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The Generation and Functional Characterization of Human Microglia From Episomally Reprogrammed iCell ® Hematopoietic Progenitor Cells Eugenia Jones, Deepika Rajesh, Sarah Burton, Christie Munn, Michael Hancock, Kwi Hye Kim, Simon Hilcove & Tom Burke FUJIFILM Cellular Dynamics, Inc., Madison, WI USA Abstract Microglia maintain immunological balance within the central nervous system by decreasing inflammation due to injury and buildup of cytotoxic substances and infectious material. Microglia research has been largely confined to rodents because human primary microglia are difficult to acquire and stably maintain in culture conditions. Here, we describe the generation, functional characterization and cryopreservation of human induced pluripotent stem cell-derived microglia (iMGL) from episomally reprogrammed iCell ® Hematopoietic Progenitor Cells (HPCs; FCDI proprietary technology) under defined conditions based on technology developed by the Blurton-Jones laboratory exclusively licensed to FCDI from the University of California-Irvine. iMGL retain purity, secrete immunomodulatory cytokines and phagocytose pHrodo-labelled bacterial bioparticles and amyloid beta (Aβ) fibrils. The ability to produce essentially limitless quantities of iMGL holds great promise for accelerating human neuroscience research into the role of microglia in normal and diseased states. Purity of Cryopreserved iPSC-derived Microglia Microglia • iCell Hematopoietic Progenitor Cells can be successfully differentiated to microglia according to the protocol outlined by Abud, et al., Neuron 2017. • Achieved successful cryopreservation and recovery of end stage mature microglia. • Cryopreserved microglia retain purity and phagocytosis function. • Ongoing efforts to scale up and generate large batches of iMGL to enable cell-based applications. Cytokine Profile of Cryopreserved iPSC-derived Microglia Origin From a mesodermal yolk sac progenitor Residence 10-15% of brain Appearance Small cell body and extended processes Personality Territorial 15 - 30 µm wide Movement 1.5 µm/min and represent a sophisticated scanning system in the brain Function Professional phagocytosis Renewal 28% per year or 0.08% per day. A self sustaining population in the brain Life span 4.2 years iPS Cell Culture 3D aggregate formation iCell Hematopoietic Progenitor Cells Hematopoietic Precursor cells GRAFT Purity of end stage iMGL. Before cryopreservation (Fresh) and cryopreserved iMGL (Frozen) were stained for the presence of cell surface (CD45, CD11β and CD33) and intracellular (P2RY12, TREM-2, CX3CR1, IBA) antigens by flow cytometry. The specific staining is compared against matched isotype controls. Mesoderm Induction/ Hemangioblast formation Day 5-13 CD34+ MACs Purification Cryopreservation iPSC-derived Microglia Purity >90% P2RY12+/ IBA+/ TREM2+/ CX3CR1+ Other markers CD45+/ CD33+/ CD11b+/ PU.1+ Function Phagocytosis of bacteria and Aβ particles Cytokine profile M1 and M2 Phagocytic Function of Cryopreserved iPSC-derived Microglia 0 100 200 300 400 500 600 700 800 CFU per 1E6cells Generation of iPSC-derived Microglia from iCell Hematopoietic Progenitor Cells Day 0 Day 2-11 Day 12 Day 14-23 Day 24-26 Thaw HPCs 2D Microglia Differentiation Split, Renew Cultures 2D Microglia Differentiation 2D Microglia Maturation IL34, TGF, MCSF, CD200, Fractalkine Day 10-12 Day 13-16 IL34, TGF, MCSF IL34, TGF, MCSF Day 23-26 Day 1-6 IL34, TGF, MCSF Defined method to generate iCell Hematopoietic Progenitor Cells. iCell Hematopoietic Progenitor Cells generate multipotent colony forming units (CFUs) and megakaryocytes upon thaw. Thaw Schematic representation of a defined method to generate iMGL from iCell Hematopoietic Progenitor Cells. iMGL were cryopreserved on day 26 of differentiation IBA TREM2 P2RY12 PU.1 CX3CR1 IBA TREM2 P2RY12 PU.1 CX3CR1 Frozen Fresh CD45 CD33 CD45 CD45 CD33 CD11b CD33 CD11b CD33 CD11b CD43 CD34 IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG IgG CD31/CD34 CD31/CD34/CD43/CD235/ CD41/CD45 Hoechst P2RY12 Cryopreserved iMGL were plated on Permanox chamber slides for 24 hr and stained for the presence of P2RY12, TREM-2 followed by staining with Hoechst dye and viewed under a fluorescent microscope Generation of iCell Hematopoietic Progenitor Cells Cryopreserved iMGL treated with 5μM Thiazovivin extend their processes to a ramified phenotype. Day 1-4 iMGL were plated in maturation media and allowed to recover for 48hr, then starved for 12 hr prior to stimulating them with LPS, interferon gamma or a combination of LPS and interferon gamma for 24 hr. Cell culture supernatants were collected and assayed on a multiplex Luminex system. 1 10 100 1000 10000 100000 pg/ml Control LPS Interferon Gamma LPS+Interferon Gamma CD45 CD11b IgG IgG Cryopreservation iMGL can phagocytose amyloid beta particles. Phagocytosis of pHrodo Red dye-labeled amyloid beta (1-42) particles was monitored over time using an IncuCyte S3 live-cell analysis system. TREM-2 P2RY12 / TREM / Hoechst Summary
Transcript of The Generation and Functional Characterization of Human ... › assets › SfN... · The Generation...
