WO2025170529A1

WO2025170529A1 - A method for deriving a multipotent neuromesodermal progenitor cell and/or daughter lineages, and related compositions

Info

Publication number: WO2025170529A1
Application number: PCT/SE2025/050099
Authority: WO
Inventors: Eva HEDLUND; Nigel KEE
Original assignee: Individual
Current assignee: Individual
Priority date: 2024-02-09
Filing date: 2025-02-10
Publication date: 2025-08-14
Anticipated expiration: 2026-08-09

Abstract

The disclosure relates to a cell culture method for deriving a multipotent neuromesodermal progenitor (NMP) cell and/or lineages of specific anterior-posterior positions along the human body axis and/or cell types of matched anterior-posterior position, from a human pluripotent stem cell (PSC), comprising culturing said stem cell under conditions and with reagents modulating the following signalling pathways: (i) activation of FGF signalling for a duration of about 12-120 hours; (ii) activation of WNT signalling for a duration of about 48-120 hours; (iii) inhibition of SMAD signalling, including TGF-beta/activin-Nodal signalling and BMP signalling, for a duration of about 48-120 hours, wherein said progenitor co-expresses SOX2, TBXT, CDX2 and CDX1 at RNA and/or protein level(s).The disclosure further relates to a composition for use in deriving multipotent NMPs and/or daughter lineages of a specific anterior-posterior position in the human body and/or cell types of matched anterior-posterior position from human pluripotent stem cells (PSC), a multipotent NMP, a cervical and a thoracic NMP obtained by culturing human PSCs, a composition for use in generating lumbar level NMPs from the multipotent NMPs, a lumbar level NMP obtained by culturing the multipotent NMP, a composition for use in generating tbx6 expressing presomitic mesoderm cells from the multipotent NMPs, a tbx6 expressing presomitic mesoderm cell obtained by culturing the multipotent NMP, a human cell derived from the multipotent NMP, a cell population, a kit and a cellular composition.

Description

1

A METHOD FOR DERIVING A MULTIPOTENT NEUROMESODERMAL PROGENITOR CELL AND/OR DAUGHTER LINEAGES, AND RELATED COMPOSITIONS

5 Technical field

The present disclosure is in the field of stem cell biology. More specifically, the present disclosure relates to a method for deriving a multipotent neuromesodermal progenitor cell and/or daughter lineages thereof with specific anterior-posterior positions along the human body axis and/or cell types of matched anterior-posterior position, a composition for use in

10 deriving multipotent neuromesodermal progenitor cells and/or daughter lineages thereof with specific anterior-posterior positions in the human body and/or cell types of matched anterior-posterior position from human pluripotent stem cells.

Specifically, the method generates a multipotent neuromesodermal progenitor cell of either cervical, or thoracic or lumbar level from human stem cells, and subsequent derivation of

15 cervical, or thoracic or lumbar preneural tube progenitors or cervical, or thoracic or lumbar presomitic mesoderm progenitors, or cervical, or thoracic or lumbar preneural crest progenitors derived from the multipotent neuromesodermal progenitor cell, and a kit, as defined in the claims.

Background art

20 The embryonic vertebrate body axis is built in several stages. The anterior body axis is constructed first, where pluripotent epiblast cells of the early embryo give rise to three germ layers through a process termed gastrulation, forming the ectoderm (neural and neural crest), definitive endoderm, and mesoderm. Following this, at late primitive streak stages, the posterior epiblast continues to experience WNT and FGF driven patterning and upregulates

25 the chromatin remodelers CDX1 and CDX2 (and later in time CDX4), to form the Caudal Growth Zone. The Caudal Growth Zone contains a mix of progenitor populations collectively termed Axial Progenitors, which include lateral plate mesoderm progenitors (LPMPs), notochord progenitors (NotoPs), and neuromesodermal progenitors (NMPs) (Wymeersch, 2021).

30 NMPs, which co-express SOX2, TEXT (BRACHYURY) and CDX2, self-renew and simultaneously generate posterior neural tube, neural crest and somitic mesoderm progenitor cells, which have been demonstrated either in vitro (for neural crest) or in vitro and in vivo (for mesoderm and neural tube) (Faustino, 2020; Frith, 2018; Guibentif, 2021; Guillot, 2021; Henrique, 2015; Selleck and Stern, 1991; Tzouanacou, 2009; Wymeersch, 2016, 2019). Over time, this self¬

35 renewal and differentiation leads to elongation of the post-cranial body axis. US2012/0244768, describing cell compositions and uses thereof, W02022/081501, disclosing compositions and methods for making and using differentiated neural cells, WO2022/051847, disclosing compositions and methods for generating neural progenitor cells with a spinal cord identity, WO2022/266527, disclosing method of generating sacral neural crest lineages and uses thereof, as well as US2013/0183674, disclosing a method of nociceptor differentiation of human embryonic stem cells and uses thereof, are examples of various methods in the field, wherein stem and/or progenitor cells are differentiated into various neural cells. Compositions including various factors (compounds) that have the capacity to affect certain signaling pathways by activation or inactivation are disclosed in these references. Further, W02018/090002 relates to the generation of Schwann cells via a Schwann cell precursor intermediate using activation of FGF and WNT signaling and NRG1. This protocol does not go through an NMP state. CN-A-109868258 relates to in vivo reprogramming of astrocytes to neurons. US-B2-9453198 discloses the derivation of neuronal progenitors and subsequently neural crest-derived peripheral nervous system nociceptor neurons from stem cells by first inhibiting SMAD signaling and subsequently by inhibiting FGF and NOTCH signaling and activating WNT signaling. This protocol does not go through an NMP state. AU-B2- 2018200059B relates to generation of neural crest-derived nociceptor neurons initiated by dual SMAD inhibition. This protocol does not go through an NMP state. CN-A-114149961 relates to specification of liver organoids from human iPSCs by generating definitive endoderm.

The knowledge within this field is emerging, however there are still major challenges and limitations in the extent to which development of progenitor cells to certain cell types can be controlled and predetermined.

There is thus a need for improved protocols for deriving multipotent NMPs having broader applicability and use than current ones.

Summary of the invention

It is an object of the present disclosure to mitigate, alleviate or eliminate one or more of the above-identified deficiencies and disadvantages in the prior art and solve at least one of the above-mentioned problems.

According to a first aspect there is provided a cell culture method for deriving a multipotent neuromesodermal progenitor cell and/or daughter lineage(s) thereof with specific anterior- posterior positions along the human body axis and/or cell types of matched anterior-posterior position, from a human pluripotent stem cell, comprising culturing said stem cell under conditions and with reagents modulating the following signaling pathways: activation of FGF signalling during the time period of about 12 to about 120 hours after exit from pluripotency; activation of WNT signalling during the time period of about 48 to about 120 hours after exit from pluripotency; and inhibition of SMAD signalling, including TGF-beta/activin-Nodal signalling and BMP signalling, during the time period of about 48 to about 120 hours after exit from pluripotency, wherein said progenitor co-expresses SOX2, TBXT, CDX2 and CDX1 at RNA and/or protein level(s).

Hereby, by having gained knowledge as to how the signalling pathways can be modulated, i.e., activated and/or inhibited, an in vitro method for deriving multipotent neuromesodermal progenitors, from which all three lineages, mesoderm, neural tube and neural crest can be derived, have been provided by the present inventors. This way, one multipotent NMP can be used for a range of applications, facilitating and simplifying the further development within the field.

According to some embodiments, the method further comprises modulating the following signalling pathway: inhibition of Notch signalling during the time period of about 48 to about 120 hours after exit from pluripotency.

By inhibiting Notch signalling, purity of NMPs will increase, by not allowing TBX6 positive mesoderm cells to emerge over time, which is important when deriving downstream neural cells, but less important when deriving mesoderm cells. According to some embodiments:

- activation of FGF signalling is performed using either FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof, and/or by blocking Dusp6 and/or by activating ERK1/2 signalling using small molecule inhibitors of DUSP6 chosen from £-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-lH-inden-l-one (BCI) and paralogues, homologues, derivatives and/or combinations thereof;

- activation of WNT signalling is performed using either CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, BIO, SB-216763, Wnt5A, Wnt5B, Wntl, Wnt3A and paralogues, homologues, derivatives and/or combinations thereof;

- inhibition of SMAD signalling is performed using either Hesperetin, SB431542, SB525334, Galunisertib, GW788388, LY2109761, SB505124, LDN-193189, LDN-193189 HCI, RepSox, A 83-01, DMH1, LDN-212854, ITD 1, LY364947, SD-208, EW-7197, ML347, K02288, A 77-01, SIS3, LDN-214117, R-268712, Pirfenidone, Noggin, Chordin, Gremlin, DAN proteins, GDF3, paralogues, homologues, derivatives and/or combinations thereof;

- inhibition of Notch signalling is performed using either DLK1, gamma-secretase inhibitors, including e.g., small molecules DAPT, RO4929097, PF-03084014, derivatives thereof, antibodies blocking Notch receptors and anti- JAG ligands, anti-Delta-like ligands and decoys against Notch receptors, JAG ligands, and Delta-like ligands, inhibitors of MAML1, and paralogues, homologues, derivatives and/or combinations thereof. Hereby, suitable molecules are provided that bring about the activation and/or inhibition of signalling pathways that are necessary for the generation of a multipotent NMP according to the present disclosure.

According to some embodiments, said progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1 and HOX at RNA and/or protein level(s), and/or wherein said cell does not express the mesodermal markers TBX6, or MSGN1 at RNA and/or protein level(s) and/or wherein said cell goes through colinear HOX gene activation.

According to some embodiments, culturing is performed in a medium, which lacks serum, supports stem cell growth and differentiation, and lacks TGFbeta and bFGF, such as E6 medium, which has proven to be a suitable and preferred medium in this context.

According to some embodiments, said stem cell is cultured with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 96 hours.

Hereby, a combination of preferred reagents/factors are provided for use in the method.

According to some embodiments, the factors are added at the following concentrations and durations after exit from pluripotency:

Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml from about 24 hours;

CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm, from about 48 hours;

A 83-01 at 5-20 pM, preferably about 10 pM, from about 48 hours;

SIS3 at 0,25-1 pM, preferably about 0.5 pM, from about 48 hours;

K02288 at 0,5-10 pM, preferably about 2 pM, from about 48 hours;

DAPT at 5-20 pM, preferably about 10 pM, from about 48 hours; and Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml, from about 48 hours.

Hereby, an efficient protocol is provided, wherein specific compounds (factors) and concentrations thereof have been fine-tuned, so that that the desired events are provided in vitro.

