Understanding theEffects of an Inflammatory Milieu on the...
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Lauren S. Sherman, Pranela Rameshwar Rutgers University, RBHS, Newark, NJ: Dept. of Medicine, Hematology/Oncology, NJMS; School of Graduate Studies at NJMS Mesenchymal stem cells (MSCs), multipotent cells found in various adult tissues, are an attractive source of cells for cellular therapy and drug delivery, and for regenerative medicine. Reasons include their ease to expand, plasticity to generate cells of all germ layers, reduced ethical concerns, and ability to be available as ‘off the shelf’ cells for immediate use in transplantation. Further, these cells exert anti-inflammatory functions, home to areas of inflammation, and can be used to deliver drugs and small molecules in vivo. MSCs can respond differently to varying microenvironments to perform distinct immune functions. The microenvironment can also affect the developmental state of MSCs. Better understanding of how the microenvironment influences MSC multipotency is crucial for effective translational use of these cells in the clinic. This study tested the hypothesis that the changes in an inflammatory microenvironment will influence MSC function. To study these effects, MSCs were treated with either aspirin, a pan-anti- inflammatory mediator, or conditioned media from an in vitro model of graft versus host disease (GvHD). The GvHD model was generated based on a modified two-way mixed lymphocyte reaction. The cells were then assessed for phenotype, multi-lineage differentiation capacity, proliferation, and viability. The anti-inflammatory microenvironment resulted in increased senescence and a loss of the stem cell state. This in vitro analysis will help elucidate factors within the inflammatory milieu that alter MSC multipotency. Identifying these factors will allow for more controlled and effective clinical use of MSCs. Abstract Summary • MSCs survive within a 3D system, with and without an inflammatory micro- environment • Culture in a 3D system appears to select for the more primitive MSCs, even in the absence of inflammation • MSCs proliferate within the 3D culture system • Inflammation supports MSC viability in the 3D culture system Future Direction • Determine the fate of MSCs as inflammation subsides, including changes in anti- inflammatory capacity • Determine the effects of the inflammatory milieu over time • Dissect the cellular and molecular causes of the observed effects on MSCs (role of specific cytokines, professional immune cells) • Validate with MSCs derived from other tissues • Emulate in vivo using models of traumatic injury Changes in the inflammatory microenvironment will influence MSC function. Hypothesis Recovery of Cells From a 3D Model, with and without inflammation Fold change from baseline 1 2.24 2.19 1.66 Figure 2: MSCs were stained with the cell tracker dye CMFDA for identification by flow cytometry and cultured in the 3D model in the absence (purple) or presence of peripheral blood mononuclear cells from one (pink, blue) or two (green) individuals. Recovered MSCs Form Apparent Colony Forming Unit – Fibroblasts Figure 3: (A) When returned to traditional (2D) growth conditions, the MSCs from the 3D model form apparent colony forming unit – fibroblasts (CFU-F). Once disassociated, the MSCs return to normal growth patterns. (B) The cells maintain an MSC phenotype. A. B. Acknowledgements This work has been funded by a grant from the New Jersey Commission on Cancer Research. Understanding the Effects of an Inflammatory Milieu on the Development of Mesenchymal Stem Cells: implication for clinical use Decrease in MSC Viability in 3D Model Figure 4: MSCs grown in a monolayer (2D) show a higher viability than those grown in the 3D model. This loss in viability may correlate with the PH4 positive (differentiated) population (Figure 3A). (B) The presence of an inflammatory milieu assisted in returning the 3D model culture’s viability to that of the monolayer 10 0 10 1 10 2 10 3 10 4 FL3-Height 13.70% 10 0 10 1 10 2 10 3 10 4 FL3-Height 0.60% 3D model 2D growth 7AAD 7AAD 7AAD 10 0 10 1 10 2 10 3 10 4 FL3-Height 4.58% 3D model + 2-way MLR Methods PBMC Donor 1 MSCs PBMC Donor 2 Ø Generate a 3D in vitro model of graft versus host disease (GvHD) based on a two-way mixed lymphocyte reaction (2-way MLR) Ø Treat MSCs with aspirin, a pan-anti- inflammatory mediator, or media from the