Cell-of-Origin Discovery in Infant Leukemia through Integration of 3D Models and Patient Transcriptomic Data

Özet

This protocol aims to explore the cellular composition and temporal placement of candidate cell-of-origin for leukemias that arise in utero by integrating single-cell and/or bulk RNA sequencing from hemogenic gastruloids with patient data.

Özet

Pediatric hematological malignancies remain challenging to investigate and model due to the age group-specificity of certain genetic abnormalities. In utero origin has been demonstrated for a subset of pediatric leukemias, placing their respective cell of origin (CoO) during embryonic development. We recently reported a 3D hemogenic gastruloid (haemGx) model of embryonic blood formation derived from mouse embryonic stem cells, resolving the spatio-temporal complexity of developmental hematopoiesis. Importantly, it allows genetic engineering to introduce disease-relevant mutations. Using haemGx, we modeled the most common acute myeloid leukemia exclusive to infants (infAML), subtype t(7;12)(q36;p13), which arises in utero and is characterized by MNX1 overexpression. Here, we detail a method to define susceptibility to specific mutations that integrate phenotypic and transcriptional changes in the haemGx system and compares them with patient data. By proxy of our MNX1-overexpression haemGx, we show a pipeline from cell engineering to downstream analyses of leukemogenic potential. In particular, we focus on the clinical relevance of the model by integrating single-cell and/or bulk RNA sequencing from the haemGx platform with patient data to extract cellular composition and temporal placement of the putative CoO. This method is adaptable to the introduction of other oncogenic mutations, chromosomal rearrangements, or epigenetic modifications, as well as to chemical perturbations, including drug vulnerability and growth factor dependence. This flexibility allows for broad application across diverse disease contexts, enabling mechanistic dissection of how specific alterations disrupt early developmental trajectories with clinical relevance.

Giriş

Pediatric leukemias can exhibit age-specific genetic abnormalities that distinguish them from those in older patients. Age-specific features configure distinct biological properties of the lineages from which the malignancies arise—their cell of origin (CoO)¹. In particular, identifying a CoO for infant leukemias (infAML) remains challenging. Leukemia initiation in utero^2,3,4, is confounded by the spatio-temporal complexity of developmental hematopoiesis, which utilizes yolk sac (YS), aorta-gonad mesonephros (AGM), and fetal liver (FL) niches ....

Protokol

1. Use of engineered leukemia-associated abnormalities (LAA) in haemGx and downstream analyses to assess leukemogenic features

1. Culturing mESC with LAA for haemGx aggregation
   NOTE: mESC can be edited to introduce the LAA of interest by overexpression systems (here, using lentiviral transduction, MNX1 is overexpressed using a pWPT-LSSmOrange-PQR under the EF-1α promoter²⁵), or gene editing approaches (e.g., CRISPR/Cas9 to introduce mutations). A control line (e.g., transduction by empty vector or Cas9-only control) is also generated. Various reporter lines can be used, for instance, Kdr(Flk1)-GFP

Sonuçlar

We used haemGx to model the most common infAML subtype, t(7;12)(q36;p13), via MNX1 overexpression as a proxy, and to infer the developmental window of susceptibility and its clinical relevance to patient transcriptomics using GSEA.

To introduce LAA, we used lentiviral transduction to introduce MNX1 overexpression with the pWPT-LSSmOrange-MNX1-OE-PQR vector to overexpress MNX1 (mESC-MNX1) (Supplemental File 1 Supplemental Figure S1AB) and use.......

Tartışma

This protocol is amenable to model a variety of LAA that can be investigated in the context of embryonic hematopoietic development, with the advantage of spatio-temporal resolution and compatibility with established downstream molecular, functional, and biochemical analyses. Here, we focused on gastruloids that recapitulate hemato-endothelial specification to YS-like EMP and AGM-like HSPC emergence; however, other gastruloid / developmental organoid models that recapitulate specification of different tissues and organs c.......

Malzemeler

Bu makalede kullanılan malzemelerin listesi

Ad	Şirket	Katalog numarası	Yorumlar
Activin A Plus	Qkine	Cat. #QK005	Peptide, recombinant protein
B-27 Supplement (50x), serum free	Thermo Fisher Scientific	Cat. #17504044	Medium supplement
CHIR99021 (Chiron)	BioGems	Cat. #2520691	Peptide, recombinant protein
Gibco 2-Mercaptoethanol (50 mM)	Fisher Scientific	Cat. #11528926	Reducing agent
Gibco DMEM/F-12, with GlutaMAX	Fisher Scientific	Cat. #10565018	Medium
Gibco Glasgow's MEM	Fisher Scientific	Cat. #11570576	Medium
Gibco Glutamax	Fisher Scientific	Cat. #35050038	Medium Supplement
Gibco Neurobasal Medium	Thermo Fisher Scientific	Cat. #21103049	Medium
Mouse Methylcellulose Complete Medium	R&D Systems	Cat. #HSC007	Medium
Murine FGF-basic	PeproTech	Cat. #450-33	Peptide, recombinant protein
Murine Flt3-Ligand	PeproTech	Cat. #250-31L	Peptide, recombinant protein
Murine LIF	PeproTech	Cat. #250-02	Peptide, recombinant protein
Murine SCF	PeproTech	Cat. #250-03	Peptide, recombinant protein
Murine Sonic Hedgehog (Shh)	PeproTech	Cat. #315-22	Peptide, recombinant protein
Murine TPO	PeproTech	Cat. #315-14	Peptide, recombinant protein
Murine VEGF165	PeproTech	Cat. #450-32	Peptide, recombinant protein
N-2 Supplement (100x)	Thermo Fisher Scientific	Cat. #17502048	Medium supplement