency genes (e.g., POU5F1, NANOG, KLF4) and early mesenchymal markers (e.g., NES, ACTA2) consistent with their multipotent phenotype. Strikingly, AMSCs modulate expression of WNT signaling components and switch production of WNT ligands (from WNT5A/WNT5B/WNT7B to WNT2/WNT2B), while upregulating WNT-related genes (WISP2, SFRP2, and SFRP4). Furthermore, post-proliferative AMSCs spontaneously express fibroblastic, osteogenic, chondrogenic, and adipogenic biomarkers when maintained in confluent cultures. Our findings validate the biological properties of self-renewing and multi-potent AMSCs by providing high-resolution quality control data that support their clinical versatility.

Keywords: ACTA2; ADH1B; ADIPOGENESIS; ADIPOSE-TISSUE DERIVED STROMAL CELLS; ASPN; CCNB2; CCND1; CD105; CD44; CD73; CD90; CELL CYCLE; CHI3L1; CHONDROGENESIS; CYCLIN; E2F1; E2F7; E2F8; ECM2; ENG; EXTRACELLULAR MATRIX; FIBROBLAST; FMOD; H19; HINFP; HIST1H3H; HIST1H4A; HIST2H4A; HIST2H4B; HISTONE; KLF4; LINEAGE-COMMITMENT; MESENCHYMAL STEM CELL; MULTIPOTENT; NANOG; NES; NPAT; NT5E; OCT4; OGN; OSTEOGENESIS; PLURIPOTENT; PODN; POU5F1; RARRES2; SFRP2; SFRP4; THY1; TNNT3; WISP2; WNT2; WNT2A; WNT5A; WNT5B; WNT7B.

FIGURE 1. High throughput analysis of gene expression in proliferating and quiescent confluent AMSCs

Incucyte cell density analysis showing cell confluence over time (A). Representative microscopy images of cell cultures at different levels of confluence (B). Number of genes not expressed, expressed at low levels, and expressed at high levels in proliferating and confluent AMSCs (C). Pie chart of data in panel A (D). Expression (RPKM) of the 100 most abundant genes relative to all other genes in proliferating and confluent AMSCs (E). The top 100 expressing genes divided by class in proliferating and confluent AMSCs (F). Differential expression of genes in proliferating and confluent AMSCs (G). Significant (FC > 1.4) gene expression differences between proliferating and confluent AMSCs (H). Reads per kilobasepair per million mapped reads = RPKM, Fold Change = FC. RNASeq data were analyzed as averages (and standard deviations) for three consenting normal patients (n= 3).

FIGURE 2. Expression of cell cycle regulatory genes in proliferating and confluent AMSCs by RNA-Seq

Expression of cyclins (A), cyclin-dependent kinases (B), and E2F family members (C) in proliferating AMSCs (top) and fold change expression values comparing confluent and proliferating AMSCs (bottom). Reads per kilobasepair per million mapped reads = RPKM, Fold Change = FC, n = 3.

FIGURE 3. Expression of cell cycle inhibitors, histone regulators, histones, and chromatin-associated genes in proliferating and confluent AMSCs by RNA-Seq

Expression of cyclin-dependent kinase inhibitors (A), Rb family members (B), histone regulators (C), histones (D), and chromatin-associated genes (E) in proliferating AMSCs (top) and fold change expression values comparing confluent and proliferating AMSCs (bottom). Histone mRNAs are derived from multiple distinct gene copies that essentially express the same protein. Because individual mRNA species are not efficiently detected in RNASeq due to the lack of a polyA tail, expression values are represented as the aggregate values of multiple different mRNAs for each histone gene subclass. Reads per kilobasepair per million mapped reads = RPKM, Fold Change = FC, n = 3.

FIGURE 4. Expression of pluripotent genes in proliferating and confluent AMSCs by RNA-Seq

Reads per kilobasepair per million mapped reads = RPKM, n = 3.

FIGURE 5. Gene expression analysis as proxy biomarkers for cell surface expression of proliferating and confluent AMSCs by RNA-Seq

Expression of class I and II CD markers in proliferating and confluent AMSCs (A). Comparison of CD marker detection by flow cytometry and RNA-Seq in proliferating AMSCs ([+] represents >90% positive cells and >10 RPKM awhile [−] represents <5% positive cell or <1 RPKM) (B). Expression of proliferation and viability markers in proliferating and confluent AMSCs (C). Expression of additional molecular biomarkers for MSC identity in proliferating and confluent AMSCs (D). Reads per kilobasepair per million mapped reads = RPKM, n = 3.

FIGURE 6. Comparison of CD marker expression by flow cytometry and RT-qPCR

Flow cytometry and RT-qPCR analysis with proliferating and confluent AMSCs was performed as described in methods. Flow cytometry analysis of CD markers in proliferating and confluent AMSCs (n=4, representative data shown) (A). RT-qPCR analysis of CD markers in proliferating and confluent AMSCs (B). Gene expression was normalized to GAPDH (set at 100), n=4.

FIGURE 7. Expression of extra-cellular matrix and WNT-related genes in proliferating and confluent AMSCs by RNA-Seq

Expression of cell adhesion genes (A), leucine rich repeat genes (B), WISPs (C), WNTs (D), LRPs (E) SFRPs (F), and DKKs (G) in proliferating and confluent AMSCs. Reads per kilobasepair per million mapped reads = RPKM, n = 3.

FIGURE 8. Expression of mesenchymal lineage genes in AMSCs by RT-qPCR

Cells were plated and RNA isolated and processed as described in methods. Expression of cell cycle genes (A), extra-cellular matrix genes (B), pluripotency genes (C), osteogenic transcription factors (D), chondrogenic transcription factors (E), and adipogenic transcription factors (F). X-Axis indicates day of culture where day 1 are actively dividing AMSCs, day 4 are confluent AMSCs, and day 7 are post-confluent AMSCs. Gene expression was normalized to GAPDH (set at 100), n = 3.