According to some embodiments, the multipotent neuromesodermal progenitor cell is developed into mesoderm, and/or neuroectoderm and/or neural crest lineages that bear a cervical, or thoracic, or lumbar axial identity and/or daughter cell types arising from these afore-mentioned mesoderm, and/or neuroectoderm and/or neural crest lineages. E.g. post- cranial neurons, oligodendrocytes, astrocytes, floor-plate, roof-plate, bone, cartilage, peripheral neurons including sympathetic neurons, parasympathetic neurons, sensory neurons (dorsal root ganglia) and enteric neurons, as well as melanocytes and Schwann cells.

Hereby, suitable tissues and cell types can be developed from the protocol of the present disclosure. According to some embodiments, a cervical neuromesodermal progenitor cell is generated by culturing with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 72-96 hours.

According to some embodiments, said cervical neuromesodermal progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB4, and not HOXB9 or HOXBIO, at the RNA level, and/or if said cervical NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2, HOXB4 and not HOXB9 and not HOXCIO, at the protein level.

According to some embodiments, a thoracic neuromesodermal progenitor cell is generated by culturing with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 96-120 hours.

According to some embodiments, said thoracic neuromesodermal progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB9, and not HOXBIO, at the RNA level, and/or if said thoracic NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2 and HOXB9, and not HOXB4 and not HOXCIO, at the protein level.

According to some embodiments, a lumbar level neuromesodermal progenitor cell is generated by, in a first step for a duration of about 120 hours, modulating the signalling pathways as listed above, and in a subsequent second step, for a duration of about 24-48 hours, continue modulating the signalling pathways of the first step with the exception of substituting inhibiting the TGF-beta/activin-Nodal signalling pathway for activating the same.

According to some embodiments, a lumbar level neuromesodermal progenitor cell is generated by culturing in a first step with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 120 hours, and in a second step with the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin, and GDF11 for a duration of about 24-48 hours.

According to some embodiments related to generation of lumbar level neuromesodermal progenitors, in the first step, the factors are added as disclosed above for the multipotent NMP, and for the second step, the factors are added at the following concentrations:

Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

K02288 at 0,5-10 pM, preferably about 2 pM;

DAPT at 5-20 pM, preferably about 10 pM;

Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml; and GDF11 at 50-500 pg/ml, preferably about 100 pg/ml.

According to some embodiments, said lumbar level neuromesodermal progenitor cell coexpresses SOX2, TBXT, CDX2, CDX1, and HOXBIO at the RNA level, and/or if said lumbar NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2, HOXCIO and/or HOXB9, but not HOXB4, at the protein level. According to some embodiments, a TBX6 expressing presomitic mesoderm cell is generated by, in a first step for a duration of about 120 hours, modulating the signalling pathways as listed in claim 1 and/or 2, and in a subsequent second step, for a duration of about 48-72 hours, activating the WNT and FGF pathways, and promoting EMT (Epithelial Mesenchymal Transition).

According to some embodiments, the TBX6 expressing presomitic mesoderm cell is generated by culturing in a first step with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 120 hours, and in a second step with the factors Fgf2, CHIR99021 and BME (Basal Matrix Extract), Matrigel or other cell-extracted extracellular matrix products for a duration of about 48-72 hours.

According to some embodiments, related to generation of TBX6 expressing presomitic mesoderm cells, in the first step, the factors are added as disclosed above for the multipotent NMP, and in the second step, the factors are added at the following concentrations:

Fgf2 at 10-100 ng/ml, preferably about 50 ng/ml;

CHIR99021 at 5 to 20 pm, preferably about 10 pm; and

BME, Matrigel, or other cell-extracted extracellular matrix products, preferably at about 8-12 mg/mL.

According to some embodiments, said TBX6 expressing presomitic mesoderm cell coexpresses TBX6, MSGN1, TBXT, RBP1 and GAD1, at protein and/or RNA level.

According to some embodiments, the human pluripotent stem cell (hPSC) is chosen from a human embryonic stem cell (hESC), or a human induced pluripotent stem cell (hiPSC), reprogrammed to pluripotency from a somatic cell, such as an adult somatic cell.

According to a second aspect there is provided a composition for use in deriving multipotent neuromesodermal progenitor cells (NMPs) and/or daughter lineages thereof with a specific anterior-posterior position in the human body and/or cell types of matched anterior-posterior position from human induced pluripotent stem cells, comprising reagents modulating the following signalling pathways: activation of FGF signalling during the time period of about 12 to about 120 hours after exit from pluripotency, using either FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof, and/or by blocking Dusp6 or by activating ERK1/2 signalling using small molecule inhibitors of DUSP6 chosen from (E)-2-benzylidene-3- (cyclohexylamino)-2,3-dihydro-lH-inden-l-one (BCI) and paralogues, homologues, derivatives and/or combinations thereof; activation of WNT signalling during the time period of about 48 to about 120 hours after exit from pluripotency using either CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, BIO, SB-216763, Wnt5A, Wnt5B, Wntl, Wnt3A, paralogues, homologues, derivatives and/or combinations thereof; and inhibition of SMAD signalling, including TGFbeta/activin-Nodal signalling and BMP signalling, during the time period of about 48 to about 120 hours after exit from pluripotency using either Hesperetin, SB431542, SB525334, Galunisertib, GW788388, LY2109761, SB505124, LDN-193189, LDN-193189 HCI, RepSox, A 83-01, DMH1, LDN- 212854, ITD 1, LY364947, SD-208, EW-7197, ML347, K02288, A 77-01, SIS3, LDN- 214117, R-268712, Pirfenidone, Noggin, Chordin, Gremlin, DAN proteins, GDF3, paralogues, homologues, derivatives and/or combinations thereof.

In some embodiments, the composition further comprises reagents modulation the following signalling pathway: inhibition of Notch signalling during the time period of about 48 to about 120 hours after exit from pluripotency, using either DLK1, gamma-secretase inhibitors (GSIs), including e.g., small molecules DAPT, RO4929097, PF-03084014, derivatives thereof, antibodies blocking Notch receptor and anti- JAG ligands and decoys against Notch receptor and JAG ligand, inhibitors of MAML1, and paralogues, homologues, derivatives and/or combinations thereof.

Hereby, a composition that can be used for deriving multipotent neuromesodermal progenitors, from which all three lineages, mesoderm, neural tube and neural crest can be derived, is provided.

According to some embodiments, the composition comprises the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin.

According to some embodiments, the factors are present at the following concentrations:

Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

A 83-01 at 5-20 pM, preferably about 10 pM;

SIS3 at 0,25-1 pM, preferably about 0.5 pM;

K02288 at 0,5-10 pM, preferably about 2 pM;

DAPT at 5-20 pM, preferably about 10 pM; and

Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml.

According to a third aspect there is provided a multipotent neuromesodermal progenitor cell, being derived from a human induced pluripotent stem cell, wherein the progenitor coexpresses SOX2, TBXT, CDX1 and CDX2 at RNA and/or protein level.

According to some embodiments, said progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1 and HOX at RNA and/or protein level, and/or wherein said cell does not express the mesodermal markers TBX6, or MSGN1 at RNA and/or protein level and/or wherein said cell goes through colinear HOX gene activation. Hereby, it is specified that the human NMPs have a specific marker profile and that the NMP goes through colinear HOX activation, which is a prerequisite for the definition of a true NMP.

According to a fourth aspect there is provided a cervical neuromesodermal progenitor cell obtained by culturing human induced pluripotent stem cells with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 96 hours, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB4, and not HOXB9 or HOXBIO, at the RNA level. If this cervical NMP were to be further directed to differentiate into ventral neural progenitors (e.g. through activation of the Sonic Hedgehog pathway, Li 2005) they would then co-express SOX1, SOX2 and HOXB4, and not HOXB9, and not HOXCIO, at the protein level.

Hereby, an NMP of cervical identity in terms of HOX gene expression is defined. Also, the anterior-posterior identity of NMPs is molecularly specified (which is also valid for other aspects, such as the fifth and sixth aspects).

According to a fifth aspect there is provided a thoracic neuromesodermal progenitor cell obtained by culturing human induced pluripotent stem cells with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 120 hours, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB9, and not HOXBIO, at the RNA level. If this thoracic NMP were to be further directed to differentiate into ventral neural progenitors (e.g. through activation of the Sonic Hedgehog pathway, Li 2005) they would then co-express SOX1, SOX2 and HOXB9, and not HOXB4 and not HOXCIO, at the protein level.

Hereby, a thoracic NMP molecularly and the HOX code thereof is specified and defined.

According to a sixth aspect there is provided a composition for use in generating lumbar level neuromesodermal progenitor cells from the multipotent neuromesodermal progenitor cells according to the third aspect, comprising the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin and GDF11.

Hereby, a lumbar NMP in terms of HOX code is defined.

Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

K02288 at 0,5-10 pM, preferably about 2 pM;

DAPT at 5-20 pM, preferably about 10 pM;

Hereby, lumbar level NMPs are generated with a slight modulation of the factors used in other aspects of the invention. Within this step, the timing of FGF, WNT and SMAD signalling (as well as the concentration of CHIR) is key. According to a seventh aspect there is provided a lumbar level neuromesodermal progenitor cell obtained by culturing the multipotent neuromesodermal progenitor cell according to the third aspect with the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin and GDF11 for a duration of about 24-48 hours, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1, and HOXBIO at the RNA level. If this lumbar NMP were to be further directed to differentiate into ventral neural progenitors (e.g. through activation of the Sonic Hedgehog pathway, Li 2005) they would then co-express SOX1, SOX2, HOXCIO and/or HOXB9, but not HOXB4, at the protein level.

According to an eighth aspect there is provided a composition for use in generating TBX6 expressing presomitic mesoderm cells from the multipotent neuromesodermal progenitor cells according to the third aspect, comprising the factors Fgf2, CHIR99021 and BME (Basal Matrix Extract).

Fgf2 at 10-100 ng/ml, preferably about 50 ng/ml;

CHIR99021 at 5 to 20 pm, preferably about 10 pm; and BME, Matrigel, or other cell-extracted extracellular matrix products, preferably at about 8-12 mg/mL.

According to a ninth aspect there is provided a TBX6 expressing presomitic mesoderm cell obtained by culturing the multipotent neuromesodermal progenitor cell according to the third aspect with the factors Fgf2, CHIR99021 and BME for a duration of about 48-72 hours, wherein said cell co-expresses TBX6, MSGN1, TBXT, RBP1 and GAD1, at protein and/or RNA level.

