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 up-regulating 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.

Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue–Derived Human Mesenchymal Stromal Cells

“…18,36 Furthermore, extracellular matrix proteins (e.g., ACAN, DCN, COMP, and HAPLN1) are highly expressed in human cartilage and human articular chondrocytes. 24,26,37 In our studies, both cell types respond to hypoxia by enhancing the expression of several chondrogenic markers including SOX9, HAPLN1, and COMP, but also HIST2H4 and HIF1A. These findings indicate that a low oxygen microenvironment may act to modulate cell proliferation and chondrogenic differentiation of both AMSCs and chondrocytes.…”

“…As an alternative, human adipose tissue-derived mesenchymal stem cells (AMSCs) are an attractive cellular therapeutic due to minimally invasive tissue harvest, high abundance of cells, and rapid expansion ex vivo. 24 Moreover, these cells are multipotent and can produce musculoskeletal extracellular matrix proteins once confluent and are able to differentiate into osteogenic and chondrogenic lineages. 24,25 While there are many studies on chondrogenic differentiation of bone marrow-derived MSCs, our studies address a major gap in our knowledge by investigating the chondrogenic potential of AMSCs under multiple culture conditions relevant to cartilage tissue engineering, including hypoxia and propagation on a 3-dimensional (3D) culture environment.…”

Identification of long non-coding RNA involved in osteogenic differentiation from mesenchymal stem cells using RNA-Seq data

“…COL4A4 was also up-regulated during osteogenic differentiation from rat bone marrow stromal cells (Kaur et al, 2010). Type XXI collagen (COL21A1) is highly up-regulated in human MSCsin the presence of the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor (GW9662), thereby enhancing osteoregeneration (Dudakovic et al, 2014). The Wnt signal transduction pathway is related to osteogenesis in human mesenchymal stem cells, via the canonical and non-canonical methods (Ling et al, 2009).…”

Epigenetic Control of Skeletal Development by the Histone Methyltransferase Ezh2

“…High throughput mRNA sequencing and bioinformatic analyses (mRNASeq) were performed as previously reported (47). Gene expression is expressed in reads per kilobase pair per million mapped reads).…”