A P P L I C AT I O N N O T E

Successful use ofmiRIDIAN
microRNA mimic
and inhibitor
libraries identify
microRNAs
involved in humanmesenchymal
stem cellosteogenesis.

Introduction
MicroRNAs are endogenous non-coding RNAs that modulate
gene expression through the RNA interference pathway1.
The mechanism of regulation is generally conserved acrossnature and is predicted to influence a significant portion of thehuman genome during early and adult stages of development2-5.
As microRNAs have been shown to play a role in stem cellfate determination, potentially exciting opportunities exist forapplications in regenerative medicine.
Human mesenchymal stem cells (hMSC) are capable ofdifferentiating into multiple lineages including bone, cartilage,
and tendon8. Unfortunately, while these cells can be easily
isolated and expanded, little is known about the contributions
that microRNAs make to MSC differentiation. Recently, several
approaches, including microarray and quantitative real-time
PCR (qPCR) technologies, have been used to identify microRNAs
that are disproportionately expressed in differentiated andundifferentiated states. Unfortunately, while these approaches
can provide potential insights into the role for microRNAs incell fate determination, they are restricted by two limitations.
First, in most in vitro cell differentiation assays only a fractionof the cells undergo differentiation. Thus, in the absence ofAuthors
Yuriy Fedorov
Jon Karpilow
Revvity, Inc.

enrichment by Fluorescence Activated Cell Sorting (FACS)“differentiated” cell profiles are contaminated with those ofundifferentiated cells, thereby compromising the ability toidentify relevant microRNAs.

Successful use of miRIDIAN microRNA mimic and inhibitor libraries identify microRNAs involved in human mesenchymal stem cell osteogenesis.

A second shortcoming of microarray and qPCR technologies

The primary screen utilized miRIDIAN collection ofis that these techniques only provide a snapshot of theinhibitors targeting all the known microRNAs of miRBase“before” and “after” cellular states. As it is plausible that

8.2. hMSCs (Lonza) were plated in propagation medium andmicroRNAs play a role in the transition from a multipotent tothen transfected with the miRIDIAN microRNAs inhibitor

fully differentiated state, experimental strategies that allow

collection using DharmaFECT™ 1 transfection reagent. The

investigation of the contribution of microRNAs to all stages

propagation medium was then replaced with a differentiationof cellular differentiation are necessary. In the following

cocktail and cells were cultured prior to assessing total

application note, we demonstrate how screening with miRNA

AP activity (Figure 2). In these studies, negative controls

mimic and inhibitor libraries can be successfully used toconsisted of 1) inhibitors targeting C. elegans microRNAs,

identify microRNAs that play a role in cell fate determination,and 2) a SMARTpool™ of Non-targeting siRNAs. As a positive

specifically, hMSC osteogenic differentiation.

control, a SMARTpool targeting RUNX2, a transcription factor
required for hMSC osteogenic differentiation, was employed

Assay workflow(see micrograph in Figure 2 for performance of controls).

The overall workflow used in these studies is presented inFigure 1. For the initial phase - Assay development – wechose to quantify alkaline phosphatase (AP), an early marker
of MSC osteogenic differentiation9. Alkaline phosphataselevels were assessed by one of two methods. In the primary
screen, cells were lysed and total AP was quantified using
the p-nitrophenol phosphate conversion method (BRSC, NY).
Alternatively, the fraction of AP positive cells in a population
were measured by staining the culture with the fluorogenic
ELF97 substrate, and then quantifying images using a highcontent imaging system.

Figure 2: Schematic of human microRNA inhibitor phenotypic
screening. Propagation medium = DMEM + 10% FBS.
Differentiation medium = propagation medium, dexamethasone,ascorbate, and glycerophosphate. Fluorescenct micrographs
show the relative performance. Negative (top) and postiive(bottom) controls.

Validating hits identified in microRNA inhibitor screens
requires approaches that are distinct from those used insiRNA phenotypic screens. For siRNA screens, hit validation
involves demonstrating that multiple siRNAs targeting thesame transcript induce the same phenotype. As microRNAs
are short, 17-28 basepair duplexes, the possibility ofdesigning multiple, non-overlapping inhibitors to eachmicroRNA does not exist. For this reason, validationconsisted of 1) eliminating primary screen hits that
did not exhibit concentration dependent responses
Figure 1: Experimental workflow encompassing assay
development, phenotypic screening, hit identification andvalidation of leads to identification of biologically relevant
endogenous targets.

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with the inhibitors, and 2) selecting hits in which thematching microRNA mimic and inhibitors pairs induced
opposite effects.

2

Successful use of miRIDIAN microRNA mimic and inhibitor libraries identify microRNAs involved in human mesenchymal stem cell osteogenesis.

Screen and validation results

Summary

From the original library of 396 human microRNA inhibitors,

A comparative study using microRNA microarrays identified

15 molecules (3.8%) significantly altered AP expression in

only two microRNAs that were recognized to be importantthe primary screen (data not shown). Seven of these moleculesin the inhibitor screening. Thus, the study presented above(inhibitors of miR-189, -153, -133a, -186, -27a, -148b, and -489)

clearly demonstrates the validity of using microRNA mimic

induced dose-dependent effects with six increasing AP activity

and inhibitors to identify non-coding RNAs that are essential

and a single inhibitor (i-148b) significantly decreasing AP

for biological processes.

activity. Mimics of miR-189, -153, -133a, and -186 microRNAs
had no effect on AP activity, suggesting that these genes might
be necessary but not sufficient for regulation of early hMSC
osteogenesis. In contrast, three microRNA mimics, miRs-27a,-148b, and -489 significantly affected AP activity in a manner

References
1. K.K-H. Farh, A. Grimson, The Widespread Impact ofMammalian MicroRNAs on mRNA Repression andthat was opposite of that induced by the matching inhibitor

Evolution, Science 310, 1817-1821 (2005).

(Figure 3). The opposite phenotypes observed with mimics andinhibitors support the notion that these three microRNAs play animportant and essential role in early osteogenic differentiation.

2. R.J. Jackson, N. Standart, How Do MicroRNAs Regulate
Gene Expression? Science STKE, 1-13 (2007).
3. L.P. Lim, N.C. Lau, Microarray analysis shows that somemicroRNAs downregulate larger numbers of target mRNAs,Nature 433, 769-773 (2005).
4. A. Stark, J. Brennecke, Animal MicroRNAs Confer
Robustness to Gene Expression and Have a Significant
Impact on 3’ UTR Evolution, Cell 123, 1133-1146 (2005).
5. A.E. Pasquinelli, B.J. Reinhart, Conservation of thesequence and temporal expression of let-7 heterochronic
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Med. 226, 507-520 (2001).
7. J.E. Dennis, P. Charbord, Origin and Differentiation of
Human and Murine Stroma. Stem Cells 20, 205-214 (2002).
For more information:

Figure 3: Bar graph showing the opposing effects ofmimic‑inhibitor pairs in osteogenic differentiation. AP = Alkaline
Phosphatase.

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