According to a tenth aspect there is provided a human cell derived from the multipotent neuromesodermal progenitor cell of the third aspect, the cell being of a tissue type chosen from the group comprising post-cranial nervous system tissue, neural crest derivative, skeletal muscle, bone and cartilage and/or of a lineage chosen from the group comprising ectoderm, neural crest and mesoderm.

According to an eleventh aspect there is provided a cell population comprising one or more of the progenitor or mesoderm cells according to the third, fourth, fifth, seventh or ninth aspect, derived by the method of the first aspect.

According to a twelfth aspect there is provided a kit comprising one or more of the compositions according to the second, sixth or eight aspect, and a suitable medium, which lacks serum, supports stem cell growth and differentiation, and lacks TGFbeta and bFGF, such as E6 medium or similar, optionally including suitable factors, such as the compositions comprising necessary factors as defined in other aspects of this disclosure, laminin coating of a plastic dish, and optionally other necessary reagents and tools, and instructions for use. According to a thirteenth aspect there is provided a cellular composition, comprising one or more cervical, thoracic or lumbar level NMP cell(s), wherein the cell(s) is/are present at a purity level of at least about 80%, 85%, 90% or at least 95%.

Thus, the present disclosure provides several aspects. Specifically, the present disclosure relates to a method detailing how to generate a molecularly defined NMP, that can build up the posterior body axis, from human pluripotent stem cells. Thus, the method generates a product, the NMP, that is multipotent and from which a large number of cell types in the post- cranial human body can be generated, including e.g. nervous system tissue, neural crest derivatives, skeletal muscle, bone and cartilage. Additionally, the NMP can also generate cells at distinct axial positions, i.e., cervical, thoracic, or lumbar axial levels.

One primary advantage of the technology disclosed in the present disclosure is that one protocol can be used derive a progenitor that in turn can be used to generate neural, neural crest, and somitic mesodermal lineages of a specific anterior-posterior position in the body and that cell types of matched anterior-posterior position can be made.

Further, the present disclosure may be used to develop cell culture media aimed towards generating e.g. the thoracic (7F) and lumbar (6F) NMP. Such cell culture media would typically comprise the components found in E6 media, and additionally include the cocktail of the appropriate stages, as specified in this disclosure.

The technology of the present disclosure may also be used to develop multiple clinically relevant cell types downstream of NMPs, including skeletal muscle, muscle satellite cells, spinal cord neurons including motor neurons and interneurons, sensory neurons, astrocytes, Schwann cells, oligodendrocytes, bone, and cartilage, which all have great potential in e.g., drug screening, transplantation, disease modelling.

To summarise, during development of the embryo a few signaling pathways, WNTs, FGFs, BMPs, SHH (sonic hedgehog) and retinoic acid (RA), are used over and over to give rise to different tissues and cell types. This is possible because these proteins give different effects on cells depending on the concentration the cell is exposed to, the length of time the signal is received by the cell and the timing of the exposure, that is when the cell is exposed to it.

The concentration dependence is because these proteins are morphogens, which means they give a different effect depending on the dose/concentration the cell sees.

The importance of timing; what a cell has seen prior to being exposed to any particular signal (morphogen), determines what the cell can become and how the morphogen affects them. This is due to the presence of different receptors as well as intrinsic signalling mechanisms that are modulated over time in development because of what the cell has encountered before. Thus, at one point in time a morphogen might give one response while it later on gives another or no response at all. Thus, timing and concentration is everything in development and subsequently also when developing protocols to copy such processes in a laboratory.

Thus, while it may seem that a protocol to block one signalling pathway to get a specific cell type is known, blocking a signalling pathway at one point can give rise to neurons of the central nervous system, while at another time point and/or at another concentration it will give rise to neural crest and neurons of the peripheral nervous system, or yet another time point and concentration, an NMP.

Thus, it is the timing, the concentration and also the length of time that a cell sees a particular signal, in combination with specific timing of other signals, that brings uniqueness to the protocol of the present disclosure.

The basis for the present invention, and all the various aspects included in the invention, is the cocktail of factors, and the timing and concentration thereof, that allow for the generation of NMPs as well as maintenance of NMPs in a multipotent stage as these go through colinear HOX activation. Thus, a prerequisite for the invention is that the cocktail allows NMPs to change their anterior-posterior identity over time (cervical to thoracic to lumbar), while maintaining the marker gene expression of a NMP and the potential to give rise to daughter lineages of distinct axial identities. In this context, to be classified as an NMP is related to the potential to give rise to a wide range of cell types of mesodermal and ectodermal lineages along different positions of the body axis.

Effects and features of the second through thirteenth aspects are to a large extent analogous to those described above in connection with the first aspect. Embodiments mentioned in relation to the first aspect are largely compatible with the second through twelfth aspects.

The present disclosure will become apparent from the detailed description given below. The detailed description and specific examples disclose preferred embodiments of the disclosure by way of illustration only. Those skilled in the art understand from guidance in the detailed description that changes and modifications may be made within the scope of the disclosure.

Hence, it is to be understood that the herein disclosed disclosure is not limited to the particular parts of component of the device described or steps of the methods described since such device and method may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It should be noted that, as used in the specification and the appended claim, the articles "a", "an", "the", and "said" are intended to mean that there are one or more of the elements unless the context explicitly dictates otherwise. Thus, for example, reference to "a unit" or "the unit" may include several devices, and the like. Furthermore, the words "comprising", "including", "containing" and similar wordings do not exclude other elements or steps. Definitions

The term "in vitro method" refers, in this context, to a method performed on or within cells outside the human or animal body. An alternative term would be "cell culture method".

The term "in vivo method" refers, in this context, to a method performed inside an organism.

The term "multipotent" refers to a cell having the potential to become any of several mature cell types.

"NMP" refers to a neuro-mesodermal progenitor, whereas a "progenitor cell" refers to a biological cell that can differentiate into a specific cell type.

The term "lineage" refers to the pattern of cell divisions in the development of a tissue or organ.

The term "anterior" refers to the front of the body, whereas the term "posterior" refers to the back of the body

A "human iPSC" refers to a human induced pluripotent stem cell, which is a type of pluripotent stem cell derived from somatic cells that have been reprogrammed to an embryonic stem (ES)- cell like state.

The term "factors" in the context of this disclosure relates to chemical compounds and/or reagents and/or proteins and/or polysaccharides having the ability to affect development and/or growth of a cell.

The term "duration" refers to the time of culturing and/or incubation and/or exposing of a cell for a certain factor or chemical compound, typically measured in hours.

The term "concentration" for a factor and/or chemical compound is typically measured in the unit M (molar), i.e., mol/liter, such as pM. For some concentrations, the unit pg/ml is used (such as for Fgf8 and heparin)

The term "mesoderm" refers to the middle layer of cells or tissues of an embryo, or the parts derived from this.

The term "ectoderm" refers to the outermost layer of cells or tissue of an embryo in early development, or the parts derived from this.

The term "neuroectoderm" (or neural ectoderm or neural tube epithelium) consists of cells derived from the ectoderm.

The "neural crest" cells are a temporary group of cells that arise from embryonic ectoderm germ layer. The terms "cervical", "thoracic", "lumbar" axial identities refer to different parts of the spine: neck (cervical), upper and middle back (thoracic) and lower back (lumbar).

The term "axial progenitors" is a collective term to describe the progenitor cells located in the caudal growth zone, which together drive axial elongation.

The term "lineage" refers to lineal descent from an ancestor, and the term "cell lineage" refers to the developmental history of a tissue or organ from a fertilized embryo. In this context, "daughter lineage" would be referred to as cells and/or tissues lineally descent from a certain progenitor cell.

The term "after exit from pluripotency" refers to the time of the culturing also referred to as "hours of culture" in tables and examples. At this time, cells are typically switched out of pluripotency medium and into E6 based medium.

The terms "activating" and "promoting" in the context of signalling pathways are typically used in the following way: "activating" typically refers to activating a single signalling pathway, whereas "promoting" typically refers to activating a cell behaviour (such as the EMT behaviour which is controlled by many different signalling pathways).

The term "post-cranial" refers to cell lineages that bear a gene expression profile that includes a HOX gene profile consistent with cervical, thoracic, lumbar or sacral axial identities. This is in distinction to cranial lineages, that do not express these HOX gene profiles.

The term "paralogues" refers to at least two proteins or compounds expressed by a pair of genes deriving from the same ancestral gene. Hence, "paralogue" proteins or compounds would be expected to exhibit similar behavior and effect in the context of the present disclosure.

The term "homologues" refers to compounds belonging to a homologous series, thereby typically having a fixed set of functional groups giving them similar chemical and physical properties. Hence, "homologous" compounds would be expected to exhibit similar behavior and effect in the context of the present disclosure.

The term "derivative" refers to a substance that is created via a chemical reaction from another component. Hence, "derivative" compounds would be expected to exhibit similar behavior and effect in the context of the present disclosure.

The term "combinations" refers to combination of compounds, factors, paralogues, homologous and/or derivatives of the present disclosure. Hence, "combinations" of compounds would be expected to exhibit similar behavior and effect in the context of the present disclosure as the specific compounds combined.

The term "decoy receptor" refers to a receptor that is able to recognize and bind specific growth factors or cytokines efficiently but is not structurally able to signal or activate the intended receptor complex. Thus, it acts as an inhibitor, binding a ligand and keeping it from binding to its regular receptor.

Brief descriptions of the drawings

The above objects, as well as additional objects, features and advantages of the present disclosure, will be more fully appreciated by reference to the following illustrative and nonlimiting detailed description of example embodiments of the present disclosure, when taken in conjunction with the accompanying drawings.

Figure 1 (A-l) shows how NMPs can be derived from pluripotent stem cells: (A) shows a schematic of how, in one embodiment, different factors in the cocktail are added to the media across the 0-120hr period to generate in vitro NMPs (x refers to any factor added in addition to Fgf8b, CHIR, and A 83-01); (B-C) shows how undesired off-target TBX6 expressing PSM cells can be fully eliminated by adding additional members of the 7F cocktail; (D-F) shows that, in the context of the 7F cocktail, the concentration of WNT signaling is instrumental such that at an optimized concentration of CHIR, NMPs can be defined by protein co-expression of SOX2, BRA, CDX1 and CDX2; (G) Shows that, in the context of the 7F cocktail, the distribution of TBXT protein intensity and SOX2 protein intensity are both unimodal, (Hardigan's diptest for TBXT = 0.994, and for SOX2 = 0.999. LaplacesDemon test for is.unimodal(SOX2) = TRUE, is.unimodal(TBXT) = TRUE), and that they show a positive correlation (pval < 2.2^A10-16); (H-K) shows how various markers for NMPs are upregulated on the RNA level across the 120hr period; (L) shows how NMPs undergo colinear HOX activation based on time spent in the 7F, then 6F cocktails (Le. anterior HOX genes such HOX1/2/3/4 are upregulated early in time, while posterior HOX genes such as HOX9 are upregulated first at 120hrs. Even more posterior HOX10 genes require subsequent application of the 6F cocktail and are upregulated after 120hrs).

