Patent Application
20250145961
The present invention relates to a method of generating bone marrow organoids from pluripotent stem cells, a model for bone marrow disorders including blood malignancies and uses for screening for efficacy of pharmacological and genetic manipulations for blood cancer treatment.
The bone and bone marrow are highly complex organs rich in multiple cell lineages, and are responsible for the generation and maintenance of blood cells. Current in vitro models of the bone and bone marrow lack both the architecture and cellular diversity to effectively model these systems and are typically composed of chips containing individual cell lineages obtained from primary human or mouse tissues or differentiated from stem cells. 3D cell culture allows for the development of a system that more accurately mimics the cellular complexity of native bone marrow. Mouse models are also often used to model human hematopoiesis. However, mouse bone marrow has certain differences to the human equivalent, and for many blood cancer types, the available mouse models fail to accurately reproduce the human pathologies. This limits their application for the study of mechanisms of disease and the identification and validation of new therapies.
A physiologically accurate bone model is therefore needed. While substantial advances have been made in the treatment of some blood cancers, many blood cancer remain incurable and there is a huge unmet need for new biological systems that may enable improved disease modelling and target screening. This is particularly true for bone marrow fibrosis, for example that occurring in a bone marrow malignancy called myelofibrosis, where a cancer clone drives excessive deposition of reticulin fibrosis resulting in scarring and destruction of the haematopoietic tissue, bone marrow failure, and typically death within 5-10 years from diagnosis. In addition, it is not currently possible to keep cells isolated from patients with certain types of blood cancer alive for sufficient time following sampling for mechanistic study or pharmacological or genetic manipulation, as they are poorly viable following sampling once they are not supported by the bone marrow niche.
Existing bone marrow models generally include minimal cell lineages or component parts, for example the co-culture of endothelial cells with immune cell subtypes in channels or microfluidic chips. However, this approach is limited in that it does not include the many specialist lineages of the bone marrow, or its unique architectural tissue organisation.
Therefore, there is a clear need for an improved model for bone marrow diseases, and in particular blood cancers—for example, to understand how the bone marrow stroma supports production of blood from healthy haematopoietic stem cells, to study the interactions between the malignant haematopoietic cells and bone marrow stroma and to identify and validate new targets for therapy.
The bone marrow organoids described here provide a transformative solution for modelling bone marrow function, blood production and blood cancers as well as target screening.
According to a first aspect of the present invention, provided herein is a method of generating bone marrow organoids from pluripotent stem cells wherein said method comprises:
Advantageously, the methods of the present invention lead to the generation of a physiologically accurate bone marrow niche that replicates the cellular, molecular and architectural features of hematopoietic tissues including key specialist lineages of the bone marrow. A physiologically accurate bone marrow will facilitate effective studies into bone marrow biology, bone marrow cancers, and for the development of novel therapeutics.
The skilled person will be familiar with routine methods to form mesodermal aggregates from iPSCs. In one embodiment, the mesodermal aggregates are formed by:
Alternative methods of mesoderm induction are known to the skilled person, for example culturing iPSCs in low oxygen (e.g. 1-5%) with BMP4, with or without a WNT inhibitor, such as CHIR99021.
The skilled person will recognise that the term “mesodermal aggregates” may also be referred to as “embryoid bodies (EBs)”.
In one embodiment, the induced pluripotent stem cells are human iPSCs (hiPSCs). The provided iPSCs, such as hiPSCs, may be undifferentiated.
Prior to the induction of iPSC aggregates, the iPSCs may be maintained and cultured on a basement/basal matrix (such as hESC-qualified Matrigel™) in mTeSR1 medium. Alternatively, the iPSCs may be cultured on other gel substrates such as, Geltrex™ (LDEV-Free Reduced Growth Factor Basement Membrane Matrix, which is a soluble form of basement membrane extracted from murine Engelbreth-Holm-Swarm (EHS) tumors), vitronectin or laminin cell culture matrix (Biolamina™) and maintained in other stem cell differentiation culture media such as StemFlex™ or E8 media, or equivalents thereof. The iPSCs may be cultured on tissue culture plates coated with the collagen matrix or other gel substrate.
In one embodiment, the iPSCs are passaged one or more times, for example at about 20% or more confluence. In one embodiment, the iPSCs are passaged one or more times, for example at about 50% or more confluence. In another embodiment, the iPSCs are passaged one or more times, for example at about 20-90% confluence. Preferably, the iPSCs may be passaged one or more times, for example at about 70-80% confluence. The iPSCs may be detached for passaging and/or aggregate formation, for example using physical/mechanical detachment and/or non-physical/mechanical detachment, such as using EDTA or enzymatic detachment. Preferably EDTA detachment is used together with mechanical dissociation, for example by pipetting action. In another embodiment, iPSCs may be detached using a method which generates a single cell suspension, for example using TRYPLE or Accutase.
For iPSC aggregate formation, detached iPSCs may be cultured in stem cell differentiation medium for a period of at least 8 hours. In another embodiment, the detached iPSCs may be cultured in stem cell differentiation medium for a period of at least 12 hours. In another embodiment, the detached iPSCs may be cultured in stem cell differentiation medium for a period of about 8-24 hours.
The stem cell differentiation medium may comprise basal media supplemented with a ROCK inhibitor, such as Y-27632. In one embodiment, the stem cell differentiation media comprises or consists of StemFlex™ (or equivalents thereof) optionally supplemented with ROCK inhibitor supplements, such as RevitaCell™ (Thermo), or equivalents thereof.
In one embodiment, the iPSCs for aggregate formation are cultured on ultra-low adhesion plates, for example comprising a covalently bound hydrogel layer that inhibits cellular attachment.
Following iPSC aggregate detachment, the iPSC aggregates may be incubated for a period before adding them to mesoderm induction medium. The incubation may be for a period of 8-24 hours, preferably about 8-12 hours. The incubation may be under standard cell maintenance conditions, such as 5% CO2 at 37° C. The resulting iPSC aggregates may be collected by gravitation or centrifugation and resuspended in the mesoderm-inducing medium (phase I medium) of step b. In one embodiment, the day iPSC aggregates are transferred for culture mesoderm-inducing medium is day 0. Culturing the iPSC aggregates in the mesoderm-inducing medium may be under suitable cell growth conditions, for example at 5% O2, 5% CO2, and 37° C.
The early mesoderm induction step may comprise incubation of the iPSC aggregates over a period of time sufficient to form mesoderm. Culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period of about 72 hours. In another embodiment, culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period of about 3 days. In another embodiment, culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period of about 2-5 days. In another embodiment, culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period of about 3-5 days. In another embodiment, culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period of about 3-7 days. In another embodiment, culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period of between about 60 hours and about 90 hours.
Additionally, or alternatively, culturing the iPSC aggregates in the mesoderm-inducing medium may be for a period until aggregates reached an average size of about 200-250 μm. The size of the aggregate is understood to be the average of the largest diameter of the iPSC aggregates.
The mesoderm may be considered formed (i.e. for moving onto the next step) when at least 60% of the cells are mesodermal cells. In another embodiment, the mesoderm may be considered formed when at least 70% of the cells are early mesodermal cells. In another embodiment, the mesoderm may be considered formed when at least 80% of the cells are mesodermal cells. The skilled person will understand that the formation of mesoderm cells may be provided as much as necessary, with the understanding that the number or percentage of mesoderm cells formed at this stage will have an impact on the end yield of bone marrow organoids. In particular, the higher number of aggregates will give a higher number of bone marrow organoids, but a higher number of mesodermal cells will give a more efficient differentiation into bone marrow lineages. Early mesodermal cells or tissue may be identified by the expression of key marker genes, such as one or more, or all of, Brachyury, Snail, TBX6 and N-cadherin, which then subsequently mature into definitive mesoderm. Therefore, in one embodiment, the production of mesodermal tissue from the iPSC aggregates may be identified by detecting the presence of mesodermal markers using any method known in the art. The mesodermal markers may comprise or consist of one or more, or all, of Brachyury, Snail, TBX6 and N-cadherin. The cell marker may be detected by protein expression, for example using immunofluorescence or mRNA expression (e.g. using qRT-PCR).
The mesoderm-inducing medium may be a chemically defined medium (CDM). In one embodiment, the mesoderm-inducing medium comprises a basal medium, preferably a stem cell differentiation culture media. In one embodiment the mesoderm-inducing medium comprises a stem cell differentiation media, such as APEL2 or StemPro, or equivalents thereof. The mesoderm-inducing medium may comprise a ROCK inhibitor, such as Y-27632.