inflammatory GvHD model Ø Assess MSC phenotype, multi-lineage differentiation capacity, proliferation, and viability Background Modified from J Chan et al. Blood 2006. K Tang et al. J Immunol 2008. JA Potian et al. 2003. • Primary tissue sources utilized for clinical use: bone marrow, adipose, umbilical cord • Home to areas of inflammation • Act as anti-inflammatory cells in the presence of inflammation clinicaltrials.gov • Used in >1300 clinical trials • Current “treatment to consider” for respiratory failure in COVID-19 patients (approved under FDA expanded access compassionate use) • But what happens to the MSCs as inflammation subsides? Aspirin Initiates Senescence in MSCs Figure 1: (A) MSCs were treated with varying concentrations of aspirin for 24 hours to determine the minimum concentration needed to initiate senescence. (B) Overnight treatment with 25 nM aspirin was sufficient to lose MSC morphology, (C) and subsequent days show loss of stem cell associated markers and increased markers of differentiation. (D) Increased senescence activity occurs within 2 hours of initial treatment in both bone marrow-derived MSCs (p4 MSCs) and immortalized adipose- derived MSCs (hTert MSCs). A. B. D. C. MSC Phenotype Differs with inflammation in a 3D Model Figure 3: (A) Cells recovered from the 3D culture model were phenotypically MSCs as defined as CD45 - , CD73 + , CD29 + , PH4 - . (B) However, MSC phenotype differs between MSCs grown with or without the presence of inflammation in a 3D model. A. 10 0 10 1 10 2 10 3 10 4 CD45 FITC 10 0 10 1 10 2 10 3 10 4 CD73 PE 10 0 10 1 10 2 10 3 10 4 PH4 FITC 15.49% Pre 3D Model 10 0 10 1 10 2 10 3 10 4 CD73 PE 10 0 10 1 10 2 10 3 10 4 CD45 FITC 10 0 10 1 10 2 10 3 10 4 PH4 FITC 0.19% 3D Model *no immune cells present B.
Transcript of Understanding theEffects of an Inflammatory Milieu on the...
Lauren S. Sherman, Pranela RameshwarRutgers University, RBHS, Newark, NJ:
Dept. of Medicine, Hematology/Oncology, NJMS; School of Graduate Studies at NJMS
Mesenchymal stem cells (MSCs), multipotent cells found invarious adult tissues, are an attractive source of cells forcellular therapy and drug delivery, and for regenerativemedicine. Reasons include their ease to expand, plasticity togenerate cells of all germ layers, reduced ethical concerns, andability to be available as ‘off the shelf’ cells for immediate usein transplantation. Further, these cells exert anti-inflammatoryfunctions, home to areas of inflammation, and can be used todeliver drugs and small molecules in vivo. MSCs can responddifferently to varying microenvironments to perform distinctimmune functions. The microenvironment can also affect thedevelopmental state of MSCs. Better understanding of how themicroenvironment influences MSC multipotency is crucial foreffective translational use of these cells in the clinic. This studytested the hypothesis that the changes in an inflammatorymicroenvironment will influence MSC function. To study theseeffects, MSCs were treated with either aspirin, a pan-anti-inflammatory mediator, or conditioned media from an invitro model of graft versus host disease (GvHD). The GvHDmodel was generated based on a modified two-way mixedlymphocyte reaction. The cells were then assessed forphenotype, multi-lineage differentiation capacity, proliferation,and viability. The anti-inflammatory microenvironment resultedin increased senescence and a loss of the stem cell state. Thisin vitro analysis will help elucidate factors within theinflammatory milieu that alter MSC multipotency. Identifyingthese factors will allow for more controlled and effectiveclinical use of MSCs.
Abstract Summary• MSCs survive
within a 3D system, with and without an inflammatory micro-environment
• Culture in a 3D system appears to select for the more primitive MSCs, even in the absence of inflammation
• MSCs proliferate within the 3D culture system
• Inflammation supports MSC viability in the 3D culture system
Future Direction• Determine the
fate of MSCs as inflammation subsides, including changes in anti-inflammatory capacity
• Determine the effects of the inflammatory milieu over time
• Dissect the cellular and molecular causes of the observed effects on MSCs (role of specific cytokines, professional immune cells)
• Validate with MSCs derived from other tissues
• Emulate in vivousing models of traumatic injury
Changes in the inflammatorymicroenvironment will influence MSCfunction.