Figure 2 shows that when Fgf2 is used in the 7F cocktail (instead of the usual Fgf8b), cells on the edge of the colony spuriously differentiate into TBX6 positive, off-target, unwanted presomitic mesoderm cells. This highlights the importance of correct FGF signalling, which in certain embodiments is achieved using Fgf8b.

Figure 3 shows that the combination of CHIR, and Fgf2, and BME together (and after the 7F cocktail) yield the highest proportion of TBX6 positive presomitic mesoderm cells. Moreover, the concentration of CHIR in the two phases (48-120hrs, or 120-192hrs, after exit from pluripotency) is critical, i.e. the highest TBX6 proportion is yielded when CHIR is at 4.5pM from 48-120hrs, and at lOpM from 120-192hrs. Decreasing these to 3pM during 48-120hrs, or to 4.5pM during 120-192hrs, dramatically decreases the proportion of TBX6 positive presomitic mesoderm cells.

Figure 4 shows how NMPs can generate cervical, thoracic or lumbar level daughter neural progenitors by initiating neural differentiation from either early (7F.NMPs at 96hrs/Day4), later (7F.NMPs at 120hrs/Day5), or later again (7F-then-6F 168hrs/Day7) stage NMPs. Detailed description

The present disclosure will now be described in more detail, whereby preferred example embodiments of the disclosure are shown. The disclosure may, however, be embodied in other forms and should not be construed as limited to the herein disclosed embodiments. The disclosed embodiments are provided to fully convey the scope of the disclosure to the skilled person.

As briefly discussed in the summary, the present inventors have successfully developed an efficient protocol that recreates the events of generating all expected lineages of the post- cranial human body in vitro, where the inventors derive NMPs from human induced pluripotent stem cells (iPSCs), and subsequently pattern these into different lineages (mesoderm, ectoderm and neural crest) to build tissues of specific axial identity (cervical, thoracic or lumbar).

A cell culture method for deriving a multipotent NMP

In a first aspect, this disclosure relates to a cell culture method for deriving a multipotent neuromesodermal progenitor (NMP) cell and/or lineages of specific anterior-posterior positions in the post-cranial human body and/or cell types of matched anterior-posterior position, from a human pluripotent stem cell (PSC).

Typically, the human pluripotent stem cell (hPSC) is chosen from a human embryonic stem cell (hESC), or a human induced pluripotent stem cell (hiPSC), reprogrammed to pluripotency from a somatic cell.

In some embodiments, the method comprises culturing the human pluripotent stem cell under conditions and with reagents modulating one or more of the following signalling pathways (durations are from initiation of post-pluripotency culturing):

• activation of FGF signalling for a duration of about 12-120 hours;

• activation of WNT signalling for a duration of about 48-120 hours; and

• inhibition of SMAD signalling, including TGF-beta/activin-Nodal signalling and BMP signalling, for a duration of about 48-120 hours.

Also, in some embodiments, the method may also comprise the following signalling pathway:

• inhibition of Notch signalling for a duration of about 48-120 hours.

Time durations mentioned are typically from initiation of post-pluripotency culturing. Modulations and changes to these intervals are conceivable as long as the desired effect (e.g., activation or inhibition of a signalling pathway) is obtained, and results in an NMP consistent with the embodiments of this disclosure. To modulate each pathway, the addition of one or more factor(s) (chemical compound(s)) is necessary. The following factors are examples having the required effect, as they act on the same signalling pathways as the factors used in the examples of this disclosure. The specific concentration and/or timing for each compound may be different compared to the factors tested out in the examples. For example, each compound (or factor) may have distinct affinities for receptors, and therefore different optimal concentration and/or timing:

Activation of FGF signalling can be performed using FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof, and/or by blocking Dusp6 or by activating ERK1/2 signalling using small molecule inhibitors of DUSP6 chosen from E-2-benzylidene-3- cyclohexylamino-2,3-dihydro-lH-inden-l-one BCI and paralogues, homologues, derivatives and/or combinations thereof. These molecules and proteins listed can be anticipated to act as Fgf8 (or at least in part very similar) as they either bind to the same receptor or act downstream of Fgf binding to its receptor.

More specifically, FGF 1-18, and especially FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof bind to the same receptor (Fgf R 1) and thus are anticipated to elicit similar downstream effects, even if not identical. Therefore, it is very plausible that these would work in the setting of the present disclosure.

Dusp6 acts downstream of FgfR signalling, blocking the action of phosphorylated ERK1/2, and thus acts as an inhibitor. Thus blocking its (Dusp's) activity, is expected to elicit a response similar to something that binds to the FgfRl.

By either blocking Dusp6 or activating ERK1/2 signalling using small molecules, this will basically have the same effect as binding to the FgfRl (as these are cross repressive downstream signalling pathways - pERKl/2 is activated by Fgf binding to FgfR and then Dusp is activated by ERK1/2, and consequently represses ERK1/2 signalling (to get a balanced response to Fgf signalling).

Activation of WNT signalling can be performed using CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, BIO, SB-216763, Wnt5A, Wnt5B, Wntl, Wnt3A, paralogues, homologues, derivatives and/or combinations thereof. More specifically:

CHIR98014 activates WNT signalling similar to CHIR99021;

AMBMP activates WNT signalling, like CHIR99021;

LP 922056 is an inhibitor of Notum, which in turn is an inhibitor of WNT signalling, thus using LP 22056 is anticipated to activate WNT signalling similar to what CHIR99021 would, and this has been demonstrated, and this compound is sold as a WNT activator; Lithium inhibits GSK3b, just as CHIR90021 does and in doing so activates WNT signalling;

BIO is a small molecule that activates WNT signalling and is anticipated to have the same effect as CHIR99021; and SB-216763 inhibits GSK3b just like CHIR99021 and in this way activates WNT signalling; Wnt5A, Wnt5B, Wntl and Wnt3A proteins bind to Frizzled (receptor) and activate the same signalling pathways which downstream (of binding of a Wnt protein to its receptor) leads to inhibition of GSK3b, thus the same effect as CHIR99021.

Inhibition of SMAD signalling can be performed using Hesperetin, SB431542, and SB431542 hydrate, SB525334, Galunisertib, GW788388, LY2109761, SB505124, LDN-193189, LDN- 193189 HCI, RepSox, A 83-01, DMH1, LDN-212854, ITD 1, LY364947, SD-208, EW-7197, ML347, K02288, A 77-01, SIS3, LDN-214117, R-268712, Pirfenidone, Noggin, Chordin, Gremlin, DAN proteins, GDF3, paralogues, homologues, derivatives and/or combinations thereof. More specifically:

A83-01 blocks ALK5 which leads to inhibition of Smads;

Hesperetin decreases the affinity of TGFbeta to its receptor thus acting similarly to A83-01, leading to the same result in the end on blocking Smad signalling;

SB431542 is another ALK5 inhibitor, like A83-01;

SB525334 is another ALK5 inhibitor, like A83-01;

Galunisertib is a selective TGFbetaR antagonist, thus acting similar to A83-01 and leading to inhibition of SMAD signalling.

Inhibition of Notch signalling can be performed using DLK1, gamma-secretase inhibitors GSIs, including e.g., small molecules DAPT, RO4929097, PF-03084014, derivatives thereof, antibodies blocking Notch receptor and anti- JAG ligands and decoys against Notch receptor and JAG ligand, inhibitors of MAML1, and paralogues, homologues, derivatives and/or combinations thereof. More specifically,

DAPT (GSI-IX) is a potent and orally active y-secretase inhibitor . DAPT inhibits the activation of Notch 1 signaling and induces cell differentiation;

DLK1: act as an inhibitor of Notch signalling; gamma-secretase inhibitors: these will all block Notch signalling, and DAPT is a gamma- secretase inhibitor;

RO4929097, PF-03084014 are gamma-secretase inhibitors just like DAPT and will thus act the same; anti-JAG ligands: JAG interacts with Notch and activates Notch signalling, thus ligands that block this activation will block Notch signalling similar to what the downstream gamma-secretase inhibitors like DAPT do; a nti-Delta-li ke ligand: Delta - Notch activates Notch signalling and thus anti-delta will block Notch signalling in neighboring cells, acting upstream of a gamma-secretase inhibitor such as DAPT; similar to comments above, decoys against Notch receptor, JAG ligands, Delta-like ligands would block Notch signalling, similar to the gamma-secretase inhibitor, DAPT, but acting slightly upstream; and inhibitors of MAML1: MAML1 is a known component of the active Notch transcriptional complex, and is required for Notch-induced transcriptional activation of downstream signalling, and thus inhibitors would inhibit downstream regulation of NOTCH signalling and act similar to DAPT (just more downstream in the signalling pathway) and thus have similar effects.

For example, culturing can be performed in a medium that is serum-free, not containing transforming growth factor |3 (TGF- ) or basic fibroblast growth factor (bFGF) and which supports the growth of pluripotent stem cells and their differentiation. One example is the E6 medium (Essential 6 Medium), which is a defined medium, lacks serum and does not contain transforming growth factor |3 (TGF-|3) or basic fibroblast growth factor (bFGF), E6 medium can be purchased from: (https://www.thermofisher.com/order/catalog/product/A1516401). Further information on the E6 medium: Chen et al. (Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011 8(5 ) :424-9) However, other suitable media of similar type, supporting stem cell growth and differentiation and lack TGF- or bFGF are also conceivable for use in this context. In one embodiment, said stem cell is cultured with a sevenfactor cocktail ("7F", see also the examples), i.e., the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin, for a duration of about 96 hours.

In some embodiments, BME (Basal Matrix Extract), being a mix of different proteins in an extracellular matrix extract, could be substituted with one or more components related to pathways such as promoting EMT and/or more cellular adhesion. See e.g. the following link: https://www.rndsystems.com/research-area/emt-induction. Also, such pathways may include WNT, Fgf, SMAD, Akt, Notch, MEK, JNK, among others. Moreover, BME could in some embodiments be substituted with other complex extracellular matrix extracts, e.g., Matrigel.

