Paracrine effects of haematopoietic cells on human mesenchymal stem cells.

Zhou S. Paracrine effects of haematopoietic cells on human mesenchymal stem cells. Sci Rep. 5:10573, 2015.

Prepared by: Donald Ohioma, Advance Stem Cell Graduate Course, Fall 2015

 

 

LAYMAN ABSTRACT

Studying the microenvironment of the bone marrow (BM) has revealed the relationship between hematopoietic (HSCs) and mesenchymal stem cells (MSCs). MSCs act as precursors to bone-forming and reabsorbing cells. It is known that MSCs also have a regulatory effect on the HSCs in the bone marrow when; however the inverse relationship remains unclear. The author hypothesizes that soluble proteins and small molecules released by hematopoietic cells are responsible for the regulation of MSC’s function in bone formation. In order to determine the validity of this hypothesis, a series of experiments were developed to grow both cell types within the same system and observe the growth and proliferation of osteoblasts (bone forming cells) and osteoclast (bone reabsorbing cells). 

Human mononuclear cells (MNCs), a derivative of HSC, were taken from young and aged discarded femoral tissue and grown in vitro with expanded human MSCs. These cells were stained to determine the osteoblast activity level after being grown with the HSC derivative. The activity level was reported to have increased, implying that some factor from the MNCs stimulated the change. The factors were then identified by first comparing the active genes in both cell types and then by allowing MSCs to grow in the presence of these factors. One soluble molecule, TNF-α, was observed to have an inhibitory effect on bone production and a stimulatory effect on bone reabsorption. This interplay was shown to increase with age.

It was concluded that intervention in the path of the molecule TNF-α can be utilized as a therapeutic measure in aging humans. By targeting a product of the microenvironment rather than the cells themselves, there may be less risk of harmful side effects in treating skeletal disease. Targeting these small molecules may also be used in bone repair and rejuvenation.

 

 

SCIENTIFIC ABSTRACT

The tissue microenvironment, known as the niche, is a key component in understanding the function of human stem cells. The Bianco Hypothesis describes a dual sinusoidal niche shared by both mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) in the bone marrow (BM). This shared niche allows for the interaction of cytokines, produces by immune cells, with human MSCs. In studying this relationship, evidence has emerged to demonstrate the immune-mediated nature of age related skeletal disease. The author of this study hypothesized that an in vitro co-culture of hMSCs and human bone marrow mononuclear cells (hMNCs) would simulate the BM niche; allowing for the analysis of the relationship between hematopoietic cell soluble factors and skeletal aging and disease.

To assess the effects of human BM hematopoietic cells (MNCs) on proliferation and senescence of elderly MSCs, the author establishes a co-culture system using a polycarbonate membrane with 4μm pores to allow the transfer of proteins and small molecules and human cells taken from discarded femoral tissue. Staining for senescence-associated β-galactosidase (SA-β-Gal) revealed MNCs to a stimulatory effect on MSC ALP (alkaline phosphatase) activity and thereby osteoblast proliferation and differentiation. To confirm this data, a similar experiment was performed using mouse embryonic fibroblasts (MEFs). This co-culture system was shown to inhibit ALP activity of hMSCs. This indicates that the positive effects of MNC+MSC co-culture are related to a soluble factor released by the MNCs. RT-PCR was then performed to evaluate the gene profiles of MNCs and MSCs. This data would be used to determine the identity of the released MNC cytokine/soluble factor. The analysis revealed growth PDGF-β (a growth factor), Wnt related factors, and TNF-α to be important in the MNC gene profile. TNF-α signaling was then analyzed in both elderly and young samples to reveal an increase in expression with age. hMSCs were then cultured with TNF-α to determine it’s effects on senescence, cell death, proliferation, and osteoblast differentiation. The results showed TNF-α to increase senescence and cell death percentage as well as decrease cell number and osteoblast proliferation/differentiation. Further analysis of increased levels of TNF- on MSCs revealed a activating effect on NF-κB (an osteoclast differentiation regulatory molecule) and an increase in β-catenin, a molecule known to prevent osteoblast differentiation. Finally the effects of β-catenin on senescence were determined using knockdown by β-catenin siRNA in MSCs. The suppression of β-catenin increased ALP activity, which implies that activation of the β-catenin pathway decreases osteoblast proliferation and differentiation.

The data from this study reveals that there are, in fact, paracrine interactions occurring between hematopoietic cells and mesenchymal stem cells in the BM niche. This implies that interventions in signaling pathways that respond to this relationship can be targeted when treating age-related skeletal diseases like osteoporosis. Such treatments would greatly diminish the risk related to targeting osteoclast proliferation and differentiation.

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