The BMP4 may be provided in the mesoderm-inducing medium at a concentration of between about 10 and about 100 ng/ml. In another embodiment, the BMP4 may be provided in the mesoderm-inducing medium at a concentration of between about 40 and about 60 ng/ml. In a preferred embodiment, the BMP4 is provided in the mesoderm-inducing medium at a concentration of 50 ng/ml.
The FGF2 may be provided in the mesoderm-inducing medium at a concentration of between about 10 and about 50 ng/ml. In a preferred embodiment, the FGF2 is provided in the mesoderm-inducing medium at a concentration of 50 ng/ml. In one embodiment, the medium comprises FGF2 in an amount suitable to promote mesoderm differentiation and priming for early lineage fibroblast.
The VEGFA may be provided in the mesoderm-inducing medium at a concentration of between about 10 and about 50 ng/ml. In a preferred embodiment, the VEGFA is provided in the mesoderm-inducing medium at a concentration of 50 ng/ml. In one embodiment, the medium comprises VEGFA in an amount suitable to promote mesoderm commitment and prime early endothelial/haematopoeitic bipotent progenitors.
In one embodiment, BMP4 is used at a concentration of 50 ng/ml, FGF2 is used at a concentration of 50 ng/ml, and VEGFA is used at a concentration of 50 ng/ml.
The step of inducing vascular and haematopoietic commitment may comprise culturing the mesodermal aggregates from step a. in mesoderm-inducing medium (Phase II media) comprising, BMP4, FGF2, VEGFA, Flt-3 and SCF. Preferably, the medium is replaced between steps a and b, for example by harvesting the mesodermal aggregates by gravitation or centrifugation and resuspension in the mesoderm-inducing medium. In an alternative embodiment, the media may not be replaced, but is may be further supplemented with the required cytokines, such as BMP4, FGF2, VEGFA, Flt-3 and SCF.
The medium may be capable of inducing mesoderm. The medium may be a chemically defined medium (CDM). In one embodiment, the medium comprises a basal medium, preferably a stem cell differentiation culture media. In one embodiment the medium comprises stem cell differentiation medium, such as APEL2™ (as described by Ng et al (Nat Protoc. 2008; 3(5):768-76. doi: 10.1038/nprot.2008.42), which is herein incorporated by reference.
The BMP4 may be provided in the medium at a concentration of between about 10 and about 100 ng/ml. In another embodiment, the BMP4 may be provided in the medium at a concentration of between about 40 and about 60 ng/ml. In a preferred embodiment, the BMP4 is provided in the medium at a concentration of 50 ng/ml.
The FGF2 may be provided in the medium at a concentration of between about 10 and about 50 ng/ml. In a preferred embodiment, the FGF2 is provided in the medium at a concentration of 50 ng/ml. In one embodiment, the medium comprises FGF2 in an amount suitable to promote mesoderm differentiation and priming for early lineage fibroblast.
The VEGFA may be provided in the medium at a concentration of between about 10 and about 50 ng/ml. In a preferred embodiment, the VEGFA is provided in the medium at a concentration of 50 ng/ml. In one embodiment, the medium comprises VEGFA in an amount suitable to promote mesoderm commitment and prime early endothelial/haematopoeitic bipotent progenitors.
The Flt-3 may be provided in the medium at a concentration of between about 10 and about 100 ng/ml. In another embodiment, the Flt-3 may be provided in the medium at a concentration of between about 10 and about 40 ng/ml. In a preferred embodiment, Flt-3 is provided in the medium at a concentration of 25 ng/ml. In one embodiment, the medium comprises Flt-3 in an amount suitable to promote haematopoeitic commitment.
The SCF (stem cell factor) may be provided in the medium at a concentration of about 1-100 ng/mL. In another embodiment, the SCF is provided in the medium at a concentration of about 10-30 ng/mL. In one embodiment, the SCF is provided in the medium at a concentration of 25 ng/ml. In one embodiment, the medium comprises SCF in an amount suitable to promote haematopoeisis (generation of haematopoeitic lineages).
In one embodiment, BMP4 is used at a concentration of 50 ng/ml, FGF2 is used at a concentration of 50 ng/ml, VEGFA is used at a concentration of 50 ng/ml, SCF is used at a concentration of 25 ng/ml and Flt3 is used at a concentration of 25 ng/ml.
The medium may be further supplemented with one or more additional cytokines, such as IL7 or other interleukins to induce lymphopoeisis. Additionally or alternatively, the additional cytokines may include one or more of IL11, FLT3L, GM-CSF, IL1, IL12, IL13, IL33, TPO, IL3, IL6, IL2, IL10 and IL8.
The vascular and haematopoietic commitment step may comprise culturing the mesodermal aggregates for a period of between about 36 and 72 hours, preferably for a period of about 40-60 hours, more preferably for about 48 hours, or until the mesodermal aggregates achieve an average size of about 350-400 um (which may be about d4-6, preferably d5). Additionally, or alternatively, the vascular and haematopoietic commitment step may comprise culturing the mesodermal aggregates for a period until there are markers of early endothelial and haematopoeitic differentiation. Such markers may include one or more, or all of CD144, CD31, RUNX1, and GATA1. Markers may be detected by any suitable means, such as by qRT-PCR.
Following vascular and haematopoietic commitment, the mesodermal aggregates may be collected, for example via centrifugation, prior to embedding in the hydrogel (step c).
The culture conditions of step b may be standard cell culture conditions, such as 5% O2, 5% CO2, and 37° C. The culture conditions of step b may be normoxic conditions (e.g. about 20-21% O2), or with 5% O2.
The media for stem cell maintenance of step c. may comprise stem cell differentiation medium, such as APEL2™ (as described by Ng et al (Nat Protoc. 2008; 3(5):768-76. doi: 10.1038/nprot.2008.42), which is herein incorporated by reference, or equivalents thereof.
In one embodiment, the sprouting medium comprises a cytokine and/or growth factor cocktail for generating the desired mix of haematopoietic cell lineages. In one embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with growthfactors, for example selected from VEGFA, VEGFC, FGF2, SCF, Flt3, IL-3, IL-6, TPO, EPO, G-CSF, FBS and heparin, or combinations thereof. In one embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGFA, VEGFC, FGF2, SCF, Flt3, IL-3, IL-6, TPO, EPO, and G-CSF, and optionally FBS and/or heparin, to form bone marrow organoids. In another embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGFA, FGF2, SCF, Flt3, IL-3, IL-6, TPO, EPO, and G-CSF, and optionally FBS and/or heparin, to form bone marrow organoids. In another embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGF, FGF2, TPO, SCF, EPO, G-CSF. In another embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGFA, VEGFC, FGF2, SCF, Flt3, TPO, EPO, and G-CSF, and optionally FBS and/or heparin, to form bone marrow organoids. In one embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGFA, VEGFC, FGF2, SCF, Flt3, IL-3, IL-6, EPO, and G-CSF, and optionally FBS and/or heparin, to form bone marrow organoids. In another embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGFA, FGF2, SCF, Flt3, IL-3, IL-6, EPO, and G-CSF, and optionally FBS and/or heparin, to form bone marrow organoids. In another embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGF, FGF2, SCF, EPO, G-CSF. In another embodiment, the sprouting medium comprises a media for stem cell maintenance supplemented with VEGFA, VEGFC, FGF2, SCF, Flt3, EPO, and G-CSF, and optionally FBS and/or heparin, to form bone marrow organoids. The media may additionally or alternatively comprise IL3, IL6, IL2, IL8, IL10, IL7 other fibroblast growth factors and VEGFs. In another embodiment, sprouting medium comprises a media for stem cell maintenance supplemented with VEGF, FGF2, TPO, SCF, Flt3 and optionally one or more of EPO, G-CSF, M-CSF, GM-CSF, IL3, IL6, and IL7, and optionally FBS and/or heparin, to form bone marrow organoids. Any sprouting medium disclosed herein may comprise one or more small molecule (e.g. less than 900 Da) enhancers of hematopoietic or vascular differentiation. Enhancers of hematopoietic or vascular differentiation may comprise one or more of Forskolin, UM171 and SR1.