In some embodiments, the factors of the 7F cocktail are added from about 12-24 hours, and/or from about 24-48 hours and/or later, and for a duration of about 72-96 hours, about 96-120 hours or more than 120 hours, such as up to 240 hours, or longer. Thus, various factors affect different signaling pathways, and depending on choice of specific compound and concentration, the optimal time point for adding the factor, and the duration for it to be in the culture, may vary and needs to be tested out.

In one embodiment, the factors of the 7F cocktail are added at the following concentrations and durations:

• Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml from about 24 hours;

• CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm, from about 48 hours;

• A 83-01 at 5-20 pM, preferably about 10 pM, from about 48 hours;

• SIS3 at 0,25-1 pM, preferably about 0.5 pM, from about 48 hours;

• K02288 at 0,5-10 pM, preferably about 2 pM, from about 48 hours;

• DAPT at 5-20 pM, preferably about 10 pM, from about 48 hours; and

• Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml, from about 48 hours. In one embodiment, it has been observed that a multipotent neuromesodermal progenitor cell generated this way co-expresses SOX2, TBXT, CDX2 and CDX1 at RNA and/or protein levels. Typically, a purity of at least 80% is obtained.

RNA level is typically quantified using RNA sequencing, RNA scope, RNA FISH and/or quantitative RT-PCR (qPCR).

Protein level is typically measured using immunofluorescence with antibodies targeted against the proteins in question and visualised using microscopy, including confocal microscopy.

In one embodiment, it has been observed that the multipotent neuromesodermal progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1 and HOX at RNA and/or protein levels, and that the multipotent neuromesodermal progenitor cell does not express the mesodermal markers TBX6 or MSGN1 at RNA and/or protein levels.

In some embodiments, these expressions are observed at the 120hr timepoint, and some genes are expected to be expressed earlier than that.

The multipotent neuromesodermal progenitor cell can be developed into mesoderm, ectoderm, neuroectoderm and/or neural crest lineages in order to generate tissues of cervical, thoracic and/or lumbar axial identity and/or cell types chosen from nervous system tissue, neural crest derivative, skeletal muscle, bone and cartilage.

In one embodiment, a cervical neuromesodermal progenitor cell is generated by culturing with the 7F cocktail (Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin) for a duration of about 72-96 hours.

In one embodiment, a thoracic neuromesodermal progenitor cell is generated by culturing with the 7F cocktail (Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin) for a duration of about 96-120 hours. In this embodiment, thoracic NMPs can be observed from around 96 hrs, even though pure thoracic NMPs, with no contamination from cervical NMPs, typically can be observed from around 120hrs.

In one embodiment, a lumbar level neuromesodermal progenitor cell is generated by culturing in a first step with the 7F cocktail (Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin) for a duration of about 120 hours, and in a second step with a six-factor cocktail ("6F", see also examples), i.e., Fgf8b, CHIR99021, K02288, DAPT, Heparin and GDF11, for a duration of about 24-48 hours.

When generating lumbar level neural progenitor cells, in the second step, the factors can be added at the following concentrations:

• Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

• CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

• K02288 at 0,5-10 pM, preferably about 2 pM; DAPT at 5-20 pM, preferably about 10 pM;

The suggested composition of the 6F cocktail can be changed. For example, Fgf8b, CHIR99021, and K02288 could be substituted with other molecules.

In one embodiment, a TBX6 expressing presomitic mesoderm cell is generated by culturing in a first step with the 7F cocktail (Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin) for a duration of about 120 hours, and in a second step with a three-factor cocktail ("3F", see also example), i.e., the factors Fgf2, CHIR99021 and BME (Basal Matrix Extract), for a duration of about 48-72 hours.

When generating a TBX6 expressing presomitic mesoderm cell, in the second step, the factors are added at the following concentrations:

• Fgf2 at 10-100 ng/ml, preferably about 50 ng/ml;

• CHIR99021 at 5 to 20 pm, preferably about 10 pm; and

• BME, Matrigel, or other cell-extracted extracellular matrix products, preferably at about 8-12 mg/mL.

A composition for use in deriving multipotent NMPs

In a further aspect, the present disclosure relates to a composition for use in deriving multipotent neuromesodermal progenitor cells NMPs and/or lineages derived thereof of a specific anterior-posterior position in the human body and/or cell types of matched anterior- posterior position from human induced pluripotent stem cells. The composition comprises reagents modulating the following signalling pathways:

• activation of FGF signalling for a duration of about 12-120 hours, or 24-120 hours, for example one or more of the factors FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof, and/or by blocking Dusp6 and/or by limiting the activity of ERK1/2 using small molecule inhibitors of DUSP6 chosen from E-2-benzylidene-3- cyclohexylamino-2,3-dihydro- lH-inden-l-one BCI and paralogues, homologues, derivatives and/or combinations thereof;

• activation of WNT signalling for a duration of about 48-120 hours, for example one or more of the factors CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, BIO, SB-216763, Wnt5A, Wnt5B, Wntl, Wnt3A, paralogues, homologues, derivatives and/or combinations thereof;

• inhibition of SMAD signalling, including TGFbeta/activin-Nodal signalling and BMP signalling, for a duration of about 48-120 hour, for example one or more of the factors Hesperetin, SB431542, SB525334, Galunisertib, GW788388, LY2109761, SB505124, LDN-193189, LDN-193189 HCI, RepSox, A 83-01, DMH1, LDN-212854, ITD 1, LY364947, SD-208, EW-7197, ML347, K02288, A 77-01, SIS3, LDN-214117, Fl- 268712, Pirfenidone, Noggin, Chordin, Gremlin, DAN proteins, GDF3, paralogues, homologues, derivatives and/or combinations thereof; and

• inhibition of Notch signalling for a duration of about 48-120 hours, for example one or more of the factors DLK1, gamma-secretase inhibitors GSIs, including e.g., small molecules DAPT, RO4929097, PF-03084014, derivatives thereof, antibodies blocking Notch receptor and anti- JAG ligands and decoys against Notch receptor and JAG ligand, inhibitors of MAML1, and paralogues, homologues, derivatives and/or combinations thereof.

In one embodiment, the composition comprises the seven factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin, i.e., the 7F cocktail (see also examples).

With regard to Fgf8b, it can be replaced with other members of the Fgf8 subfamily, including FGF8a, as well as by FGF17, FGF18, though at different concentrations. However, Fgf2 is not expected to work in the context of this cocktail. Using Fgf2 results in precocious mesodermal differentiation of 7F NMPs.

With regards to the CHIR99021 concentration, it results in in vitro NMPs that express at the protein and RNA level the NMP marker CDX1 in at least roughly 80% of cells and does not eliminate the expression of SOX2 in at least 95% of cells. CHIR99021 can be replaced with other potentiators of WNT signalling.

With regards to K02288, it is seen as blocking the same pathway as the molecule LDN-193189. So, LDN-193189 (or a LDN-193189 replacement) can be used as a substitute for K02288.

With regards to A 83-01, it is a derivative of SB431542. SB431542 and other derivatives of SB431542, including RepSox, can be used as a substitute.

With regards to SIS3, it is a cell permeable inhibitor of SMAD3 signalling. Unlike A 83-01 and K02288 (which inhibit SMAD signalling through cell surface receptors) SIS3 inhibits the intercellular downstream mediation of SMAD3 signalling. This adds an additional point of SMAD signalling inhibition on top of A 83-01 and K02288.

For example, the factors are present at the following concentrations:

• Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

• CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

• A 83-01 at 5-20 pM, preferably about 10 pM;

• SIS3 at 0,25-1 pM, preferably about 0.5 pM;

• K02288 at 0,5-10 pM, preferably about 2 pM;

• DAPT at 5-20 pM, preferably about 10 pM; and

• Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml. The factors of this disclosure have the following chemical definitions:

CHIR99021: 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-l/7-imidazol-2-yl)-2- pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile

CHIR98014: A/6-[2-[[4-(2,4-Dichlorophenyl)-5-(l/7-imidazol-l-yl)-2-pyrimidinyl]amino]ethyl]- 3-nitro-2,6-pyridinediamine

AMBMP hydrochloride: A/4-(l,3-Benzodioxol-5-ylmethyl)-6-(3-methoxyphenyl)-2,4- pyrimidinediamine hydrochloride

LP 922056: 2-[(6-Chloro-7-cyclopropylthieno[3,2-d]pyrimidin-4-yl)thio]acetic acid

SB-216763: 3-(2,4-Dichlorophenyl)-4-(l-methyl-lH-indol-3-yl)-lH-pyrrole-2, 5-dione

SB431542: 4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-l/7-imidazol-2-yl] benzamide (obtained from TOCRIS) or 4-(5-Benzol[l,3]dioxol-5-yl-4-pyrldin-2-yl-lH-imidazol-2-yl)-benzamide hydrate, 4-[4-(l,3-Benzodioxol-5-yl)-5-(2-pyridinyl)-lH-imidazol-2-yl]-benzamide hydrate, 4-[4- (3,4-Methylenedioxyphenyl)-5-(2-pyridyl)-lH-imidazol-2-yl]-benzamide hydrate (obtained from Merck (Sigma-Aldrich)).

