Priming of hMSCs to Improve Potency

In this blog, we will present internal data and information on priming RoosterBio’s bone marrow-derived human MSCs (hMSC) with pro-inflammatory molecules and the impact of these priming protocols on hMSC immunomodulatory function and angiogenic cytokine secretion.


Human Mesenchymal Stem/Stromal Cells, or hMSCs, are key components of future therapeutics, engineered tissues, and medical devices. There are currently over 400 clinical trials investigating hMSCs as therapies (1). The trials have produced some promising results, with hMSCs generally deemed safe, and in some cases effective (2). It is believed that these versatile cells achieve their biologic and therapeutic effects by secreting a plethora of biomolecules (referred to as the MSC secretome) that moderate a variety of processes including angiogenesis, immunosuppression, and overall “tissue repair” (3-6). As the secretome is one of the likely Mechanisms of Actions (MOA) of hMSC therapies, there is a significant amount of recent work on engineering hMSC preparations to enhance secreted factors by genetic modification, by culture strategies, or by engineering the hMSC microenvironment (5, 7-11). In addition, a recent ISCT paper (12) advances the concept of “priming” hMSCs by exposing the cells to pro-inflammatory cytokines prior to implantation.

Thus, priming of hMSCs can have two primary purposes:

  1. To assess human MSC preparations in vitro as recommended by the ISCT and the FDA (12, 13), and
  2. To enhance hMSC potency (survival, immunosuppression, homing) prior to implantation (8, 9, 14).

As part of our standard quality control (QC) testing, RoosterBio analyzes the immunosuppressive capability of our cell lots through priming with IFN-γ. The hMSC response (i.e. immunomodulatory potential) is reported as a measure of indoleamine 2,3-dioxygenase (IDO) activity, determined by measuring the amino acid kynurenine in the culture supernatant.  The IDO enzyme converts L-tryptophan to N-formylkynurenine (or kynurenine), an immunosuppressive molecule that acts as an inhibitor of immune cell proliferation – including T cells (12, 15, 16).  Testing every hMSC lot for inducible IDO activity provides a quality assurance that the cells we release have some level of functional potency as it relates to immunomodulation– which we consider a key quality attribute of hMSCs.

While researchers are beginning to implement testing of hMSC preparations for inducible IDO activity prior to implantation, few are looking at the impact of priming on other hMSC functions.  Here, we present information on priming of RoosterBio’s hMSCs with IFN-γ ± TNF-α across multiple lots and donors and the impact of such treatment on hMSC IDO activity and angiogenic cytokine secretion.  The goal of this blog post is to demonstrate that priming has impacts on several functional properties of hMSCs, and that researchers should consider priming regimens to (a) understand the potency of their specific cell products, especially in inflammatory environments, and (b) to potentially increase potency of these cell products upon therapeutic administration.

Methods & Experimental Design

Materials & Reagents

Cell culture reagents were purchased from Life Technologies, chemicals and reagents for kynurenine measurement were from Sigma, and cultureware was from Corning.  Other products are: RoosterVial™ (BM-hMSC, part # MSC-001, RoosterBio) and RoosterBio RoosterNourish™-MSC (SKU KT-001).

hMSC Priming

RoosterBio bone marrow-derived hMSCs from multiple manufacturing cell lots and several donors were used for these experiments. All testing was performed on hMSCs of PDL range 10-14.

  1. Frozen hMSC vials were thawed and expanded in fully supplemented RoosterBio RoosterNourish-MSC according to the RoosterBio customer expansion protocol. Briefly, cells were plated in a T75 flask at 3000-3500 viable cells/cm2, incubated with no media change for 5-6 days, and harvested using TrypLE (LifeTech).
  2. For priming studies, hMSCs were seeded at confluence at 40,000 cells/cm2 in 12- or 6-well plates (Falcon) in 1 or 2 ml  RoosterNourish. After 18-24 hr, media was changed, to exclude RoosterBio RoosterBooster™, by replacing media with RoosterBio basal media (SU-005) +2% FBS or DMEM +2% FBS.
  3. After 1 hr, cells were (a) left alone as a non-primed control, or were treated with (b) Interferon (IFN-γ, LifeTech), at a final concentration of 10 ng/ml, (c) Tumor Necrosis Factor-α (TNF-α, LifeTech), at 10 ng/ml, or (d) IFN-γ and TNF-α together (10 ng/ml each).
  4. After 24 ± 1hr, culture supernatant was collected and frozen at -80˚C until assayed for IDO activity and cytokine secretion.

