MULTI-LINEAGE DIFFERENTIATION OF HUMAN INDUCED … · et al. (2017). Figure 1. (A) The MECP2 iPSC...

transcript

HPCsPrimitive Erythroid Expansion

Erythroid Maturation

Erythroblasts4A

MULTI-LINEAGE DIFFERENTIATION OF HUMAN INDUCED PLURIPOTENT STEM CELLS ENGINEERED TO EXPRESS TRUNCATED MeCP2 PROTEIN

Sarah Burton, Christie Munn, Madelyn Goedland, Anne Strouse, Beatriz Freitas, Simon Hilcove, Eugenia Jones, Deepika Rajesh FUJIFILM Cellular Dynamics, Inc., Madison, WI USA

Hematopoietic precursor cells (HPCs) derived from human induced pluripotent stem cells (iPSCs) are an excellent resource to study the onset of hematopoiesis in vitro and explore treatment options for hematological disorders and cancer. We have developed a defined in vitro differentiation protocol for the generation of iCell® Hematopoietic Progenitor Cells 2.0 from episomally reprogrammed iPSCs from a healthy donor. Cryopreserved iCell Hematopoietic Progenitor Cells 2.0 are >90% CD34 positive, express CD43, CD31, CD45, CD41, CD235, generate multipotent/mixed colonies in serum-free methylcellulose-based colony assay and megakaryocyte colonies in collagen-based colony assays. Cryopreserved iCell Hematopoietic Progenitor Cells 2.0 can be successfully differentiated to iCell Microglia, erythroblasts and lymphoid cells. Cryopreserved isogenic HPCs were generated from engineered iPSCs harboring a frame shift mutation to create loss of function of Methyl-CpG-binding protein 2 (MeCP2) to mimic disease modelling for Rett Syndrome (RTT). HPCs from the MeCP2 engineered iPSC matched phenotypic purity and functional requirements of iCell Hematopoietic Progenitor Cells 2.0. Further downstream differentiation of HPCs with impaired MeCP2 function generated a relatively mature erythroid cell (CD71+, CD235+, beta globin+) than the parental, apparently healthy normal (AHN), iCell Hematopoietic Progenitor Cells 2.0. MeCP2-engineered cells also revealed an increase in the efficiency of generating lymphoid (CD3+, CD8+ and CD56+) cells. Additionally, MeCP2 microglia exhibited a dysregulated inflammatory response compared to cells derived from the parental line. These findings identify a novel role for MeCP2 function in the onset of definitive hematopoiesis.

5 Figure 4. (B) Schematic representation of theemergence of beta-globin genes and thedevelopmental switches in expression from thecluster, from embryonic-to-fetal during the firsttrimester of conception, and from fetal-to-adultaround the time of birth as described by Orkin et al(2016).

CFU assays

CFU-GM

CFU-GEMM

4 Figure 1. (B) Structure of the MECP2gene, with the primary (E1) and minor,alternate (E2) transcripts. The insertionsite is downstream of the start codons forboth transcripts, resulting in truncation ofboth proteins and inactivation of the MBD(mutation site indicated by the black star).MBD: Methyl Binding Domain; ID: InterDomain; TRD: Transcriptional RepressionDomain; CTD: C-Terminal Domain.

Engineering Strategy

5 Figure 5. (E) Phagocytosis of S.aureus particles in iCell Microglia was monitored overtime using an IncuCyte® S3 live-cell analysis system. Controls shown in red (bioparticlesalone), brown (cell lines alone), and blue (cell lines treated with Cytochalasin D +bioparticles).

iPSC Characterization

Hematopoietic Differentiation – iCell® Hematopoietic Progenitor Cells 2.0

5 Figure 5. (C) Cryopreserved iCell Microglia wereplated in microglia maintenance medium and allowedto recover for 3 days, before stimulation with LPS for24 hours. Supernatants were assayed using themultiplex Luminex system. Heat map generated usingthe Standard curve for each analyte provided in the kit.

5 Figure 5. (B)Cryopreserved iCellMicroglia were thawedand labeled for thepresence of cell surface(CD45, CD11b, CD11c,TREM2 and CD33) andintracellular (P2RY12,TMEM119, CX3CR1,IBA1) antigens by flowcytometry. The specificstaining is comparedagainst matchedisotype controls.

4 Figure 5. (D) sTREM2 levels from conditioned mediawere quantified using a SimpleStep ELISA. The TREM2antibody recognizes the extracellular portion of TREM2.The iCell Microglia AHN control and MeCP2 werethawed and plated at the same density in maturationmedia in a 96-well Primaria plate. The absolute levels ofsTREM2 were quantified by collecting the supernatantson days 3 and 7 DIV from separate wells.

Erythroid Differentiation Myeloid Differentiation – iCell® Microglia

5 Figure 3. (A) iCell Hematopoietic Progenitor Cell 2.0 (HPCs) generation from iPSCs. Release assays forcharacterization of HPCs include serum-free methylcellulose CFU Methocult assay (Stem Cell Technologies).

5 Figure 4. Hemoglobin expression patternsduring erythroid differentiation and expansionto erythroblasts from (C) cord blood (CB), (D)parental apparently healthy normal (AHN) and(E) MeCP2 lines.

4 Figure 4. Expression of surfacedifferentiation markers during erythroiddifferentiation and expansion to erythroblastsfrom (F) cord blood (CB), (G) parentalapparently healthy normal (AHN) and (H)MeCP2 lines.