SB431542 hydrate: 4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-l/7-imidazol-2-yl] benzamide (same as above)

SB525334: 6-[2-(l,l-Dimethylethyl)-5-(6-methyl-2-pyridinyl)-l/7-imidazol-4-yl]quinoxaline

Galunisertib: LY2157299,

GW788388: 4-{4-[3-(Pyridin-2-yl)-lH-pyrazol-4-yl]-pyridin-2-yl}-N-(tetrahydro-2H-pyran-4- yl)benzamide hydrate

LY2109761: 4-[5,6-Dihydro-2-(2-pyridinyl)-4H-pyrrolo[l,2-b]pyrazol-3-yl]-7-[2-(4- morpholinyl)ethoxy]-quinoline,

SB505124: 2-[4-(l,3-Benzodioxol-5-yl)-2-(l,l-dimethylethyl)-l/7-imidazol-5-yl]-6-methyl- pyridine

LDN-193189: 4-[6-[4-(l-Piperazinyl)phenyl]pyrazolo[l,5-o]pyrimidin-3-yl]quinoline dihydrochloride

LDN-193189 HCI: 4-[6-[4-(l-Piperazinyl)phenyl]pyrazolo[l,5-o]pyrimidin-3-yl]quinoline dihydrochloride (basically same compound as above)

RepSox: 2-(3-(6-Methylpyridine-2-yl)-l/7-pyrazol-4-yl)-l,5-naphthyridine

A 83-01: 3-(6-Methyl-2-pyridinyl)-/V-phenyl-4-(4-quinolinyl)-l/7-pyrazole-l-carbothioamide

DMH1: 4-[6-[4-(l-Methylethoxy)phenyl]pyrazolo[l,5-o]pyrimidin-3-yl]-quinoline LDN-212854: 5-[6-[4-(l-Piperazinyl)phenyl]pyrazolo[l,5-a]pyrimidin-3-yl]-quinoline

ITD 1: 4-[l,l'-Biphenyl]-4-yl-l,4,5,6,7,8-hexahydro-2,7,7-trimethyl-5-oxo-3- quinolinecarboxylic acid ethyl ester

LY364947: 4-[3-(2-pyridinyl)-lH-pyrazol-4-yl]-quinoline

SD-208: 2-(5-Chloro-2-fluorophenyl)-4-[(4-pyridyl)amino] pteridine

EW-7197: N-((4-([l,2,4]triazolo[l,5-a]pyridin-6-yl)-5-(6-methylpyridin-2-yl)-lH-imidazol-2- yl)methyl)-2-fluoroaniline

ML347: 5-[6-(4-Methoxyphenyl)pyrazolo[l,5-a]pyrimidin-3-yl]quinoline

K02288: 3-[(6-Amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol, 3-[6-Amino-5-(3,4,5- trimethoxyphenyl)-3-pyridinyl]-phenol

A 77-01: 4-[3-(6-Methyl-2-pyridinyl)-l/7-pyrazol-4-yl]quinoline

SIS3: Smad3 Inhibitor, SIS3, SIS3, 6,7-Dimethoxy-2-((2E)-3-(l-methyl-2-phenyl-lH-pyrrolo[2,3- b]pyridin-3-yl-prop-2-enoyl))-l,2,3,4-tetrahydroisoquinoline,

LDN-214117: l-(4-(6-Methyl-5-(3,4,5-trimethoxyphenyl)pyridin-3-yl)phenyl)piperazine

R-268712: 4-[2-Fluoro-5-[3-(6-methyl-2-pyridinyl)-l/7-pyrazol-4-yl]phenyl]-l/7-pyrazole-l- ethano

Chemical definitions for listed compounds are also known in the art.

A multipotent NMP

In a further aspect of this disclosure, a multipotent neuromesodermal progenitor cell is disclosed, being derived from a human induced pluripotent stem cell, iPSC, wherein the progenitor co-expresses SOX2, TBXT, CDX2 and CDX1 at RNA and/or protein levels.

In one embodiment, said progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1 and HOX at RNA and/or protein levels, and/or wherein said cell does not express the mesodermal markers TBX6, or MSGN1 at RNA or protein levels and/or wherein said cell upregulates at early time points 3' HOX genes, which are expressed more anteriorly in the body axis, and at progressively later time points more 5' HOX genes, which are expressed more posteriorly in the body axis.

Axial (cervical/thoracic/lumbar) neuromesodermal progenitors

In a further aspect of this disclosure, a cervical neuromesodermal progenitor cell is disclosed, being obtained by culturing human induced pluripotent stem cells, iPSCs, with any of the compounds disclosed above for the first aspect, and in one embodiment the 7F cocktail (factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin) for a duration of about 72-96 hours.

In one embodiment, said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB4, and not HOXB9 or HOXBIO at the RNA level.

In a further aspect of this disclosure, a thoracic neuromesodermal progenitor cell is disclosed, being obtained by culturing human induced pluripotent stem cells iPSCs with any of the compounds disclosed above for the first aspect, and in one embodiment the 7F cocktail (factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin) for a duration of about 120 hours.

In one embodiment, said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB9, and not HOXBIO, at the RNA level.

In a further aspect of this disclosure, a lumbar level neuromesodermal progenitor cell is disclosed, being obtained by culturing the multipotent neuromesodermal progenitor cell according to the third aspect with any of the compounds disclosed above for the first aspect, and in one embodiment the 6F cocktail (factors Fgf8b, CHIR99021, K02288, DAPT, Heparin, GDF11) for a duration of about 48 hours.

In one embodiment, said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXC10 at the RNA level.

Wichterle et al (2002) and Li et al. (2005) are references of the art describing dorsal and neural tube differentiation protocols that are incorporated herein as references. an axial (cervica umbar) neuromesodermal

In a further aspect of this disclosure, a composition for use in generating lumbar level neuromesodermal progenitor cells from the multipotent neuromesodermal progenitor cells according to the third aspect is disclosed, comprising the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin, GDF11 (6F cocktail).

In one embodiment, the factors are present at the following concentrations:

(i) Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

(ii) CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

(iii) K02288 at 0,5-10 pM, preferably about 2 pM;

(iv) DAPT at 5-20 pM, preferably about 10 pM;

(v) Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml; and

(vi) GDF11 at 50-500 pg/ml, preferably about 100 pg/ml.

In a further aspect of this disclosure, a composition for use in generating TBX6 expressing presomitic mesoderm cells from the multipotent neuromesodermal progenitor cells according to the third aspect is disclosed, comprising the 3F cocktail (factors Fgf2, CHIR99021 and BME Basal Matrix Extract).

In one embodiment, the factors are present at the following concentrations:

(i) Fgf2 at 10-100 ng/ml, preferably about 50 ng/ml;

(ii) CHIR99021 at 5 to 20 pm, preferably about 10 pm; and

(iii) BME, Matrigel, or other cell-extracted extracellular matrix products, preferably at about 8-12 mg/mL.

A TBX6-1 itic mesoderm

In a further aspect of this disclosure, a TBX6 expressing presomitic mesoderm progenitor cell is disclosed, obtained by culturing the multipotent neuromesodermal progenitor cell according to the third aspect with the 3F cocktail (factors Fgf2, CHIR99021 and BME (Basal Matrix Extract)) for a duration of about 48-72 hours.

In some embodiments, BME (basal matrix extract, which is a complex mix of different proteins in an extracellular matrix extract), which promotes EMT signalling pathway and /or more cellular adhesions, could be substituted with factors or extracts affecting alternative pathways obtaining similar effects, such as WNT, Fgf, SMAD, Akt, Notch, MEK, JNK, among others. For example, Matrigel is an alternative, as well as other complex extracellular matrix extracts. In one embodiment, said cell co-expresses TBX6, MSGN1, TBXT, RBP1 and GAD1, at protein and/or RNA level.

A cell population

In a further aspect of this disclosure, a cell population comprising one or more of the NMPs, the daughter neural, or neural crest or mesoderm progenitor cells according to any of the other aspects is disclosed. Such cell population could further be used for subsequent cel I- and/or tissue related applications and would essentially be derived by the method of the first aspect of this disclosure.

A kit

In a further aspect of this disclosure, a kit, primarily for use in the cell culture method of this disclosure, is disclosed, said kit comprising one or more of the compositions according to the other aspects, suitable media, such as a medium that is serum-free, not containing transforming growth factor |3 (TGF-|3) or basic fibroblast growth factor (bFGF) and which supports the growth of pluripotent stem cells and their differentiation. One example is the E6 medium (Essential 6 Medium), which is a defined medium, lacks serum and does not contain transforming growth factor |3 (TGF-|3) or basic fibroblast growth factor (bFGF). A suitable medium or suitable media for use in a kit could also be chosen from the cell culture media disclosed in this disclosure, thus comprising a media suitable for the NMP and/or cell to be generated, further comprising suitable factors, if required, as disclosed and defined elsewhere in this disclosure. The factors can e.g., be included as one or more of the compositions disclosed herein, for use in deriving multipotent neuromesodermal progenitor cells (NMPs) and/or daughter lineages thereof with a specific anterior-posterior position in the human body and/or cell types of matched anterior-posterior position from human induced pluripotent stem cells. This includes e.g. a thoracic, cervical or lumbar level neuromesodermal progenitor cells, preneural tube progenitor, neural crest progenitor cells, as well as TBX6 expressing presomitic mesoderm progenitor cells.

Optionally, the kit may further comprise suitable human stem cells, such as human induced pluripotent stem cells, and optionally other necessary reagents and tools, such as laminin coating of a plastic culture dish used for culturing. Typically, laminin coating is used at a minimum amount of 0.05 pg/cm² surface area of the culture dish. The typical laminin coating procedure would be: Coat tissue culture dishes for 4-24hrs at 4°C in a 2pg/ml solution of laminin-521 in saline (e.g. PBS, HBSS) without Magnesium or Calcium. Any other technique that supports the growth of the cells could also work. For example, other laminins, or Matrigel, in other saline solutions could also coat the dishes in a suitable way to support the growth of the cells. Other possible coatings could also be vitronectin, or Synthemax ll-SC Substrate (Cat# 3535, Corning) referred to as Synthemax in protocols or Geltrex (Cat # A1413302, Gibco). Also, instructions for use can be included, which e.g. can include protocols including concentrations, culturing conditions and durations of included components and steps in order to optimise development of the NMP and/or cell to be generated.

Such kit could be used for performing any of the methods of this disclosure thereby obtaining any of the progenitors and/or cells and/or tissues related to this disclosure.

Cell culture media

Further, the present disclosure may be used to develop cell culture media aimed towards generating the thoracic (7F) and lumbar (6F) NMP. Such cell culture media would typically comprise the components found in E6 media, including the cocktail of the appropriate stages, as specified in this disclosure. For example:

• Medial would be only E6 media and would be used for 24hrs.

• Media2 would be E6 media + Fgf8b and would be used for 24hrs.

• Media3 would be E6 media + all 7 factors and would be used for 72hrs (with daily changes).

• Media4 would be E6 media + 6 factors and would be used for 24-48hrs.

Variations in terms of components and suggested durations may be made, in line with the details and embodiments of the present disclosure. Cellular composition

According to yet another aspect there is provided a cellular composition, comprising one or more cervical, thoracic or lumbar level NMP cell(s), wherein the cell(s) is/are present at a purity level of at least roughly 80%. Subsequent daughter neural progenitor cells, and/or neural crest progenitor cells, and/or presomitic mesoderm progenitor cells are derived from the neuromesodermal progenitor cell as defined in this disclosure.

The invention will now be described in relation to the following working examples, wherein examples of the methods and compositions are practiced.

Examples

Example 1 - Experimental details

1. Stem cells are thawed from liquid nitrogen (however, vapour nitrogen is also an alternative) into mTeSR-plus medium, or other appropriate medium supporting stem cell growth, on tissue culture dished coated with laminin, matrigel, vitronectin, geltrex or other appropriate coating for stem cells.