Hypothesis Recovery of Cells From a 3D Model, with and
without inflammationFold change from baseline12.242.191.66
Figure 2: MSCs were stained with thecell tracker dye CMFDA foridentification by flow cytometry andcultured in the 3D model in the absence(purple) or presence of peripheralblood mononuclear cells from one(pink, blue) or two (green) individuals.
Recovered MSCs Form Apparent Colony Forming
Unit – Fibroblasts
Figure 3: (A) When returned to traditional (2D) growthconditions, the MSCs from the 3D model form apparentcolony forming unit – fibroblasts (CFU-F). Once disassociated,the MSCs return to normal growth patterns. (B) The cellsmaintain an MSC phenotype.
A.
B.
AcknowledgementsThis work has been funded by a grant from the New JerseyCommission on Cancer Research.
Understanding the Effects of an Inflammatory Milieu on the Development of Mesenchymal Stem Cells: implication for clinical use
Decrease in MSC Viability in 3D Model
Figure 4: MSCs grown in a monolayer (2D) show a higherviability than those grown in the 3D model. This loss inviability may correlate with the PH4 positive (differentiated)population (Figure 3A). (B) The presence of an inflammatorymilieu assisted in returning the 3D model culture’s viabilityto that of the monolayer
100 101 102 103 104
FL3-Height
7AAD_MSC.013
13.70%
100 101 102 103 104
FL3-Height
7AAD_plate.002
0.60%
3D model2D growth
7AAD 7AAD7AAD100 101 102 103 104
FL3-Height
7AAD_PBMCmix+MSC.011
4.58%
3D model + 2-way MLR
Methods
PBMC Donor 1
MSCsPBMC Donor 2
Ø Generate a 3D in vitro model of graft versushost disease (GvHD) based on a two-waymixed lymphocyte reaction (2-way MLR)
Ø Treat MSCs with aspirin, a pan-anti-inflammatory mediator, or media from theinflammatory GvHD model
Ø Assess MSC phenotype, multi-lineagedifferentiation capacity, proliferation, andviability
Background
Modified from J Chan et al. Blood 2006. K Tang et al. J Immunol 2008. JA Potian et al. 2003.
• Primary tissue sources utilized for clinical use:bone marrow, adipose, umbilical cord• Home to areas of inflammation
• Act as anti-inflammatory cells in the presence ofinflammation
clinicaltrials.gov
• Used in >1300 clinical trials• Current “treatment to consider” for respiratory
failure in COVID-19 patients (approved underFDA expanded access compassionate use)• But what happens to the MSCs as inflammation
subsides?
Aspirin Initiates Senescence in MSCs
Figure 1: (A) MSCs were treated with varyingconcentrations of aspirin for 24 hours todetermine the minimum concentrationneeded to initiate senescence. (B) Overnighttreatment with 25 nM aspirin was sufficientto lose MSC morphology, (C) and subsequentdays show loss of stem cell associatedmarkers and increased markers ofdifferentiation. (D) Increased senescenceactivity occurs within 2 hours of initialtreatment in both bone marrow-derivedMSCs (p4 MSCs) and immortalized adipose-derived MSCs (hTert MSCs).
A.
B.
D.
C.
MSC Phenotype Differs with inflammation in a 3D
Model
Figure 3: (A) Cells recovered from the 3D culture modelwere phenotypically MSCs as defined as CD45-, CD73+,CD29+, PH4-. (B) However, MSC phenotype differs betweenMSCs grown with or without the presence of inflammationin a 3D model.
A.
100 101 102 103 104
CD45 FITC
P3_panel1 cells.001
100 101 102 103 104
CD73 PE
P3_panel1 cells.001
100 101 102 103 104
PH4 FITC
P3_panel3 cells.004
15.49%
Pre
3D M
odel
100 101 102 103 104
CD73 PE
Teflon_panel1 cells.005
100 101 102 103 104
CD45 FITC
Teflon_panel1 cells.005
100 101 102 103 104
PH4 FITC
Teflon_panel3 cells.1.001
0.19%
3D M
odel
*no immune cells presentB.
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