The VEGFA in the sprouting medium may be human VEGFA. In one embodiment VEGFA is present in the sprouting medium at a concentration of at least 2 ng/ml. In another embodiment, VEGFA is present in the sprouting medium at a concentration of between about 2 and 100 ng/ml. In one embodiment, VEGFA is present in the sprouting medium at a concentration of between about 5 and 100 ng/ml. In another embodiment, VEGFA is present in the sprouting medium at a concentration of between about 5 and 50 ng/ml. In another embodiment, VEGFA is present in the sprouting medium at a concentration of between about 40 and 60 ng/ml. In a preferred embodiment, VEGFA is present in the sprouting medium at a concentration of about 50 ng/ml. In one embodiment, the sprouting medium comprises VEGFA in an amount suitable to promote vascular commitment and endothelial sprouting.
The FGF2 in the sprouting medium may be human FGF2. In one embodiment FGF2 is present in the sprouting medium at a concentration of at least about 1 ng/ml. In another embodiment FGF2 is present in the sprouting medium at a concentration of 1-100 ng/ml. In another embodiment FGF2 is present in the sprouting medium at a concentration of 1-500 ng/ml. In one embodiment FGF2 is present in the sprouting medium at a concentration of 10-100 ng/ml. In another embodiment FGF2 is present in the sprouting medium at a concentration of 10-500 ng/ml. In one embodiment, the sprouting medium comprises FGF2 in an amount suitable to promote growth of fibroblast, mesenchymal stromal cells, and support vasculogenesis.
The sprouting medium may comprise between about 1 and 20% Foetal Bovine Serum (FBS). In one embodiment, the sprouting medium comprises at least about 1% Foetal Bovine Serum (FBS). In a preferred embodiment, the sprouting medium comprises about 5% Foetal Bovine Serum (FBS). In one embodiment, the sprouting medium comprises FBS in an amount suitable to promote endothelial sprouting and vasculogenesis. The FBS may be substituted with knock-out serum.
The sprouting medium may comprise at least about 1 U/mL heparin sulfate. The sprouting medium may comprise about 1-100 U/mL heparin sulfate. The sprouting medium may comprise about 1-500 U/mL heparin sulfate. In another embodiment, the sprouting medium may comprise about 2-10 U/mL heparin sulfate. The sprouting medium may comprise about 2-500 U/mL heparin sulfate. In one embodiment, the sprouting medium comprises at least about 5 U/mL heparin sulfate. In one embodiment, the sprouting medium comprises heparin sulfate in an amount suitable to promote FGF signalling, and optionally proplatelet formation from megakaryocytes.
The sprouting medium may comprise at least about 1 ng/ml Interleukin 3 (IL3). The sprouting medium may comprise about 1-50 ng/ml Interleukin 3 (IL3). In one embodiment, the sprouting medium comprises at least about 10 ng/ml Interleukin 3 (IL3). In one embodiment, the sprouting medium comprises about 1-100 ng/ml Interleukin 3 (IL3). In one embodiment, the sprouting medium comprises about 1-500 ng/ml Interleukin 3 (IL3). In one embodiment, the sprouting medium comprises IL3 in an amount suitable to promote haematopoeitic cell development and haematopoeitic commitment.
The sprouting medium may comprise at least about 1 ng/ml Interleukin 6 (IL6). The sprouting medium may comprise about 1-50 ng/ml Interleukin 6 (IL6). In another embodiment, the sprouting medium may comprise at least about 10 ng/ml Interleukin 6 (IL6). The sprouting medium may comprise about 1-100 ng/ml Interleukin 6 (IL6). The sprouting medium may comprise about 1-500 ng/ml Interleukin 6 (IL6). In one embodiment, the sprouting medium comprises IL6 in an amount suitable to promote haematopoeitic cell development and haematopoeitic commitment.
The sprouting medium may comprise at least about 1 ng/ml SCF The sprouting medium may comprise about 1-100 ng/ml SCF. In another embodiment, the sprouting medium may comprise about 1-500 ng/ml SCF. In another embodiment, the sprouting medium may comprise about 10-30 ng/ml SCF. In another embodiment, the sprouting medium may comprise about 10-500 ng/ml SCF. In another embodiment, the sprouting medium may comprise at least about 25 ng/ml SCF. In one embodiment, the sprouting medium comprises SCF in an amount suitable to promote haematopoeitic cell development and haematopoeitic commitment.
The sprouting medium may comprise at least about 1 ng/ml Flt3. The sprouting medium may comprise about 1-100 ng/ml Flt3. The sprouting medium may comprise about 1-500 ng/ml Flt3. In another embodiment, the sprouting medium may comprise about 10-30 ng/ml Flt3. The sprouting medium may comprise about 10-500 ng/ml Flt3. In another embodiment, the sprouting medium may comprise at least about 25 ng/ml Flt3. In one embodiment, the sprouting medium comprises Flt3 in an amount suitable to promote haematopoeitic cell development and haematopoeitic commitment.
The sprouting medium may comprise at least about 1 ng/ml thrombopoietin (TPO). The sprouting medium may comprise about 1-50 ng/ml thrombopoietin (TPO). The sprouting medium may comprise about 1-500 ng/ml thrombopoietin (TPO). In another embodiment, the sprouting medium may comprise about 10-30 ng/ml thrombopoietin (TPO). The sprouting medium may comprise about 10-500 ng/ml thrombopoietin (TPO). In another embodiment, the sprouting medium may comprise at least about 25 ng/ml thrombopoietin (TPO). In one embodiment, the sprouting medium comprises TPO in an amount suitable to promote haematopeoisis and the development of megakaryocytes (megakaryo- and thrombopoiesis).
The sprouting medium may comprise at least about 1 ng/ml Erythropoietin (EPO). The sprouting medium may comprise about 1-100 ng/ml Erythropoietin (EPO). The sprouting medium may comprise about 1-500 ng/ml Erythropoietin (EPO). In another embodiment, the sprouting medium may comprise about 10-30 ng/ml Erythropoietin (EPO). The sprouting medium may comprise about 10-500 ng/ml Erythropoietin (EPO). In another embodiment, the sprouting medium may comprise at least about 25 ng/ml Erythropoietin (EPO). In one embodiment, the sprouting medium comprises EPO in an amount suitable to promote development of erythroid cells (erythropoiesis).
The sprouting medium may comprise at least about 1 ng/ml Granulocyte Colony Stimulating Factor (G-CSF). The sprouting medium may comprise about 1-100 ng/ml Granulocyte Colony Stimulating Factor (G-CSF). The sprouting medium may comprise about 1-500 ng/ml Granulocyte Colony Stimulating Factor (G-CSF). In another embodiment, the sprouting medium may comprise about 10-30 ng/ml Granulocyte Colony Stimulating Factor (G-CSF). The sprouting medium may comprise about 10-500 ng/ml Granulocyte Colony Stimulating Factor (G-CSF). In another embodiment, the sprouting medium may comprise at least about 25 ng/ml Granulocyte Colony Stimulating Factor (G-CSF). In one embodiment, the sprouting medium comprises G-CSF in an amount suitable to promote commitment to neutrophil/monocyte lineage (development of this arm of immune cells).
The sprouting medium may comprise about 1-100 ng/ml BMP4. The sprouting medium may comprise about 1-500 ng/ml BMP4. In another embodiment, the sprouting medium may comprise about 10-30 ng/ml BMP4. The sprouting medium may comprise about 10-500 ng/ml BMP4. In another embodiment, the sprouting medium may comprise at least about 25 ng/mL BMP4. In one embodiment, the sprouting medium comprises BMP4 in an amount suitable to promote haematopoeisis.
In one embodiment, the embedded mesoderm aggregates are maintained until about d12. In one embodiment, the embedded mesoderm aggregates are maintained in sprouting medium for at about 1 week. In another embodiment, the embedded mesoderm aggregates are maintained in the sprouting medium for at about 5-10 days, preferably about 7 days.
The sprouting medium may be changed during the maintenance period, for example the sprouting medium may be changed every 48-84 hours, preferably every 72 hours
Sprouting medium changes may comprise 100% fresh medium or may comprise a mixture of fresh and conditioned sprouting medium. A mixture of fresh and conditioned sprouting medium may be used at d5 onwards. The fresh and conditioned sprouting medium may comprise between 20:80 and 80:20 fresh:conditioned media. In one embodiment, the fresh and conditioned sprouting medium may comprise about 60:40 fresh:conditioned media.
The conditioned media can provide a benefit as differentiating cells are secreting factors (cytokines and matrix components) which can help the niche develop, as well as support haematopoiesis.