2. Protein measurements are made using immunofluorescence, fluorescent confocal microscopy, and bioinformatic imaging software (e.g. Stardust, custom R scripts).

3. RNA is measured using bulk or single-cell RNA sequencing (e.g. SmartSeq or Drop-seq or Nuc-seq chemistries) along with downstream bioinformatic analysis (published and custom computational scripts).

4. Cells are grown at 5% CO2, in a humidified incubator at atmospheric pressure.

Example 2 - Protocol for deriving NMPs

The inventors have conducted experiments that demonstrate that NMPs can be derived from human pluripotent stem cells (Fig. 1). Previous reports of in vitro derived NMP marker profiles describe co-expression of SOX2, TBXT, and CDX2. The present disclosure relates to a protocol using a cocktail of 7 factors (7F) that can derive NMPs that co-express SOX2, TBXT, CDX2 and CDX1 at the RNA and protein levels.

Importantly, the inventors demonstrate that robust CDX1 expression requires higher levels of WNT signalling (4.5pM CHIR, Fig. 1D-F) than in previously published protocols (3pM CHIR), as well as 72hrs of 7F exposure (Fig. IK). In other cocktail contexts, this higher level of WNT signalling can provoke NMPs to differentiate down the mesoderm lineage (TBX6+ve, Fig. 1B- C), but in the context of the present 7F, cells can be held in a stable NMP state that now expresses CDX1. Lastly, the inventors found that within the 7F cocktail, Fgf8b cannot be substituted for Fgf2, as this leads to unwanted differentiation down the mesoderm lineage (TBX6+ve, Fig. 2), and decreased purity of NMPs. Moreover, the inventors demonstrate that the higher level of WNT signalling (4.5pM) in the 7F cocktail is required for efficient downstream generation of the presomitic mesodermal lineage (Fig. 3). Specifically, at 12Ohrs, 7F.NMPs are first treated with neat Basal Matrix Extract (BME) (8-12mg/ml), and then switched into a media containing lOpM CHIR, and 50ng/ml Fgf2 for an additional 72hrs. These protocols yield high proportions of Tbx6 expressing presomitic mesoderm progenitors. If NMPs are instead generated with lower WNT signalling (3 pM CHIR) between 48-120hrs, the subsequent treatment with BME (8-12 mg/ml), FGF2 (50 ng/ml) and CHIR (10 pM) from 120-192hrs does not induce a high proportion of presomitic mesoderm (as defined by the % of TBX6+ cells). The FGF2 and BME are needed to induce a high proportion of TBX6+ cells as leaving these out and treating with 10 pM CHIR only after exiting the 7F.NMP state results in a significantly lower proportion of TBX6+ presomitic mesoderm cells. Furthermore, treatment of 7F.NMPs from 120-192hrs with a lower CHIR concentration (4.5 pM) in combination with FGF2 (50 ng/ml) and BME (8-12 mg/ml) is not sufficient to induce a high proportion of TBX6+ cells.

The inventors have used bulk RNA-sequencing to molecularly define 7F.NMPs (Fig. 1, G-l), which shows that at 120hrs in vitro, 7F.NMPs express a molecular code that includes SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1, and numerous HOX genes.

The inventors have demonstrated that 7F.NMPs are multipotent and can generate post-cranial cell types of three lineages; neurectoderm, neural crest and mesoderm.

The inventors have demonstrated that the anterior-posterior identities of NMP-derived ventral neural progenitors (specified through stimulation of the SHH pathway) are determined based on the length of the time NMPs are exposed to FGF8b and WNT signalling in the cocktail (Fig. 4). In more detail, 96hr 7F.NMPs generate cervical neural progenitors, 120hr 7F.NMPs generate thoracic neural progenitors, and 168hr 6F.NMPs generate lumbar level neural progenitors. Thus, the inventors have shown that the present 7F and subsequent 6F cocktails generate NMPs of all axial positions (cervical, thoracic, lumbar) in a time-dependent manner.

The inventors are using a cocktail of 7 factors to generate and maintain multipotent NMPs (7F.NMPs) over a 5 day period that can build up the posterior human body axis at the thoracic level. This cocktail, including the timing and concentrations of the 7 factors, is specified below in Table 1.

Subsequent to this, the inventors transition 7F.NMPs into a different cocktail of 6 factors, termed 6F.NMPs, over an additional 2 days, as outlined in Table 2. These 6F.NMPs are competent to generate cells at more posterior lumbar levels.

Lastly, the inventors use a cocktail of three factors (Table 3) to transition 7F.NMPs into TBX6 expressing presomitic mesoderm. GDF11 (pg/ml) (R&D systems, Cat No: 1958-GD-010)

The person skilled in the art realizes that the present disclosure is not limited to the preferred embodiments described above. The person skilled in the art further realizes that modifications and variations are possible within the scope of the appended claims. Additionally, variations to the disclosed embodiments can be understood and effected by the skilled person in practicing the claimed disclosure, from a study of the drawings, the disclosure, and the appended claims.

References :

Faustino Martins JM et al. (2020) Cell Stem Cell 26:172.

Frith TJR et al. (2018) ELife 7:e35786.

Guibentif C et al. (2021) Dev Cell 56(1):141.

Guillot C et al. (2021) ELife 10:e64819.

Henrique D et al. (2015) Development 142(17):2864

Li XJ et al. (2005) Nat Biotechnol 23, 215-221

Selleck MA and Stern CD (1991) Development 112(2):615.

Tzouanacou E et al. (2009) Dev Cell 17(3):365.

Wichterle H et al, Cell, 2002; Aug 9; 110(3):385-97. doi: 10.1016/s0092-8674(02)00835-8

Wymeersch FJ et al. (2016) ELife 5:el0042

Wymeersch FJ et al. (2019) Development 146(l):devl68161

Wymeersch FJ et al. (2021) Development 148(4):devl80612.

Claims

1. A cell culture method for deriving a multipotent neuromesodermal progenitor (NMP) cell and/or daughter lineages thereof with specific anterior-posterior positions along the human body axis and/or cell types of matched anterior-posterior position, from a human pluripotent stem cell (PSC), comprising culturing said stem cell under conditions and with reagents modulating the following signalling pathways:

(i) activation of FGF signalling during the time period of about 12 to about 120 hours after exit from pluripotency;

(ii) activation of WNT signalling during the time period of about 48 to about 120 hours after exit from pluripotency; and

(iii) inhibition of SMAD signalling, including TGF-beta/activin-Nodal signalling and BMP signalling, during the time period of about 48 to about 120 hours after exit from pluripotency; wherein said progenitor co-expresses SOX2, TBXT, CDX2 and CDX1 at RNA and/or protein level(s).

2. The method according to claim 1, further including modulating the following signalling pathway:

(iv) inhibition of Notch signalling during the time period of about 48 to about 120 hours after exit from pluripotency.

3. The method according to claim 1 or 2, wherein:

(i) activation of FGF signalling is performed using either FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof, and/or by blocking Dusp6 or by activating ERK1/2 signalling using small molecule inhibitors of DUSP6 chosen from e-2-benzylidene-3- (cyclohexylamino)-2,3-dihydro-lH-inden-l-one (BCI) and paralogues, homologues, derivatives and/or combinations thereof;

(ii) activation of WNT signalling is performed using either CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, BIO, SB-216763, Wnt5A, Wnt5B, Wntl, Wnt3A, paralogues, homologues, derivatives and/or combinations the reof;(iii) inhibition of SMAD signalling is performed using either Hesperetin, SB431542, SB525334, Galunisertib, GW788388, LY2109761, SB505124, LDN-193189, LDN-193189 HCI, RepSox, A 83-01, DMH1, LDN-212854, ITD 1, LY364947, SD-208, EW-7197, ML347, K02288, A 77-01, SIS3, LDN-214117, R-268712, Pirfenidone, Noggin, Chordin, Gremlin, DAN proteins, GDF3, paralogues, homologues, derivatives and/or combinations thereof; and/or

(iv) inhibition of Notch signalling is performed using DLK1, gamma-secretase inhibitors (GSIs), including e.g., small molecules DAPT, RO4929097, PF-03084014, derivatives thereof, antibodies blocking Notch receptor and anti-JAG ligands, a nti-Delta-li ke ligands and decoys against Notch receptor, JAG ligands, Delta-like ligands, inhibitors of MAML1, paralogues, homologues, derivatives and/or combinations thereof.

4. The method according to any of the preceding claims, wherein said progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1 and HOX at RNA and/or protein level (s), and/or wherein said cell does not express the mesodermal markers TBX6, or MSGN1 at RNA and/or protein level (s) and/or wherein said cell goes through colinear HOX gene activation.

5. The method according to any of the preceding claims, wherein culturing is performed in a medium, which lacks serum, supports stem cell growth and differentiation, and lacks TGFbeta and bFGF, such as E6 medium.

6. The method according to any one of the preceding claims, wherein said stem cell is cultured with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 96 hours.

7. The method according to claim 6, wherein the factors are added at the following concentrations and durations after exit from pluripotency:

(i) Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml from about 24 hours;

(ii) CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm, from about 48 hours;

(iii) A 83-01 at 5-20 pM, preferably about 10 pM, from about 48 hours;

(iv) SIS3 at 0,25-1 pM, preferably about 0.5 pM, from about 48 hours;

(v) K02288 at 0,5-10 pM, preferably about 2 pM, from about 48 hours;

(vi) DAPT at 5-20 pM, preferably about 10 pM, from about 48 hours; and

(vii) Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml, from about 48 hours.

8. The method according to any of the preceding claims, wherein the multipotent neuromesodermal progenitor cell is developed into mesoderm and/or neuroectoderm and/or neural crest lineages that bear a cervical or thoracic or lumbar axial identity and/or daughter cell types arising from mesoderm, and/or neuroectoderm and/or neural crest lineages, chosen from post-cranial neurons, oligodendrocytes, astrocytes, floor-plate, roof-plate, bone, cartilage, peripheral neurons including sympathetic neurons, parasympathetic neurons, sensory neurons (dorsal root ganglia) and enteric neurons, as well as melanocytes and Schwann cells.

9. The method according to any of the preceding claims, wherein a cervical neuromesodermal progenitor cell is generated by culturing with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 72-96 hours.

10. The method according to claim 9, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB4, and not HOXB9 or HOXBIO, at the RNA level, and/or if said cervical NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2, HOXB4 and not HOXB9 and not HOXCIO, at the protein level.

11. The method according to any of the preceding claims, wherein a thoracic neuromesodermal progenitor cell is generated by culturing with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 96-120 hours.

