Method for preparing BAP or BA cells

Abstract

The invention relates to a method for preparing BAP or BA cells, obtained BAP or BA cell populations and their use as a medicament.

Claims

1. A method for preparing Brown Adipocyte Progenitor (BAP) or Brown Adipocyte (BA) cells, said method comprising the following steps: a) Culturing pluripotent cells in a culture medium comprising an activator of the Wnt signaling pathway to obtain induced paraxial mesoderm progenitor (iPAM) cells; b) Culturing said iPAM cells in a myogenic culture medium c) Optionally further culturing cells obtained at the end of step b) in a culture medium with serum or an equivalent thereof, optionally further comprising FGF2 or an equivalent thereof; d) Selecting BAP cells by passaging the cells obtained at the end of step b) or c) and seeding them into culture dish; and e) Optionally culturing selected BAP cells preferably those obtainable at the end of step d) in an adipogenic culture medium comprising serum or an equivalent thereof obatining BA cells.

2. (canceled)

3. The method according to claim 1, wherein step a) is carried out in a culture medium further comprising an inhibitor of the Bone Morphogenetic Pathway (BMP) signaling pathway and optionally DMSO.

4. The method according to claim 1, wherein: a) the Wnt signaling pathway is the canonical Wnt/beta catenin signaling pathway and/or the Wnt/PCP signaling pathway, b) the inhibitor or the BMP signaling pathway is selected from the group consisting of: Noggin, Chordin, Chordin-like 1-3, Follistatin, Follistatin-like 1-5, a member of the Dan family and variants and fragments thereof.

5. The method, according to claim 1, wherein step b) is carried out using a myogenic culture medium that comprises or consists of or essentially consists of a culture medium, serum or an equivalent thereof, an inhibitor of a BMP receptor, an activator of the c-MET receptor and an activator of an IGF or insulin receptor

6. The method according to claim 1, wherein step e) is carried out using an adipogenic culture medium that comprises or essentially consists of a culture medium, an inhibitor of the TGFbeta/Activin/NODAL pathway (preferably SB431542), an activator of the EGF (Epidermal Growth Factor) receptor (preferably EGF), ascorbic acid, and an activator of a corticoid receptor (preferably hydrocortisone).

7. The method according to claim 1, wherein BA cells are characterized by the expression of UCP1

8. The method according to claim 1, wherein BAP cells are characterized by their ability to be converted into BA cells expressing UCP1.

9. A population of BA or BAP cells obtainable by the method of claim 1, wherein the population of BA cells comprises at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of cells expressing UCP1 or wherein the population of BAP cells is characterized by the ability to be converted into a population of BA cells comprising at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% of cells espression UCP1.

10.-12. (canceled)

13. A method for treating a disease or condition linked with BA or BAP cell activity and preferably being a metabolic disease or condition such as obesity-related pathologies, metabolic syndrome, diabetes mellitus, hyperlipidemia, NASH (Non-Alcoholic Steato Hepatitis), Energy balance (intake versus expenditure), comprising administering a population of BA or BAP cells defined in cliam 9.

14. (canceled)

Description

FIGURES AND TABLES

[0209] FIG. 1. Process to derive human brown adipocyte progenitors (hBAP) and generate human brown adipocytes (hBA) from hiPS cells. After generation of induced paraxial mesoderm (iPAM), cells undergo myogenic differentiation. BAP are then derived from these cells and induced to differentiate into brown adipocytes.

[0210] FIG. 2. Experimental scheme to derive human brown adipocyte progenitors from hiPSC. From day 0 to day 22 a sequence of differentiation media allows to induce successively iPAM and myogenic lineages from hiPS cells on matrigel-coated culture plate. From day 12 to day 22, cells are dissociated using tryspin and seeded on uncoated culture plate. After several passages, cell population is enriched in BAP.

[0211] FIG. 3. BAP derivation from hiPS cells. Phase contrast light pictures of cell population during derivation of BAP. Cells acquire homogeneous morphology throughout successive passages (scale bar=1,000 m).

[0212] FIG. 4. Adipogenic potential of BAP. mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days at different passages (P5, P6, P7, P8 and P9). Six adipogenic markers have been analyzed by qPCR.

[0213] FIG. 5. Characterization of brown adipocytes. BAP differentiation has been induced for 17 days at different passages (P5, P6, P7, P8 and P9). UCP1, lipid droplets and nuclei are visualized by immunostaining (scale bar=50 m).