The skilled person may adjust the concentration and/or composition of cytokines in order to alter the composition of the resulting bone marrow organoid. For example, part way (e.g. about half way) through the sprouting phase the concentration of EPO may be increased to produce more erythrocytes, or EPO may be reduced along with an increase in the SCF/FLT3 content to produce more HSPCs. The increase or decrease may be sufficient to allow the production of a desired cell composition in the bone marrow organoids. The decrease of a given cytokine may be about a 10%, 30%, 50%, 80%, or 90% decrease. The increase of a given cytokine may be about a 10%, 30%, 50%, 80%, 100%, 150% or 200% increase.
In one embodiment, the sprouting medium may further comprise VEGFC, for example for developing sinusoid specific endothelial vasculature. In an embodiment wherein VEGFC is provided into the sprouting medium the VEGFC may be added at a later period, for example from d3 (from the overall differentiation timeline). The sprouting medium may comprise VEGFC at a concentration of about 10-100 ng/ml. In another embodiment, the sprouting medium may comprise VEGFC at a concentration of about 20-30 ng/ml. In a preferred embodiment, the sprouting medium may comprise VEGFC at a concentration of about 25 ng/ml.
The hydrogel may be a mixed matrix hydrogel. In one embodiment, the mixed matrix hydrogel comprises collagen I and collagen IV. The ratio of collagen I to collagen IV may be 1:1 to 3:1. In one embodiment, the ratio of collagen I to collagen IV is 1:1. In one embodiment the hydrogel comprises or consists of collagen I or collagen IV.
The collagen may be provided at a concentration of between about 0.1 and 3.5 mg/ml. The collagen may be provided at a concentration of between about 1 and 3.5 mg/ml. In one embodiment, the collagen is provided at a concentration of about 1 mg/ml. In one embodiment, the mixed matrix hydrogel comprises Matrigel.
In one embodiment, the hydrogel comprises a natural polymer, such as collagen or fibrin. In another embodiment, the hydrogel may be a synthetic hydrogel, for example comprising or consisting of synthetic peptides, or peptide/adhesion functionalized polysaccharides.
Advantageously, the mix of collagen I and collagen IV in the hydrogel yields a high proportion of myeloid cells and a population of mesenchymal stromal cells, which are important for remodelling the bone marrow space.
Once the mesodermal aggregates are embedded into the hydrogel, vascular sprouts may form which function as primitive blood vessels.
In one embodiment, the mesoderm aggregates are allowed to sprout until an optimal size of between 800 μm and 2 mm is observed, which may be between d10-12. Once the optimal aggregate size has been reached, the organoids may be extracted from the mixed matrix hydrogel and media. The extracted organoids can be further cultured on tissue plates, such as ultra-low attachment plates (e.g. tissue culture plates or wells comprising a layer of covalently bonded hydrogel) or other tissue culture ware, and/or flow cells or chips. The extracted organoids can be further cultured under media flow conditions.
Sprouted bone marrow organoids may be extracted from the mixed matrix hydrogel and resuspended in basal media (Phase IV medium). The sprouted bone marrow organoids may be cultured individually, for example in multi-well ultra-low attachment dishes. The bone marrow organoids may be cultured in media, such as basal media. In one embodiment, the basal media (Phase IV medium) comprises the same media, including supplements, as the sprouting medium (Phase III medium) of step b.; or the same media, but with reduced cytokine content, such as about 50% reduction in cytokine content. The cytokine concentration may be maintained or reduced. The cytokine concentrations may be reduced to 10 ng/mL. In another embodiment, the cytokine concentrations may be reduced to 1 to 10 ng/mL.
The bone marrow organoids may be extracted from the mixed matrix hydrogel by physically/mechanically scraping them from the mixed matrix hydrogel and/or the extracted bone marrow organoids may be suspended in a media, such as basal media, and centrifuged to separate the bone marrow organoids from the mixed matrix hydrogel and form free bone marrow organoids.
The bone marrow organoids may be extracted at any suitable time, such as from day 12. The bone marrow organoids may be extracted at any suitable time, such as from day 12 to 30 or more, such as for as long as the cells of the bone marrow organoids are viable. The bone marrow organoids may be obtained at d18 or more for validation experiments. Validation may be performed by any means known to the skilled person, for example by immunofluorescence imaging of whole, ethyl cinnamate cleared bone marrow organoids, or by imaging of the bone marrow organoids embedded in optical cutting temperature (OCT) solution and frozen, or by embedding in paraffin. Validation may be performed by genetic analysis such as RNA sequencing and/or qRT PCR.
According to another aspect of the present invention, there is provided a model of a bone marrow organoid, wherein the bone marrow organoid is formed by the methods described herein.
According to another aspect of the present invention, there is provided bone marrow organoids formed by the methods described herein.
In one embodiment, the bone marrow organoids comprise haematopoietic stem/progenitor cells (CD34+), neutrophils and monocytes (CD11b+, Lin+), megakaryocytes (CD41+, CD34−, Lin−), erythroid cells (CD71+, CD235+, Lin−), endothelial cells (CD31+, CD144+), fibroblasts and bone marrow mesenchymal stromal cells (PDGFRb+, LepR+, VCAM1+). Preferably, the bone marrow organoid further comprises a vasculature network and/or sinusoidal cells. In one embodiment, the bone marrow organoid does not comprise osteoprogenitors and/or lymphoid cells. In an alternative embodiment, the bone marrow organoid comprises osteoprogenitors and/or lymphoid cells. In one embodiment, the bone marrow organoid is synthetically produced (i.e. in vitro) and does not comprise bone marrow tissue extract. In another embodiment, the bone marrow organoid is entirely comprised from cells derived from native bone marrow and assembled in the support matrix described herein.
The bone marrow organoids may further comprise cancer cells, for example from a cancer donor. The cancer cells may be engrafted cancer cells (e.g. cancer cells isolated from patients that are added to the cultures). The cancer may be a blood malignancy. The cancers cells may be from patients with myeloid or lymphoid blood malignancies, such as myeloma, acute or chronic lymphoblastic leukaemias, acute or chronic myeloid leukaemias, myelodysplastic syndrome, myeloproliferative neoplasms, lymphomas, or mast cell neoplasms. The bone marrow organoids may further comprise cells from a healthy donor, which may be engrafted. The cells from a healthy donor may be one or more of CD34+ cells, peripheral blood mononuclear cells (PBMCs), donor derived fibroblast or endothelium cells.
According to another aspect of the present invention, there is provided a model for fibrosis, wherein the model for fibrosis comprises a bone marrow organoid according to the invention that has been treated with an agent to induce collagen deposition in the bone marrow organoid.
According to another aspect of the present invention, there is provided a method of producing a model for fibrosis, wherein the method comprises treating a bone marrow organoid according to the invention with an agent that is capable of inducing collagen deposition in the bone marrow organoid.
In one embodiment, an agent is used to induce fibrosis in the bone marrow organoids. In one embodiment, an agent is used to induce extracellular matrix deposition in the bone marrow organoids, which respond to fibrosis-promoting factors by increasing smooth muscle actin and collagen expression, resulting in fibrosis. In one embodiment the agent comprises or consists of a growth factor or cytokine. The agent, such as a cytokine, may be TGFβ. In one embodiment the bone marrow organoids are treated with TGFβ to emulate fibrosis. In another embodiment, fibrosis may be induced genetically, such as by genetic modification, genetic overexpression smooth muscle actin and/or collagen, or by siRNA silencing, or by treatment with other proteins or pharmacological agents. In another embodiment, fibrosis may be induced genetically, such as by overexpression of genes, such as TGFB1 or other fibrosis-promoting genes, that induce expression of αSMA/collagen. Fibrosis may be induced by using an iPSC line from a patient suffering from fibrosis, or iPSCs may be gene edited to harbour a known fibrosis causing gene.
The amount of agent and incubation time with the agent may be an amount and time sufficient to cause collagen deposition in the bone marrow organoid, such as at least 2 ng/ml TGFβ, or such as at least 10 ng/ml, for at least 24 hours.
In another embodiment, the amount and incubation time with TGFβ may be at least 2 ng/ml, such as at least 5 ng/ml for at least 2 hours, such as at least 72 hours. In another embodiment, the amount and incubation time with TGFβ may be between about 2 ng/ml and 500 ng/ml, such as between about 5 ng/ml and 500 ng/ml, for a period of between about 2 and 96 hours or more, such as between about 24 and 96 hours or more. In another embodiment, the amount and incubation time with TGFβ may be between about 2 ng/ml and 500 ng/ml, such as between about 5 ng/ml and 500 ng/ml for a period of between about 2 and 72 hours, such as between about 24 and 72 hours. In another embodiment, the amount and incubation time with TGFβ may be between about 2 ng/ml and 50 ng/ml, such as between about 5 ng/ml and 50 ng/ml, for a period of between about 24 and 72 hours. In another embodiment, the amount and incubation time with TGFβ may be between about 2 ng/ml and 50 ng/ml, such as between about 5 ng/ml and 50 ng/ml for a period of between about 36 and 72 hours. The skilled person will appreciate that where a higher concentration is used the incubation time may be shorter, and vice versa.