12. The method according to claim 11, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB9, and not HOXBIO, at the RNA level, and/or if said thoracic NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2 and HOXB9, and not HOXB4 and not HOXCIO, at the protein level

13. The method according to any of the preceding claims, wherein a lumbar level neuromesodermal progenitor cell is generated by, in a first step for a duration of about 120 hours, modulating the signalling pathways as listed in claim 1 and/or 2, and in a subsequent second step, for a duration of about 24-48 hours, continue modulating the signaling pathways of the first step with the exception of substituting inhibiting the TGF-beta/activin-Nodal signalling pathway for activating the same.

14. The method according to claim 13, wherein the lumbar level neuromesodermal progenitor cell is generated by culturing in a first step with the factors Fgf8b, CHIR99021, A 83- 01, SIS3, K02288, DAPT and Heparin for a duration of about 120 hours, and in a second step with the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin, GDF11 for a duration of about 24- 48 hours.

15. The method according to claim 13 or 14, wherein, in the second step, the factors are added at the following concentrations:

(i) Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

(ii) CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

(iii) K02288 at 0,5-10 pM, preferably about 2 pM;

(iv) DAPT at 5-20 pM, preferably about 10 pM;

(v) Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml; and

(vi) GDF11 at 50-500 pg/ml, preferably about 100 pg/ml.

16. The method according to any of claims 13-15, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1, and HOXBIO at the RNA level, and/or if said lumbar NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2, HOXCIO and/or HOXB9, but not HOXB4, at the protein level.

17. The method according to any of the preceding claims, wherein a TBX6 expressing presomitic mesoderm cell is generated by, in a first step for a duration of about 120 hours, modulating the signalling pathways as listed in claim 1 and/or 2, and in a subsequent second step, for a duration of about 48-72 hours, activating the WNT and FGF pathways, and promoting EMT (Epithelial Mesenchymal Transition).

18. The method according to claim 17, wherein the TBX6 expressing presomitic mesoderm cell is generated by culturing in a first step with the factors Fgf8b, CHIR99021, A 83- 01, SIS3, K02288, DAPT and Heparin for a duration of about 120 hours, and in a second step with the factors (i) Fgf2, (ii) CHIR99021 and (iii) BME (Basal Matrix Extract), Matrigel or other cell-extracted extracellular matrix products for a duration of about 48-72 hours.

19. The method according to claim 18, wherein, in the second step, the factors are added at the following concentrations:

(i) Fgf2 at 10-100 ng/ml, preferably about 50 ng/ml;

(ii) CHIR99021 at 5 to 20 pm, preferably about 10 pm; and

20. The method according to any of claims 17-19, wherein said cell co-expresses TBX6, MSGN1, TBXT, RBP1 and GAD1, at protein and/or RNA level.

21. The method according to any one of the preceding claims, wherein the human pluripotent stem cell (hPSC) is chosen from a human embryonic stem cell (hESCs), or a human induced pluripotent stem cell (hiPSC) reprogrammed to pluripotency from a somatic cell, such as an adult somatic cell.

22. A composition for use in deriving multipotent neuromesodermal progenitor cells (NMPs) and/or daughter lineages thereof with a specific anterior-posterior position in the human body and/or cell types of matched anterior-posterior position from human induced pluripotent stem cells, comprising reagents modulating the following signalling pathways:

(i) activation of FGF signalling during the time period of about 12 to about 120 hours after exit from pluripotency, using either FGF1, FGF2, FGF4, FGF6, FGF7, FGF8, FGF8a, FGF8b, FGF 17, FGF 18, derivatives and/or combinations thereof, and/or by blocking Dusp6 or by activating ERK1/2 signalling using small molecule inhibitors of DUSP6 chosen from (E)-2- benzylidene-3- (cyclohexylamino)-2,3-dihydro-lH-inden-l-one (BCI) and paralogues, homologues, derivatives and/or combinations thereof;

(ii) activation of WNT signalling during the time period of about 48 to about 120 hours after exit from pluripotency, using either CHIR99021, CHIR98014, AMBMP hydrochloride, LP 922056, Lithium, BIO, SB-216763, Wnt5A, Wnt5B, Wntl, Wnt3A, paralogues, homologues, derivatives and/or combinations thereof; and

(iii) inhibition of SMAD signalling, including TGFbeta/activin-Nodal signalling and BMP signalling, during the time period of about 48 to about 120 hours after exit from pluripotency, using either Hesperetin, SB431542, SB525334, Galunisertib, GW788388, LY2109761, SB505124, LDN-193189, LDN-193189 HCI, RepSox, A 83-01, DMH1, LDN-212854, ITD 1, LY364947, SD-208, EW-7197, ML347, K02288, A 77-01, SIS3, LDN-214117, R-268712, Pirfenidone, Noggin, Chordin, Gremlin, DAN proteins, GDF3, paralogues, homologues, derivatives and/or combinations thereof.

23. The composition according to claim 22, further comprising reagents modulating the following signalling pathway:

(iv) inhibition of Notch signalling during the time period of about 48 to about 120 hours after exit from pluripotency, using either DLK1, gamma-secretase inhibitors (GSIs), including e.g., small molecules DAPT, RO4929097, PF-03084014, derivatives thereof, antibodies blocking Notch receptor and anti- JAG ligands and decoys against Notch receptor and JAG ligand, inhibitors of MAML1, and paralogues, homologues, derivatives and/or combinations thereof.

24. The composition according to claim 22-23, comprising the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin.

25. The composition according to claim 24, wherein the factors are present at the following concentrations:

(i) Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

(ii) CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

(iii) A 83-01 at 5-20 pM, preferably about 10 pM;

(iv) SIS3 at 0,25-1 pM, preferably about 0.5 pM;

(v) K02288 at 0,5-10 pM, preferably about 2 pM;

(vi) DAPT at 5-20 pM, preferably about 10 pM; and

(vii) Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml.

26. A multipotent neuromesodermal progenitor cell being derived from a human induced pluripotent stem cell (iPSC), wherein said progenitor co-expresses SOX2, TBXT, CDX2 and CDX1 at RNA and/or protein level(s).

27. The multipotent neuromesodermal progenitor cell of claim 26, wherein said progenitor cell co-expresses SOX2, TBXT, CDX2, CDX1, NKX1-2, RBP1, WNT5B, SALL1, SALL3, GAD1 and HOX at RNA and/or protein level (s), and/or wherein said cell does not express the mesodermal markers TBX6, or MSGN1 at RNA and/or protein level(s) and/or wherein said cell goes through colinear HOX gene activation.

28. A cervical neuromesodermal progenitor cell obtained by culturing human induced pluripotent stem cells (iPSCs) with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 96 hours, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB4, and not HOXB9 or HOXBIO, at the RNA level, and/or if said cervical NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2, HOXB4 and not HOXB9 and not HOXCIO, at the protein level.

29. A thoracic neuromesodermal progenitor cell obtained by culturing human induced pluripotent stem cells (iPSCs) with the factors Fgf8b, CHIR99021, A 83-01, SIS3, K02288, DAPT and Heparin for a duration of about 120 hours, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1 and HOXB9, and not HOXBIO, at the RNA level, and/or if said thoracic NMP is further directed to differentiate into ventral neural progenitors, said cell co-expresses SOX1, SOX2 and HOXB9, and not HOXB4 and not HOXCIO, at the protein level.

30. A composition for use in generating lumbar level neuromesodermal progenitor cells from the multipotent neuromesodermal progenitor cells according to any of claims 26-29, comprising the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin, GDF11.

31. The composition of claim 30, wherein the factors are present at the following concentrations:

(i) Fgf8b at 50-100 ng/ml, preferably about 75 ng/ml;

(ii) CHIR99021 at 3,5 to 5,5 pm, preferably about 4,5 pm;

(iii) K02288 at 0,5-10 pM, preferably about 2 pM;

(iv) DAPT at 5-20 pM, preferably about 10 pM;

(v) Heparin at 0,25-5 pg/ml, preferably at about 1 pg/ml; and

(vi) GDF11 at 50-500 pg/ml, preferably about 100 pg/ml.

32. A lumbar level neuromesodermal progenitor cell obtained by culturing the multipotent neuromesodermal progenitor cell according to claim 26 or T1 with the factors Fgf8b, CHIR99021, K02288, DAPT, Heparin, GDF11 for a duration of about 48 hours, wherein said cell co-expresses SOX2, TBXT, CDX2, CDX1, and HOXBIO at the RNA level, and/or if said lumbar NMP is further directed to differentiate into ventral neural progenitors, said cell coexpresses SOX1, SOX2, HOXCIO and/or HOXB9, but not HOXB4, at the protein level.

33. A composition for use in generating TBX6 expressing presomitic mesoderm cells from the multipotent neuromesodermal progenitor cells according to claim 26 or T1 , comprising the factors Fgf2, CHIR99021 and BME (Basal Matrix Extract).

34. The composition according to claim 33, wherein the factors are present at the following concentrations:

(i) Fgf2 at 10-100 ng/ml, preferably about 50 ng/ml;

(ii) CHIR99021 at 5 to 20 pm, preferably about 10 pm; and

35. A TBX6 expressing presomitic mesoderm cell obtained by culturing the multipotent neuromesodermal progenitor cell according to claim 26 or T1 with the factors Fgf2, CHIR99021 and BME (Basal Matrix Extract) for a duration of about 48-72 hours, wherein said cell coexpresses TBX6, MSGN1, TBXT, RBP1 and GAD1, at protein and/or RNA level.

36. A human cell derived from the multipotent neuromesodermal progenitor cell of claim 26 or T1 , said cell being of a tissue type chosen from the group comprising post-cranial nervous system tissue, neural crest derivative, skeletal muscle, bone and cartilage and/or of a lineage chosen from the group comprising ectoderm, neural crest and mesoderm.

37. A cell population comprising one or more of the progenitor or mesoderm cells according to any one of claims 26-29, 32 and 35, derived by the method according to any one of claims 1-21.

38. A kit comprising one or more of the compositions according to any one of claims 22-25, 30-31 and 33-34, and a suitable medium, which lacks serum, supports stem cell growth and differentiation, and lacks TGFbeta and bFGF, such as E6 medium, optionally including suitable factors, laminin coating of a plastic dish, and optionally other necessary reagents and tools, and instructions for use.

39. A cellular composition, comprising one or more cervical, thoracic or lumbar level NMP cell(s), wherein the cell(s) is/are present at a purity level of at least about 80%, 85%, 90% or at least 95%.