The Generation and Functional Characterization of Human Microglia From Episomally Reprogrammed iCell® Hematopoietic Progenitor CellsEugenia Jones, Deepika Rajesh, Sarah Burton, Christie Munn, Michael Hancock, Kwi Hye Kim, Simon Hilcove & Tom Burke FUJIFILM Cellular Dynamics, Inc., Madison, WI USA
AbstractMicroglia maintain immunological balance within the central nervous system by decreasing inflammation due to injury and buildup of cytotoxic substances and infectious material. Microglia research has been largely confined to rodents because human primary microglia are difficult to acquire and stably maintain in culture conditions. Here, we describe the generation, functional characterization and cryopreservation of human induced pluripotent stem cell-derived microglia (iMGL) from episomally reprogrammed iCell® Hematopoietic Progenitor Cells (HPCs; FCDI proprietary technology) under defined conditions based on technology developed by the Blurton-Jones laboratory exclusively licensed to FCDI from the University of California-Irvine. iMGL retain purity, secrete immunomodulatory cytokines and phagocytose pHrodo-labelled bacterial bioparticles and amyloid beta (Aβ) fibrils. The ability to produce essentially limitless quantities of iMGL holds great promise for accelerating human neuroscience research into the role of microglia in normal and diseased states.
Purity of Cryopreserved iPSC-derived Microglia
Microglia
• iCell Hematopoietic Progenitor Cells can be successfully differentiated to microglia according to the protocol outlined by Abud, et al., Neuron 2017.
• Achieved successful cryopreservation and recovery of end stage mature microglia.
• Cryopreserved microglia retain purity and phagocytosis function.
• Ongoing efforts to scale up and generate large batches of iMGL to enable cell-based applications.
Cytokine Profile of Cryopreserved iPSC-derived Microglia
Origin From a mesodermal yolk sac progenitorResidence 10-15% of brain
Appearance Small cell body and extended processes
Personality Territorial 15 - 30 µm wide
Movement 1.5 µm/min and represent a sophisticated scanning system in the brain
Function Professional phagocytosis
Renewal 28% per year or 0.08% per day. A self sustaining population in the brain
Life span 4.2 years
iPS Cell Culture
3D aggregate formation
iCell Hematopoietic Progenitor Cells
Hematopoietic Precursor cells
GRAFT
Purity of end stage iMGL. Before cryopreservation (Fresh) and cryopreserved iMGL (Frozen) were stained for the presence of cell surface (CD45, CD11β and CD33) and intracellular (P2RY12, TREM-2, CX3CR1, IBA) antigens by flow cytometry. The specific staining is compared against matched isotype controls.
Mesoderm Induction/ Hemangioblast formation
Day 5-13
CD34+MACs
PurificationCryopreservation
iPSC-derived Microglia
Purity >90% P2RY12+/ IBA+/ TREM2+/ CX3CR1+
Other markers CD45+/ CD33+/ CD11b+/ PU.1+
Function Phagocytosis of bacteria and Aβ particles
Cytokine profile M1 and M2
Phagocytic Function of Cryopreserved iPSC-derived Microglia
0100200300400500600700800
CFU
per
1E6
cells
Generation of iPSC-derived Microglia from iCell Hematopoietic Progenitor Cells
Day 0 Day 2-11 Day 12 Day 14-23 Day 24-26
Thaw HPCs
2D Microglia Differentiation
Split, Renew Cultures
2D Microglia Differentiation
2D Microglia Maturation
IL34, TGF, MCSF,
CD200, Fractalkine
Day 10-12 Day 13-16
IL34, TGF, MCSF
IL34, TGF, MCSF
Day 23-26Day 1-6
IL34, TGF, MCSF
Defined method to generate iCell Hematopoietic Progenitor Cells.
iCell Hematopoietic Progenitor Cells generate multipotent colony forming units (CFUs) and megakaryocytes upon thaw.
Thaw
Schematic representation of a defined method to generate iMGL from iCell Hematopoietic Progenitor Cells. iMGL were cryopreserved on day 26 of differentiation
IBA
TREM
2
P2RY12 PU.1 CX3CR1
IBA
TREM
2
P2RY12 PU.1 CX3CR1
FrozenFresh
CD45
CD33
CD45
CD45
CD33
CD11
bCD
33
CD11
bCD
33CD
11b
CD43
CD34
IgG
IgG
IgG Ig
G
IgGIgGIgGIgG IgG IgG
IgG
IgG
IgG
IgG
IgG
IgG
IgG
IgG
IgG
IgG
CD31/CD34 CD31/CD34/CD43/CD235/CD41/CD45
Hoechst
P2RY12
Cryopreserved iMGLwere plated on Permanoxchamber slides for 24 hr and stained for the presence of P2RY12, TREM-2 followed by staining with Hoechst dye and viewed under a fluorescent microscope
Generation of iCell Hematopoietic Progenitor Cells
Cryopreserved iMGL treated with 5μM Thiazovivin extend their processes to a ramified phenotype.
Day 1-4
iMGL were plated in maturation media and allowed to recover for 48hr, then starved for 12 hr prior to stimulating them with LPS, interferon gamma or a combination of LPS and interferon gamma for 24 hr. Cell culture supernatants were collected and assayed on a multiplex Luminex system.
1
10
100
1000
10000
100000
pg/m
l
Control LPS Interferon Gamma LPS+Interferon Gamma
CD45
CD11
b
IgG
IgG
Cryopreservation
iMGL can phagocytose amyloid beta particles. Phagocytosis of pHrodo Red dye-labeled amyloid beta (1-42) particles was monitored over time using an IncuCyte S3 live-cell analysis system.
TREM-2
P2RY12 / TREM / Hoechst
Summary