According to another aspect of the present invention, there is provided the use of the model for fibrosis to identify agents capable of preventing or treating fibrosis, wherein the bone marrow organoid is treated with a potential agent before, during or after the bone marrow organoid is treated with a fibrosis-inducing agent.
In another embodiment, fibrosis induced by a fibrosis-promoting agent may be prevented by pharmacological or genetic manipulation of the organoid.
The use may comprise the determination of whether the treatment by the potential agent has any effect in inhibiting or reducing the development of fibrosis, or the reduction in fibrosis after it has developed. The use may comprise the determination of whether the treatment by the potential agent has any effect in inhibiting or reducing the deposition of collagen. The use may comprise the determination of whether the treatment by the potential agent has any effect in inhibiting or reducing smooth muscle actin and/or collagen expression, or other markers of fibrosis.
The prevention or treatment of fibrosis may comprise inhibiting, preventing or reducing fibrosis.
According to another aspect of the present invention, there is provided a method of screening for agents capable of preventing or treating fibrosis, for example using the bone marrow organoid described herein, wherein the bone marrow organoid is genetically manipulated or treated with a potential agent before, during or after the bone marrow organoid is treated to induce fibrosis; and
The bone marrow organoids according to the present invention may be used for one or more of:
According to another aspect of the present invention, there is provided a method for maintaining the viability of cells from a patient donor with blood cancer ex vivo, to enable mechanistic studies or screening of agents or potential agents consisting of one or more of:
The agent to be screened or investigated may be dosed at a physiological relevant amount. The agent to be screened or investigated may be dosed at a therapeutically relevant amount. Combinations of agents may be investigated.
The determination may be relative to an untreated bone marrow organoid (i.e. not treated with the potential agent) and/or relative to a control or reference value.
In one embodiment the agent to be investigated is a small molecule (e.g. less than 900 Da), nucleic acid, antibody therapy, cellular therapy, drug compound, metabolite or peptide. In one embodiment the agent to be investigated is a small molecule (e.g. less than 900 Da), nucleic acid or peptide. The peptide may comprise or consist of an antibody. In another embodiment the agent to be investigated is a genetic manipulation agent, such as siRNA, shRNA, CRISPR-CAS9, lentiviral or retroviral vectors, for example for over expression.
Another aspect of the invention is its ability to support engraftment and survival of cells from patients with a range of blood malignancies, including cancer cell types which are difficult to keep alive ex vivo in standard liquid culture systems. The cancer cell types may include, but not be limited to, cells from patients with myeloid or lymphoid blood malignancies such as myeloma, acute or chronic lymphoblastic leukaemias, acute or chronic myeloid leukaemias, myelodysplastic syndrome, myeloproliferative neoplasms, lymphomas, and mast cell neoplasms.
Therefore, in one embodiment, the organoids of the present invention may be used for an engraftment and/or survival assay for cells from a patient with a blood malignancy.
A method is provided for seeding of the organoids with cells from a donor, and tracking the cells to assay one or more of survival, proliferation and isolation of engrafted cells, for example for downstream functional testing. This model also presents a novel system to study cancer-associated pathogenic remodelling of the bone marrow niche, such as the fibrosis induced by a malignant clone in a proportion of patients with myeloproliferative neoplasms.
The cells maybe tracked by fluorescent markers or tags. Tracking the cells may comprise the use of a fluorescent cell tracking system.
The cell donor may be an adult or child donor.
The fibrosis in accordance with any aspect or embodiment herein may comprise or consist of myelofibrosis.
In one embodiment, the bone marrow organoids according to the invention may be used to produce blood platelets.
According to another aspect of the present invention, there is provided a method for producing platelets and/or erythroid cells (RBCs), the method comprising the incubation of bone organoids in accordance with the invention in vitro, and harvesting the platelets and/or erythroid cells (RBCs) produced from the bone organoids.
The platelets and/or erythroid cells (RBCs) may be produced naturally by the bone marrow organoids or induced, for example by the dosing of heparin, hirudin, and/or ROCKi to drive more proplatelet formation. The platelets and/or erythroid cells (RBCs) may be harvested by separating them from the cells of the bone organoids, such as by FACS. Additionally or alternatively, the platelets and/or erythroid cells (RBCs) may be harvested by BSA gradient and/or centrifugation.
According to another aspect of the present invention, there is provided a method of screening for biomarkers of fibrosis or other bone marrow disorders, the method comprising the monitoring of biomarkers released from the bone marrow organoids or cells engrafted therein, or biomarkers in tissue or cellular extracts of the bone marrow organoids.
A disease state may develop, or be induced in the bone marrow organoid, whereby changes to the biomarker profile may be determined and linked to the disease state.
The biomarkers may comprise proteins, glycoproteins, glycans, peptides, nucleic acids, or any cellular product which may indicate a diseased state of the bone marrow organoid or engrafted cells in the bone marrow organoid. The biomarkers may be cell markers, such as surface proteins.
In a further aspect, the invention provides one or more compositions comprising the recombinant growth factors and cytokines required to carry out the first aspect of the invention. The one or more composition may comprise or consist of two or more of, such as all of, recombinant BMP4, FGF2, VEGFA, Flt-3 and SCF. The one or more composition may comprise or consist of VEGFA and FGF2. The one or more composition may comprise or consist of VEGFA, FGF2, BMP4 and VEGFC. The one or more composition may comprise or consist of VEGFA, FGF2, SCF, TPO and Flt3. The one or more composition may comprise or consist of VEGFA, VEGFC, FGF2, SCF, TPO, BMP4 and Flt3. The one or more composition may comprise or consist of VEGFA, VEGFC, FGF2, SCF, TPO, EPO, BMP4, IL3, IL6 and Flt3. Any composition disclosed herein may further comprise IL7 and/or calcium.
The one or more composition may comprise recombinant BMP4 for use at a concentration of between about 10 and about 100 ng/ml, for example if diluted to a working concentration. When recombinant BMP4 is provided in the one or more composition for use at a concentration of between about 10 and about 100 ng/ml, recombinant FGF2 is provided for use at a concentration of between about 10 and about 50 ng/ml, recombinant VEGFA is provided for use at a concentration of between about 10 and about 50 ng/ml, recombinant Flt-3 is provided for use at a concentration of between about 10 and about 100 ng/ml, and/or recombinant SCF is provided for use at a concentration of between about 1 and about 100 ng/ml. Such a composition allows the skilled person to induce vascular and haematopoietic commitment of mesodermal aggregates before embedding in a hydrogel.
In another aspect, there is provided a kit comprising one or more, such as all of, recombinant BMP4, FGF2, VEGFA, Flt-3 and SCF. The kit may comprise or consist of VEGFA and FGF2. The kit may comprise or consist of VEGFA, FGF2, BMP4 and VEGFC. The kit may comprise or consist of VEGFA, FGF2, SCF, TPO and Flt3. The kit may comprise or consist of VEGFA, VEGFC, FGF2, SCF, TPO, BMP4 and Flt3. The kit may comprise or consist of VEGFA, VEGFC, FGF2, SCF, TPO, EPO, BMP4, IL3, IL6 and Flt3. Any kit disclosed herein may further comprise IL7 and/or calcium.
The kit may further comprise a set of instructions. The instructions will enable the reader to perform any method disclosed herein. The recombinant growth factors and/or cytokines may be provided in one solution in the kit, or two, three, four, five, six, seven or more separate solutions in the kit. In this way, one or more than one of the recombinant growth factors and/or cytokines may be provided in each solution, if required. The kit may also comprise a mesoderm-inducing medium, sprouting medium and/or hydrogel required to carry out any method disclosed herein.
In a further aspect, there is a method of producing hematopoietic and/or stromal cells, wherein said method comprises:
The method my employ any step of any other method recited herein.
In yet a further aspect, there is provided hematopoietic and/or stromal cells obtained by any method disclosed herein.
