Cellular and Molecular Bioengineering
Chun Hei Ryan Chan (he/him/his)
Undergraduate Research Assistant
Johns Hopkins University, Cahan Lab
Baltimore, Maryland, United States
Ray Cheng
Ph.D. Candidate
Johns Hopkins University, Cahan Lab, United States
Patrick Cahan
Primary Investigator
Johns Hopkins University, Cahan Lab, United States
The bone marrow niche is responsible for many physiologically essential functions such as bone homeostasis, supporting hematopoiesis, and adipogenesis. These functions are subject to age-related changes and demonstrate pathological differences such as reduced bone density and the marrow space gradually being filled with adipose tissue [1]. One significantly affected cell population in this niche is bone-marrow stromal cells (BMSC) – these stem cells demonstrate self-renewal capabilities as well as the ability to differentiate into adipocytes, osteoblasts, and chondrocytes. However, human BMSCs isolated from aged populations demonstrate significantly diminished self-renewal and differentiation potential and similar aging trends are observed in murine models [2][3]. And although recent research has attempted to reverse the aging of murine tissue with transient expression of the Yamanaka factors - Oct4, Sox2, Klf4, and c-Myc - also known as OSKM, similar approaches have not been employed with BMSCs [4]. We look to investigate the potential rejuvenation of aged murine BMSCs via in vitro transient OSKM reprogramming. We have demonstrated that this transient reprogramming significantly increases self-renewal capabilities, measured by colony forming efficiency (CFE), without compromising their trilineage differentiation potential. This research will highlight relevant aging mechanisms and pathways specific to BMSCs and provide insights on how to reverse, minimize, or even prevent the aging process for BMSCs in the bone marrow niche.
To study the effects of OSKM transient expression in BMSCs, we acquired the R26rtTA;Col1a14F2A strain mice from the Jackson laboratories, which has a dox-inducible gene cassette containing OSKM incorporated into the Col1a1 locus and reverse tetracycline controlled transactivator inserted in the ROSA locus. The transgene system can be activated by tetra-doxycycline (dox) and will transiently express OSKM until dox has been removed. Cells are isolated from the bone marrow flushed from the femur on day 0 and the culture media is supplemented with 2μg/ mL dox. After 7 days, cells are passaged into regular culture media without dox and allowed to recover for 5 days. On day 12, cells can be passaged again for experimental use. To assess the self-renewal capabilities, we will use a colony forming efficiency assay and quantify the number of colonies formed with crystal violet staining. To assess the trilineage differentiation potential, we will use established differentiation protocols and stain with Oil red O, alizarin red, and alcian blue to detect the presence of lipid accumulation, calcium deposition, and acidic polysaccharides respectively as well as immunohistochemistry staining; Positive stains will demonstrate the presence of mature and functional adipocytes, osteoblasts, and chondrocytes respectively. Once the mice have reached an elderly state (18 mo+), we will compare the self-renewal capabilities and differentiation potential of young, old-untreated, and old-treated BMSCs. Finally, we will utilize single-cell RNA sequencing and computational tools developed by the Cahan lab to assess the transcriptomic differences between the three populations.
The transient expression of OSKM was first validated via qPCR. We observe a significant increase in colony forming efficiency when comparing dox-treated and untreated young BMSCs (Fig 1). There was no significant difference observed in directed differentiation into adipocytes, osteoblasts, and chondrocytes between dox-treated and untreated cells when assessed by functional staining (Fig 2a) or immunohistochemistry staining (Fig 2b). We collected a middle-aged BMSC sample (12 mo) and observed a significant increase in CFE when middle-aged samples were treated with dox, indicating the transient expression system is still intact for aged mice (Fig 1).
To conclude, the transient reprogramming in vitro does not disturb the cellular identity of 4TF BMSCs as demonstrated by the ability to differentiate into the three respective lineages. We now await the maturation of our 4TF mice such that we can observe age-associated changes in the BMSCs. We will utilize intravital imaging techniques such as bone scans to inform our decisions for when to harvest our aged mice. We plan to conduct a multi-omics sequencing on our samples, including ChIP-seq to assess the binding sites of transcriptional factors, single-cell RNA-seq to assess the transcriptomic differences, and bisulfite sequencing to assess methylation patterns.
Our work will highlight significant transcriptomic and methylation differences between young, untreated aged, and transiently reprogrammed aged BMSCs. These differences point toward gene regulatory networks that are essential for in vitro self-renewal and fate decision for BMSCs but are significantly hindered by aging. Follow up experiments will isolate these networks and investigating if inhibiting or upregulating certain components will lead to an improvement or diminishing of BMSCs’ in vitro self-renewal capabilities. This project will provide insights into potential therapeutic targets and can benefit the aging population of our world.