Download Notch Activation of Notch2 Selected Mesenchymal Stem Cells

Notch Activation of Notch2 Selected Mesenchymal Stem Cells Demonstrate Superior
Stem Cell Multipotency, Proliferation, and Enhanced Differentiation Ex Vivo
+Dong, Y; Rutkowski, T; O’Keefe, RJ; Hilton, MJ
University of Rochester Medical Center, Rochester, NY
[email protected]
Introduction: Human mesenchymal stem cells (hMSCs) have been
studied with great interest due to their therapeutic potential for treating
skeletal disease and facilitating skeletal repair, although maintaining their
multipotency and expanding this heterogeneous group of cells ex vivo has
proven to be very difficult limiting their use in clinical settings. Recently,
the Notch signaling pathway was identified as an important regulator of
MSC proliferation and differentiation during mouse skeletogenesis
leading to questions whether Notch signaling can be utilized as a tool to
maintain stem cell mutipotency and expand cell numbers ex vivo. Notch
signaling is initiated when the ligands Jagged1, 2 or Delta-like 1, 3, 4 bind
to the single-pass transmembrane cell surface Notch receptors (Notch1-4)
on neighboring cells. This interaction induces cleavage of the Notch
receptors, via the gamma-secretase complex, releasing the Notch
intracellular domain (NICD) to the nucleus activating the canonical Notch
transcriptional effector, RBPJk. Canonical Notch signaling is well
recognized as a critical factor controlling stem cell differentiation and
self-renewal in the hematopoietic, neural, pancreatic, intestinal, and
skeletal muscle systems. Therefore, we set out to demonstrate that
specific components of the Notch signaling pathway can be utilized to
select a subpopulation of hMSCs that can maintain greater multipotency
over multiple passages and can be expanded more readily ex vivo.
Materials and Methods: To determine the role of Notch signaling on
hMSC maintenance and expansion ex vivo, we cultured Lonza bone
marrow derived hMSCs and Notch2 selected hMSCs on IgG or Jagged1
(Jag1) coated plates for mutiple passages. Real-time RT-PCR and flow
cytometric analyses were used for measuring gene expression and stem
cell surface marker expression. Notch2 positive hMSCs were selected
using both flow cytometry and immonomagentic bead sorting methods.
BrdU labeling was performed for cell proliferation analysis and colony
forming unit – fibroblast (cfu-f) assays were used to evaluate hMSC
multipotency following several cell passages. hMSCs were initially
cultured on Jagged1 or IgG protein-coated plates for stem cell
maintenance and expansion assays, although the cells were removed from
the coated dishes and seeded on standard cell culture plates for osteogenic
differentiation assays. Alkaline phosphatase (AP) staining and real-time
RT-PCR assays for osteogenic markers Col1a1, AP, and Oc were
performed.
Results: To determine whether the Notch pathway could be utilized to
promote the maintenance and expansion of hMSCs derived from bone
marrow, we first analyzed the expression of all Notch receptors and each of
the Hes/Hey target genes using hMSCs cultured over multiple passages.
We identified Notch2 and Hes1 as being the most highly expressed
components of the Notch pathway, data consistent with work from our
previous developmental mouse studies. Interestingly, Notch2 and Hes1
expression levels were significantly reduced as hMSCs were cultured for
multiple passages (p1 – p10), concurrent with the dramatic reduction in
stem cell regulatory gene expression (Sox2, Oct4, and Nanog). Flow
cytometric analyses of these cells at p2 and p10 demonstrates that p10
hMSCs only retain about 40% of the hMSCs cell surface marker CD105,
suggesting that hMSCs lose their stem cell phenotype during normal
culturing and passaging of cells. To identify whether Notch activation can
both enhance hMSCs proliferation and maintain multipotency, we cultured
hMSCs for up to 10 passages on plates coated with recombinant protein for
the extracellular domain of the Notch ligand, Jagged-1, or control proteins.
Gene expression analyses showed that Jagged1 induced Notch activation
enhances the expression of stem cell transcriptional regulators, Sox2, Oct4
and Nanog, as well as, the Notch target gene Hes1 (Figure 1A). Cell
proliferation assays using BrdU labeling also demonstrated that Jag1
mediated Notch activation increases proliferation by more than 20% as
compared to controls using total hMSCs. On the other hand, when we
cultured Notch2 selected hMSCs on Jag1 coated plates; cell proliferation
rates were increased by 2.5 fold compared to control using total hMSCs
(Figure 1B). Cfu-f assays indicate that following Jagged1 mediated Notch
activation the number hMSC colonies was increased following p1, p5, and
p10 for Notch2 selected hMSCs as compared to total and Notch2 negative
A: Real time PCR
B: BrdU ELISA
Figure 1. Notch activation maintains hMSC mutipotency and
enhances cell proliferation. A: Real-time RT-PCR shows Jagged1
activated Notch signaling induces the expression of stem cell
transcriptional regulators, Sox2, Oct4 and Nanog, and the Notch target
gene, Hes1, and Notch receptor, Notch2. B: BrdU ELISA shows
enhanced hMSC proliferation of Notch2 selected hMSCs (Notch2+)
cultured on Jagged1 coated plates as compared to total and Notch2
negative (Notch2 -) hMSCs.
Notch2Passage 2
Notch2+
Notch2-
Notch2+
Passage 5
Figure 2: Notch2 selected hMSCs show enhanced osteogenesis.
Notch2 selected hMSCs were cultured on Jagged1 coated plates for 2
and 5 passages. Alkaline phosphatase staining was performed when
the cells were reseeded in osteogenic media for 12 days in the absence
of the Jagged1 recombinant protein.
hMSCs, suggesting an enhanced maintenance of multipotency. Osteoblast
differentiation assays demonstrate that when Notch2 selected hMSCs are
removed from Jagged1 mediated Notch activation they have enhanced
skeletal differentiation as compared to Notch2 negative hMSCs regardless
of how long the cells were maintained on Jagged1 coated plates (Figure 2).
Discussion: Our data clearly indicates that the Notch pathway is an
important regulator of hMSC maintenance and expansion ex vivo. These
data further suggest that the Notch2 positive sub-set of hMSCs represents a
uniquely multipotent and expandable population of hMSCs with a greater
differentiation capacity. We therefore believe that Notch signaling
activation can be used as a tool for hMSC maintenance and expansion ex
vivo and that Notch2 selected stromal cells have enhanced osteoblast
differentiation capacity and can be rapidly expanded ex vivo for the use in
bone tissue repair. We are currently trying to identify whether Notch2
selected and expanded hMSCs may also show enhanced chondrogenic
differentiation, as our previous work in the mouse has demonstrated that
Notch activation maintains and expands MSCs without affecting lineage
allocation. These data will be crtical to determine all of the potential
clinical uses for Notch2 selected, maintained, and expanded hMSCs.
Additional, studies are also underway to determine whether these cells can
demonstrate enhanced bone formation in various mouse models as
compared to traditionally isolated bone marrow derived hMSCs.
Poster No. 1762A • ORS 2011 Annual Meeting