References to amounts or concentrations of agents provided in the medium of incubation steps may refer to the amount or concentration provided at the start of incubation (i.e. prior to cell consumption during incubation). In another embodiment, references to amounts or concentrations of agents provided in the medium of incubation steps may refer to the total amount provided during the incubation, or the level of exposure of the agent to the cells, for example where a sustained release or drip feed system is used to continuously or periodically provide the agent during incubation.
References to “inhibition” or similar, may comprise a reduction in activity or presence of a molecule or the block of a biological pathway, such as a signaling pathway. The inhibition may be total (i.e. 100%) or at least a substantial inhibition. The inhibition may be partial inhibition. Partial inhibition may comprise significant inhibition in order to affect the desired outcome of the inhibition.
It is understood that pluripotent stem cells (PSCs) have indefinite capacity to self-renew and can differentiate into three primary germ layers of early embryo, thus differentiating into any adult cell type except extra-embryonic lineage such as placenta. Pluripotent stems cells may comprise embryonic stem cells or induced pluripotent stem cells (iPSC) made from adult somatic cells such as blood or skin cells by reprogramming technology. In one embodiment, the invention herein may not use embryonic stem cells. PSCs can be isolated or generated from non-human species. Human iPSC have the advantage harbor the patient genetic signature are useful in investigating genotype-phenotype links when differentiated into relevant cell types. This is useful for investigating disease mechanism or identifying new drug targets or investigating patient specific changes.
As used herein, the term “iPSC aggregate refers” to a 3D mass of cells, which may not grow as a spreading monolayer on a plate surface.
It is understood that “chemically defined medium” is a growth medium suitable for the in vitro cell culture of human or animal cells in which all of the chemical components are known.
It is understood that the terms “cell induction” or “cell differentiation” is the promotion of an iPSC to differentiate into a particular cell type, such that it is no longer pluripotent. The induction/differentiation may comprise the stimulation, upregulation or downregulation of specific biological pathways, which may be provided by growth conditions; agents; delivery of genes by an expression system such as plasmids; activation of genes using genome engineering approaches, reduction or knock-out of genes by an expression system such as RNAi or genome engineering approaches; or media components. It is understood therefore that the term “mesodermal induction” refers to the promotion of a pluripotent stem cell to differentiate into mesodermal tissue.
As used herein, the term “bone marrow organoid” may be taken to describe a 3-dimensional, multi-lineage cellular structure. The structure is composed of human, iPSC-derived cell types with high homology to those found in bone marrow, including stromal cells (comprised of but not limited to mesenchymal stromal cells, fibroblasts), endothelial cells, and hematopoietic cells (including but not limited to hematopoietic stem and progenitor cell types as well as myeloid cellular subtypes). The cells making up the structure are organized in highly reproducible fashion including but not limited to lumen-forming vessels with perivascular stroma and myeloid cells. The organoids can be generated in vitro, reproducibly at scale, for in vitro or in vivo experimentation e.g. to test candidate or tool compounds, cellular therapies, with or without engraftment of cells isolated from blood or bone marrow of human donors. A bone marrow organoid as defined herein may contain engrafted cells from a cancer patient or healthy patient.
Unlike naturally occurring bone marrow, bone marrow organoids are grown ex vivo, in miniature and in multiples, typically forming multiple structures that are a millimeters in diameter, such as the size of between 800 μm and 5 mm, or between 800 μm and 2 mm. The bone marrow organoids may be less than 5 mm, or less than 3 mm in size (measured as the largest diameter). In contrast, human bone marrow exists in large bony structures. A key distinguishing feature of bone marrow organoids is that they are grown reproducibly with multiple replicates, enabling experimental studies. In normal human physiology, the bone marrow is populated by cells from the fetal liver during gestation. In the present system, we differentiate mesodermal and bipotent hematopoietic-endothelial progenitors from iPSCs, which form hematopoietic, vascular as well as stromal lineages in the organoid within the culture system. Using iPSCs and differentiating the cell lineages in a dish enables multiple, reproducible replicates of bone marrow organoids for experimental manipulation which incorporate haematopoietic, stromal and vascular elements. This is not possible using native human tissue. The iPSC-derived bone marrow organoids can contain cell types that are transcriptionally representative of fetal liver, fetal bone marrow and adult bone marrow cells, in contrast to human tissues.
The skilled person will recognise that the term “organoid” may alternatively be termed “spheroid” or “microtissue”.
The skilled person will understand that optional features of one embodiment or aspect of the invention may be applicable, where appropriate, to other embodiments or aspects of the invention.
Embodiments of the invention will now be described in more detail, by way of example only, with reference to the accompanying drawings.
A human induced pluripotent stem cell line (iPSCs) purchased from Gibco (Thermo) was cultured on GelTrex (Thermo) coated 6-well tissue culture plates (Corning). Cells were passaged at approximately 70% confluence using EDTA detachment. Briefly, wells were washed once with PBS and once with EDTA before a 3-minute incubation at 37° C. and 5% CO2. EDTA was then aspirated, and cells removed through gentle mechanical dissociation by pipetting with StemFlex (Thermo) basal media. Cells were then diluted and maintained in StemFlex medium.
A step wise differentiation protocol was applied to generate vascularised bone marrow organoids. Firstly, baseline iPSCs grown to 70-80% confluence before detachment using the EDTA method described above. Detached iPSCs were cultured overnight in StemFlex supplemented with
RevitaCell (Thermo) on 6 well ultra-low attachment plates. The resulting iPSC aggregates were then collected either by gravitation or low speed centrifugation on the following day (d0), and resuspended in Phase I medium. Phase I medium was comprised of APELII (Stem Cell Technologies) supplemented with 50 ng/ml of Bone Morphogenic Protein-4 (BMP4) (Thermo), Fibroblast Growth Factor-2 (FGF2) (Stem Cell Technologies) and Vascular Endothelial Growth Factor A (VEGFA-165) (Stem Cell Technologies). Cells were then maintained at 5% O2, 5% CO2, and 37° C. for 72 hours until mesodermal aggregates reached an average size of approximately 200-250 um (typically d3).
After 72 hours, cells were collected via gravitation before resuspension in Phase II medium, which was comprised of APELII with 50 ng each BMP4, FGF2, and VEGFA, supplemented with 25 ng Fms Related Receptor Tyrosine Kinase-3 (Flt-3) and stem cell factor (SCF) (25 ng/mL). Cells were cultured for a further 48 hours, or until they achieve an average size of 350-400 um, under these conditions (typically d5).
At d5 cells were collected via centrifugation and prepared for embedding in mixed matrix hydrogels. Initially, different compositions of hydrogels were tested to determine the best conditions for the generation of myeloid and bone marrow specific lineages. Each matrix was comprised of 40% reduced growth factor Matrigel (Corning) and 60% either Collagen Type I or Collagen Type IV (Cell Systems), or a mixed Collagen I Collagen IV gel. All gels were prepared with Collagen at a concentration of 1 mg/mL. Gel preparation began on d4 when Matrigel aliquots were thawed overnight at 4° C. Gel mixtures were prepared on ice, with Collagen mixes neutralised with 1N NaOH prior to distribution in 12 well cell culture plates. Each hydrogel was allowed a minimum of 90 minutes to polymerise. An initial cell free layer was prepared before cells were collected by gravitation and resuspended in the remaining gel volume.
Once fully polymerised, 3D cultures were supplemented in Phase III sprouting medium which was comprised of APELII medium supplemented with 5% Foetal Bovine Serum (FBS), 5 U/mL Heparin Sulfate, 50 ng VEGFA, 10 ng each Interleukin 3 (IL3) and Interleukin 6 (IL6), as well as 25 ng each of SCF, Flt3, Thrombopoietin (TPO), Erythropoietin (EPO), Granulocyte Colony Stimulating Factor (G-CSF) (Stem Cell Technologies), FGF2, and BMP4. Cells were maintained in this media formulation until d12, with media changes every 72 hours. From d5 onwards, media changes are performed as 60:40 splits fresh: conditioned medium. For experiments developing sinusoid specific endothelial vasculature Vascular Endothelial Growth Factor C (VEGFC, Stem Cell Technologies) was supplemented into d3 media.
Cultures are allowed to sprout until an optimal size of between 800 um and 1.5 mm is observed, typically between d10-12. At this stage sprouted bone marrow organoids were extracted from hydrogels, resuspended in Phase IV medium, and cultured individually in 96-well ultra-low attachment dishes. Organoids were harvested first by scraping with a sterile cell scraper, before pipetting in an excess of media into a 15 mL Falcon (Corning). Samples were then spun down at 500 G for 5 minutes to separate organoids from both the media and collagen. The free organoids are then resuspended in the desired volume of media and collected for individual culture in 96-well ultra-low attachment plates. Phase IV medium was formulated similarly to Phase III medium, however with all cytokine concentrations reduced to 10 ng/mL.