Measuring of Priming (IDO activity and Cytokines)

IDO activity measurement. 
IDO activity was assayed by quantifying kynurenine in the culture supernatant using a published colorimetric assay (16-19).

  1.   N-formylkynurenine was hydrolyzed to kynurenine by trichloroacetic acid (TCA) and treated with DMBA (1% (w/v) p-dimethylaminobenzaldehyde in acetic acid).
  2.   Kynurenine was measured by absorbance at 480 nm. A standard curve of 0-10 μg/ml L- kynurenine (Sigma) in media was used to calculate kynurenine levels.
  3.  IDO activity rates were calculated by normalizing kynurenine levels to the number of cells in the well and days of incubation. IDO activity is expressed as pg kynurenine/cell/day.

Cytokine secretion.

  1.   Culture supernatant was collected and assayed for basic FGF (bFGF, FGF2), HGF, IL-8, TIMP-1, TIMP-2 and VEGF by MultiPlex ELISA (Quansys, Angiogenesis Panel).
  2.   Secreted cytokine concentration was calculated and normalized to initial number of cells seeded and days of incubation to obtain specific cytokine secretion rates (presented as pg/cell/day).


Priming can be tested in different basal media. Data presented in Figure 1 show that (a) hMSC can be primed in RoosterBio basal medium or DMEM and (b) that the up-regulation of IDO activity between cell lots and different hMSC donors is highly consistent, demonstrated by the small SEM error bars.

IFN-γ and TNF-α demonstrate synergism in hMSC priming. Data in Figure 1 also show that the IDO response of hMSC to IFN-γ + TNF-α is synergistic, which is consistent with other publications with hMSCs; i.e. hMSCs responding to IFN-γalone, no response to TNF-α alone, and a synergistic response to IFN-γ and TNF-α together (12, 20-22).

priming of hMSCs

Angiogenic cytokine secretion is also variably regulated during priming. When angiogenic cytokine secretion was assayed under priming conditions, we found a complicated pattern of secretion (Figure 2).  None of the angiogenic factors were greatly altered (>30% change up or down) by IFN-γ alone. Basic FGF (FGF2) increased with TNF-α; this change was IFN-γ independent. HGF increased with IFN-γ/TNF-α co-treatment, but not with either treatment alone. There was a very large increase in IL-8 secretion with TNF-α treatment. Interestingly IL-8 was decreased by IFN-γ (+/- TNF-α). There was a slight decrease in secretion of VEGF, an important trophic factor, with IFN-γ/TNF-α co-treatment. We have only performed cytokine testing across 2 donors, but consistent patterns were seen across lots in both RoosterBio basal medium and DMEM basal media.

priming of hMSCs
Figure 2. Changes in cytokine secretion in IFNy and IFNy/ TNFa treated hMSC


Here, we present information on priming of RoosterBio hMSCs across various lots and donors. The current data demonstrates that:

  1.  hMSCs from multiple donors can be primed in multiple basal media types.
  2. The effect of IDO induction by IFN-γ is synergistically amplified by co-treatment with TNF-α.
  3. There are complicated changes in hMSC angiogenic cytokine secretion with priming.

These results reinforce the need for more complete (global) expression studies of any treatments to hMSCs. In addition, biological results need to be interpreted carefully, and any biological effects of priming cannot be solely attributed to changes in immunomodulatory cytokines/chemokines as they may be a function of hMSC cytokine secretion as well.

The take-home message is that hMSC functional properties can be altered in vitro in response to various stimuli. Monitoring the various hMSC biological functions is an important part of quality control testing on final cell products.  As demonstrated here, the simple addition of 2 factors can greatly alter hMSC function.  Clearly, introducing cells into a living host will provide many more micro-environmental cues that will modulate hMSC response and function upon therapeutic administration.  Extensive in vivo testing, and eventual clinical testing, will be required to understand if priming of hMSCs prior to delivery to the patient is a robust approach to enhance product potency. Thus, the impact of priming of hMSCs may go beyond simply using the approach as a standardization assay.

Readers, any comments or questions?


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