Lymphoid Differentiation

Conclusion

Marker

Expression of Pluripotency Markers

Figure 5. (A)Schematic of thedifferentiationprocess from iCellHematopoieticProgenitor Cells2.0 to iCellMicrogliaaccording to Abudet al. (2017).

Figure 1. (A) The MECP2 iPSC linecontains an insert directly after Serine49 (refers to Ensembl transcript MECP2-201 / ENST00000303391.11). The insertconsists of a stop codon for all 3reading frames, generating a non-functional truncated protein before themethyl-CpG-binding domain with theeffect of the protein being p.(Ala50Ter).

Figure 2. Engineered MeCP2iPSCs and the parental AHNisogenic control line expressed highlevels of both surface andintracellular markers whencharacterized by flow cytometry.Both lines maintained a normalkaryotype upon passaging (datanot shown). AHN = apparentlyhealthy normal

iPSCsAggregate Formation

Mesoderm Induction

Hematopoietic Precursor Cells

CD34+ MACS Purification

HPCs3A

Figure 4. (A) Diagram of differentiationof hematopoietic progenitor cells (HPCs) toerythroid cells.

2 4 5 6 7 8 10

CB Hemoglobin Expression

Hemoglobin Beta

Hemoglobin Gamma

Hemoglobin Epsilon

2 3 4 5 6 8

Parental Line Hemoglobin Expression

Hemoglobin Beta

Hemoglobin Gamma

Hemoglobin Epsilon

2 4 5 6 7

MeCP2 Line Hemoglobin ExpressionHemoglobin Beta

Hemoglobin Gamma

Hemoglobin Epsilon

1 2 4 5 6 7 8

CB Surface Expression of Erythrocyte Differentiation Markers

CD235a

CD71/235a

CD235a/36

CD71/36

1 2 3 4 5 6 8Perc

Parental Line Surface Expression of Erythrocyte Differentiation Markers

CD235a

CD71/235a

CD235a/36

CD71/36

1 2 4 5 6 7

MeCP2 Line Surface Expression of Erythrocyte Differentiation Markers

CD235a

CD71/235a

CD235a/36

CD71/36

Secretion of Soluble TREM2 by iCell Microglia

ositiv

Marker

iCell Microglia Marker Expression

iCell Microglia MECP2

iCell Microglia AHN

5 Figure 6. Phenotypic profile of MeCP2 and isogenic control apparently healthy normal (AHN) cell lineswhen differentiated to (A) lymphoid progenitors and (B) T- and NK-cells. HPCs were differentiatedtoward the lymphoid lineage and assayed at 2 and 6-8 weeks, respectively. Purity values gated using thelymphoid population on FSC vs. SSC.

CD45 CD7 CD5

Marker

Expression of Lymphoid Progenitor Markers (Lymphoid Gate)

AHN MeCP2

CD8/CD3 CD56/CD3-

Marker

Expression of T- and NK-Cell Markers (Lymphoid Gate)

AHN MeCP2

4 Figure 3. (B) Surfaceexpression of hematopoieticprogenitor cell markers onHPCs generated from theMeCP2 engineered line andisogenic, apparently healthynormal (AHN) control line.Flow cytometry analysis ofsurface markers CD34, CD43,CD45, CD41 and CD235a wasperformed.

CD34 CD43 CD45 CD41 CD235a

Marker

Expression of Hematopoietic Markers

AHN MeCP2

Rett Syndrome (RTT) is a devastating neurodevelopmental disorder, caused mainly bymutations in the MECP2 gene responsible for DNA methylation. Here we describe thederivation of an engineered MeCP2 iPSC line, along with the isogenic, apparently healthynormal (AHN) parent line, allowing for unparalleled investigation into the development andmanifestation of Rett Syndrome. While HPC derivation of MeCP2 engineered cells appearsnormal, downstream differentiation of many lineages was altered. Our studies demonstratedisordered maturation timelines in the hematopoietic lineage during erythropoiesis, suchas the enhanced emergence of CD235a in HPCs, and increased beta-hemoglobin expressionin erythroblasts. Myeloid differentiation to microglia was also successfully performed withthe MeCP2 engineered HPCs. However, these cells revealed functional differences such asdysregulated cytokine secretion to inflammatory stimuli, reduced TREM2 secretion, anddecreased phagocytosis of bioparticles when compared to isogenic control. These resultssupport a critical role of MeCP2 function in microglia in the brain microenvironment. Lastly,lymphoid differentiation of MeCP2 engineered HPCs was performed. Enhanced expressionof both lymphoid progenitor and T- and NK-cell markers was observed in the MeCP2 linewhen compared to isogenic control, suggesting a lymphoid bias during definitivehematopoiesis.MECP2 is a highly conserved, master switch gene responsible for transcriptional regulation.This study highlights its important role in developmental systems throughout the body, notjust in the brain microenvironment. Lack of a functional MeCP2 protein can lead to thedisruption of temporal development, manifesting as an enhancement, such as in the case oferythroid maturation and lymphoid bias, or as an impairment, such as in microglia function.Utilizing the iPSC technology, cells derived from the MeCP2 engineered line and isogenic,apparently healthy normal (AHN) parent line provide a valuable model for diseasemodeling, drug discovery and understanding of multiple developmental systems.

Introduction

MULTI-LINEAGE DIFFERENTIATION OF HUMAN INDUCED … · et al. (2017). Figure 1. (A) The MECP2 iPSC...

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