Organoids were obtained at d18 for validation experiments. Validation was either performed by immufluorescence imaging of whole, ethyl cinnamate cleared organoids, or imaging of organoids embedded in optical cutting temperature (OCT) solution and frozen.
To model myelofibrosis (MF), we treated bone marrow organoids with Transforming Growth Factor β (TGFβ, Peprotech) at various concentrations for 72 hours. Treated organoids were maintained in this media before collection via pasteurette and fixation for immunofluorescence (whole organoids or embedded sections), or centrifugation for RNA extraction and subsequent quantitative Real Time Polymerase Chain Reaction (qRT PCR assessment of changes in gene expression). RNA extraction was performed using the Qiagen RNAEasy micro kit to the manufacturer's instructions.
Flow cytometry was performed using CyAn ADP High-Performance Flow Cytometer. Samples were dissociated using Collagenase Type B (Sigma) at 20 mg/mL in sterile HEPES. Samples were collected by gravitation in a 15 mL falcon tube before washing first in PBS, then in HEPES. Once washed, samples were incubated in the prepared Collagenase solution at 37° C. for 10 minutes before complete dissociation via tituration. Single cell suspensions were washed spun at 500 G and blocked in 0.5% BSA for 15 minutes before labelling with flow cytometry antibodies.
qRT-PCR was performed on an Applied Biosystems 7500 Fast Real Time PCR System. Primers were obtained from Integrated DNA Technologies (IDT) as pre-validated PrimeTime qPCR primers. Reactions were prepared from lug of isolated RNA converted to cDNA with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems) under manufacturer recommended thermal cycling conditions. Synthesised cDNA was diluted 5-fold and stored at −20° C. until required.
A master mix was prepared for each qPCR reaction which was made up of 10 μl TaqMan® Universal Primer MasterMix (thermos), 1 μl of forward and reverse primers, and 2 μl of cDNA at a final concentration of 5 ng/mL. Calculations for fold changes in expression were performed using the ΔΔCT method, with human GAPDH used as a housekeeping gene, and undifferentiated iPSC used as controls for expression levels.
Immunofluorescence was performed using a Zeiss LSM880 confocal microscope (25×0.8 LD LCI plan apo 0.8 dual immersion objective). Samples were prepared first by fixation in 10% formalin, before 3×5 min successive washes with PBS. Samples were then blocked overnight in a detergent blocking solution comprised of 2% goat serum (Thermo), 1% Bovine Serum Albumin (BSA) in 50 mL of PBS. 250 μl of Triton X100 and Tween-20 (Sigma) were added, as well as 500 μl sodium deoxycholate (Sigma) to permeabilise samples and encourage antibody preparation. Primary antibody incubations were performed overnight at 4° C., before 4×5-minute successive washes in PBS. Samples were then incubated overnight once more at 4° C. in secondary antibody mixes: DAPI for nuclear labelling, Alexa-488, Alxea-568, and Alexa-647 (Thermo) depending on the species mix of antibodies present.
Once labelled samples were embedded in a small volume of 0.5% agarose in Ibidi 8-well slides (Ibidi). Once cooled, this gel was subject to a progressive dehydration by a series of Ethanol washes adjusted to pH 9. Finally, the sample was dehydrated completely in absolute Ethanol before clearance with Ethyl Cinnamate (SLS). At this stage samples were ready for confocal imaging as described above.
Cells were isolated from cryopreserved peripheral blood or bone marrow mononuclear cells and stained with CellVue Claret Far Red Fluorescent Cell Linker Mini Kit for General Membrane Labeling (Sigma Aldrich, Cat #MINCLARET-1KT) following the instructions provided by the manufacturer. Organoids were seeded with 5000 cells per wells and cultured for 8-14 days in StemPro. On collection day, organoids were fixed for imaging or digested for assessment by flow cytometry or RNA extraction and qRT PCR.
mTeSR1 medium may comprise basal medium supplemented with recombinant human basic fibroblast growth factor (rh bFGF) and recombinant human transforming growth factor β (rh TGFβ). APEL2 medium is used for cellular differentiation protocols. APEL2 low insulin medium is used for long-term cell culture. StemPro-34 medium is used for patient cell culture and engraftment protocols. StemFlex medium (with and without Revitacell) is used for iPSC growth and/or expansion).
A Gibco Human Episomal iPSC (Thermo Fisher Scientific Cat #A 18945) line was maintained in StemFlex medium (Thermo Fisher Scientific Cat #A3349401) and on Geltrex (Thermo Fisher Scientific Cat #A1569601)-coated 6-well plates. The iPSC line was karyotyped prior to use and potency markers assessed upon expansion and freezing. Cells were passaged as clumps using EDTA at 0.02% in PBS (0.5 mM, Sigma Cat #E8008), and were freshly thawed or passaged for differentiation and maintained in StemFlex supplemented with RevitaCell (Thermo Fisher Scientific Cat #2644501). Cultures were maintained at 37° C. and 5% CO2. Differentiations were initiated with iPSCs between passages 5 and 30.
For differentiations, iPSC were dissociated using EDTA when colonies were approximately 100 μm in diameter. The resulting iPSC aggregates were incubated overnight in StemFlex supplemented with RevitaCell in 6-well Costar Ultra-Low Attachment plates (Corning Cat #3471) (day −1). After an overnight incubation, cells were collected by gravitation in a 15 mL Falcon tube (Fisher Scientific Cat #11507411) and resuspended in Phase I medium comprised of APEL2 (StemCell Technologies Cat #05275) supplemented with Bone Morphogenic Protein-4 (BMP4, Thermo Fisher Scientific Cat #PHC9531), Fibroblast Growth Factor-2 (FGF2, StemCell Technologies Cat #78134.1), Vascular Endothelial Growth Factor-A (VEGF-165, StemCell Technologies Cat #78159.1) at 50ng/mL, plated in a 6-well ULA plates and incubated at 5% O2 for 3 days (d0-3).
Cell aggregates were then collected by gravitation and re-suspended in Phase II medium for a further 48 hours (d3-5). Phase II medium (APEL2 supplemented with BMP-4, FGF2, and VEGFA at 50 ng/ml; human Stem Cell Factor (hSCF, StemCell Technologies Cat #78062) and Fms-like tyrosine kinase-3 Ligand (Flt3, StemCell Technologies Cat #78009) at 25 ng/mL.
On d5 cells were collected by gravitation for hydrogel embedding. Hydrogels were composed of 60% collagen (either type I, type IV, or an equal parts type I+IV mix) and 40% Matrigel. Hydrogels were prepared on ice as per manufacturer's instructions and were comprised of Reduced Growth Factor Matrigel (Corning, Cat #354230) supplemented with 1 mg/mL human collagen type I (Advanced Biomatrix, Cat #5007) and human collagen type IV (Advanced Biomatrix, Cat #5022) as per designated gel composition. Hydrogel mixes were neutralised with 1N NaOH. An 0.5 mL cell-free base layer was added and allowed to polymerise for 2 hours, before a further 0.5 mL layer of gel supplemented with gravitated cell aggregates was added and also left to polymerise for 2 hours at 37° C. and 5% CO2. Fully polymerised gels with cell aggregates were then supplemented with Phase III media comprised of VEGFA at either 50 ng or 25 ng/ml, VEGFC (where relevant) at 50 or 25 ng/mL, FGF2, BMP4, hSCF, Flt3, Erythropoeitin (EPO, StemCell Technologies, Cat #78007), Thrombopoeitin (TPO, StemCell Technologies, Cat #78210), Granuolocytic Colony-Stimulating Factor (G-CSF, StemCell Technologies, Cat #78012), at 25 ng/ml, and Interleukin-3 (IL3, StemCell Technologies, Cat #78194) and Interleukin-6 (IL6, StemCell Technologies, Cat #78050) at 10 ng/mL. Media was replenished every 72 hours.
Sections were blocked using 2% Goat Serum (Thermo Fisher Scientific, Cat #31872) 1% Bovine Serum Albumin (BSA) (Sigma, Cat #A9418) prior to primary antibody labelling with antibody diluted in 1% BSA, sequential PBS washes, and finally secondary labelling with AlexaFluor conjugates. Whole organoid blocking solution was further supplemented with Triton X100, Tween, and Sodium deoxycholate.
Sprouting organoids were imaged within hydrogels in 8-well microslides (Ibidi, Cat #80806), whole organoids were labelled in 15 mL Falcons before embedding in 0.5% Agarose within 8-well microslides. Whole organoids were subject to serial dehydration (50%, 70%, 90%, 100%) within microslides before clearance with Ethyl Cinnamate and subsequent imaging. Sections were prepared by embedding fixed organoids in Optimal Cutting Temperature compound (OCT, VWR Cat #361603E) before sectioning onto Poly-L-Lysine covered slides. Slides were washed in Acetone before immunofluorescence labelling.
Confocal microscopy was performed using a Zeiss LSM880 confocal AiryScan microscope with either a 25×LD LCI plan apo 0.8 NA dual immersion (420852-9871-000) or 40×C-APO NA 1.2 water immersion objective (421767-9971-711) as described previously. Confocal images were acquired as representative Z-stacks (with Z-resolution set to Nyquist requirements), and presented as maximum intensity projections (Fiji) where stated. Histological preparations (reticulin and H&E, details provided in supplementary materials and methods) were imaged using a Zeiss AxioScan.Z1 slide scanner. Image analysis was performed in Fiji. For measurements of sprout radii, brightfield images acquired on an Evos (Thermo Fisher Scientific) desktop microscope. Sprout radii were measured manually by drawing and measuring a line from the centre to the tip of the sprout across 3 independent biological replicates, with between 30-50 sprouts measured per replicate. To measure the proximity of megakaryocytes to organoid blood vessels, 250 μm×50 μm volumes of individual organoids were acquired using cleared whole mount organoids imaged by confocal microscopy, as previously described. CD41 labelled megakaryocytes within 5 μm of UEAl labelled vessels were counted as ‘vessel-associated MKs’ within a maximum intensity projection of each imaged volume
Cryopreserved cells pooled from 15 organoids from 3 differentiations from both VEGFA and VEGFA+C protocols were thawed, stained with DAPI to exclude non-viable cells, and DAPI-live cells sorted on a Becton Dickinson Aria Fusion with 100 nm nozzle as per recommendations in the 10× Genomics Single Cell Protocols—Cell Preparation Guide. 10,000 live cells per sample were sorted into 2 μL PBS/0.05% BSA (non-acetylated) and the cell number/volume adjusted to the target for loading onto the 10× Chromium Controller. Samples were processed according to the 10× protocol using the Chromium Single Cell 3′ library and Gel Bead Kits v3.1 (10× Genomics). Cells and reagents were prepared and loaded onto the chip and into the Chromium Controller for droplet generation. Reverse transcription was conducted in the droplets and cDNA recovered through demulsification and bead purification. Pre-amplified cDNA was used for library preparation, multiplexed and sequenced on a Novaseq 6000. Details on data processing ana
Demultiplexed FASTQ files were aligned to the human reference genome (GRCh38/hg38) using standard CellRanger (version 6.0.1) ‘cellranger count’ pipeline (10× Genomics). SingCellaR (https://supatt-lab.github.io/SingCellaR.Doc/) was used for the downstream analysis. Data was first subject to quality control with the maximum percentage of mitochondrial genes, maximum detected genes and max number of UMIs set to 12%, 6,000, and 50,000, respectively. Minimum detected genes and UMIs were set to 300 and 500, respectively and genes with minimum expressing cells was set as 10. Raw expression matrix was then normalised and scaled and number of UMIs and percentage of mitochondrial reads were regressed out before a general linear model (GLM) was used to identify highly variable genes were then subject to downstream analyses including principal component analysis (PCA), UMAP analysis (top 40 PCs were used, and n.neighbour=120), and clustering using the Louvain method. Differentially expressed genes were calculated using ‘identifyDifferentialGenes’ function (min.log2FC=0.3 and min.expFraction=0.25). To compare cells from the two experimental conditions (VEGFA only and VEGFA+C), cells were down-sampled so that each cell group had the same number of cells. Wilcoxon test of normalized UMIs was used to compare the gene expressions and Fisher's exact test was used to compare the cell frequency. The resulting P values from both tests were combined using Fisher's method and subsequently adjusted by Benjamini-Hochberg correction. ‘runFA2 ForceDirectedGraph’ function was used to identify the trajectories. CellPhoneDB v 2.1.1 (https://github.com/Teichlab/cellphonedb) was performed for ligand-receptor interactions using normalized expression matrix of VEGFA+C as detailed by Garcia-Alonso et al. Cell-cell interaction network between the different cell clusters from VEGFAC and Sankey plot demonstrating the interaction between TGFβ1, CXCL12, and CD44 ligands with their responding receptors from VEFGA and VEFGAC were plotted using a modified version of the CrossTalkeR R package (version 1.2.1).
We applied Symphony to map cells from VEGFA+C organoids to published scRNAseq datasets from human bone marrow and fetal liver and bone marrow cells respectively. For the human bone marrow dataset, we first built the reference data using the normalized expression matrix using ‘symphony::buildReference’. For the fetal liver dataset we used the pre-built reference provided by the Symphony developer. The ‘mapQuery’ and ‘knnPredict’ function were used to map the VEGFA+C cells onto the three reference datasets.
Organoids were fixed in neutral buffered formalin (Sigma-Aldrich, Cat #HT501128-4L) in a 15 mL Falcon tube, washed twice with PBS, and then subject to serial dehydration (30%, 50%, 70%, 100%) in ethanol before immersion in Histoclear (Geneflow, Cat #A2-0101). Samples were then embedded in paraffin and sent as blocks to C&C laboratories for staining and mounting.
Primary cells were labelled with CellTrace Far Red as indicated by the manufacturer. Briefly, cells were washed 1× with PBS and resuspended at 1×106 cells/mL in staining solution (CellTrace Far Red 2 μM in PBS). Cells were incubated in staining solution for 30 min at 37° C. After incubation CellTrace was quenched with 5 volumes of PBS with FBS (10%), spun down and resuspended in the appropriate media.
Whole organoids were processed using either the Micro RNEasy Kit (Qiagen, Cat #74004) or Qiagen Mini RNA isolation kit (Qiagen, Cat #74104) according to the manufacturer's instructions. Isolated RNA was quantified on the NanoDrop ND-100 (Thermo Scientific) and cDNA was prepared using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat #4368814) or EvoScript Universal cDNA Master (Roche, Cat #07912374001) according to the manufacturer's instructions using standard cycling conditions. cDNA was diluted to 5 ng before being combined with PowerUp SYBR Green Master Mix reagent (Applied Biosystems, Cat #A25742) and the relevant PrimeTime qRT-PCR primers (IDT), or performed using TaqMan™ Universal PCR Master Mix (Applied Biosystems) on StepOne plus machine (Applied Biosystem) (see Suppl. Table 6 for a full list of primers). The absolute expression of the respective genes was calculated using the ΔCt method using GAPDH as an internal housekeeping control.
To assess the production of growth factors, organoids were washed and cultured in StemPro-34 (L-Glutamine only) without any added supplements or growth factors for 12 days. 50:50 media changes were performed at 72 hour intervals, and media was collected for Luminex assays at day 12. Supernatant from 12 organoids was collected and pooled for each repeat.
Luminex kits (LXSHAM-03, LXSAHM-28) were used for multiplexed proteomic assays as per the manufacturer instructions. No detectable signal was observed in cell-free medium.
Primary cells were labelled with CellTrace Far Red as per kit instructions. Briefly, cells were washed and resuspended at 1×106 cells/mL in staining solution for 30 min at 37° C. After incubation, CellTrace was quenched with 5 volumes of PBS with 10% FBS.
Prior to seeding, donor cells were labelled with CellVue Claret Far Red Fluorescent Cell Linker Mini Kit for General Membrane Labelling (Sigma Aldrich, Cat #MINCLARET-1KT) following manufacturer instructions. For seeding of organoids, each well of a 96-well plate containing 1-2 individual organoids or media alone were seeded with 5000 cells per well and cultured for up to 14 days in StemPro (Thermo Fisher Scientific, Cat #10639011) supplemented with Phase IV cytokines. Wells seeded with iALL cells were further supplemented with IL7, with 50% media changes every 2-3 days.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2202025.9 | Feb 2022 | GB | national |
| 2216647.4 | Nov 2022 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/GB2023/050348 | 2/15/2023 | WO |