[0214] FIG. 6. Process to derive human brown adipocyte progenitors (hBAP) and generate human brown adipocytes (hBA) from hiPS cells. After generation of induced paraxial mesoderm (iPAM), cells undergo myogenic differentiation. BAP are then derived from these cells and induced to differentiate into brown adipocytes.

[0215] FIG. 7. Experimental scheme to derive human brown adipocyte progenitors from hiPSC. From day 0 to day 22 a sequence of differentiation media allows to induce successively iPAM and myogenic lineages from hiPS cells on matrigel-coated culture plate. From day 12 to day 22, cells are dissociated using tryspin and seeded on uncoated culture plates. After several passages, cell population is enriched in BAP.

[0216] FIG. 8. BAP derivation from hiPS cells. Phase contrast light pictures of cell population during derivation of BAP at day 16. Cells acquire homogeneous morphology throughout successive passages (scale bar=1,000 m).

[0217] FIG. 9. Adipogenic potential of BAP over early passages. mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days at different passages (P1, P2, P3, P4 and P5). Six adipogenic markers have been analyzed by qPCR.

[0218] FIG. 10. Characterization of brown adipocytes over early passages. Differentiation has been induced for 17 days for BAP derived at day 16 at different passages (P1, P2, P3, P4 and P5). UCP1, lipid droplets and nuclei are visualized by immunostaining . (B) Quantification of the cell population expressing UCP1 and presenting lipid droplets.

[0219] FIG. 11. Adipogenic potential of BAP over late passages . mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days at different passages (P5, P6, P7, P8 and P9). Six adipogenic markers have been analyzed by qPCR.

[0220] FIG. 12. Characterization of brown adipocytes over late passages. BAP differentiation has been induced for 17 days at different passages (P5, P6, P7, P8 and P9). (A) UCP1, lipid droplets and nuclei are visualized by immunostaining. (B) Quantification of the cell population expressing UCP1 and presenting lipid droplets.

[0221] FIG. 13. Characterization of brown adipocytes from BAP derived at different endpoints.

[0222] BAP were derived from day 12 to day 22 after a culturing step in medium containing serum, passaged 5 times and have undergone adipocyte differentiation for 17 days. (A) UCP1, lipid droplets and nuclei are visualized by immunostaining. (B) Quantification of the cell population expressing UCP1 and presenting lipid droplets. (C) mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days (Differentiated) and adipogenic markers have been analyzed by qPCR. (D) BAP derived form day 12 or day 16 were induced to differentiate for 17 days, then lipolysis is stimulated with 10 M forskolin.

[0223] FIG. 14. Effect of the culturing step in medium containing serum. (A)BAP were derived from day 20 with (1) or without (2) a culturing step in medium containing serum (i.e. optional step c)) and have undergone adipocyte differentiation for 17 days. UCP1, lipid droplets and nuclei are visualized by immunostaining.

[0224] FIG. 15. Comparison to method 2. hiPS cells were induced toward paraxial mesoderm lineage according to the Method 2 . At day 8, cells are maintained in myogenic medium (a) or two adipogenic media (b and c). mRNA were prepared from day 20 and day 30. BAP and BA are also generated from hiPS cells according to the method 1. mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days (Diff.) as described in method 1. Two adipogenic markers have been analyzed by qPCR.

[0225] FIG. 16. Comparison to method 3.1. After the induction of paraxial mesoderm, the cells are differentiated using the method 3.1. mRNA were prepared from day 20 and day 30. BAP and BA are also generated from hiPS cells according to the method 1. mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days (Diff.) as described in method 1. Two adipogenic markers have been analyzed by qPCR.

[0226] FIG. 17. Comparison to method 3.2. After the induction of paraxial mesoderm, the cells are differentiated using the method 3.2. At day 8, cells are maintained in two different adipogenic media (3.2. a and b). BAP and BA are also generated from hiPS cells according to the method 1. mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days (Diff.) as described in method 1. Two adipogenic markers have been analyzed by qPCR.

[0227] FIG. 18. Comparison to the method 4. BAP were generated from hiPS cells as described in the method 4 or the method 1. (A)BAP differentiation has been induced for 17 days at P5. UCP1, lipid droplets and nuclei are visualized by immunostaining. (B)mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days (Diff) at P5. Two adipogenic markers have been analyzed by qPCR.

[0228] FIG. 19. Comparison to the method 5. (A) Phase contrast light pictures of cell population during differentiation hiPS cells as described in the method 5.). BAP and BA are also generated from hiPS cells according to the method 1. (B) mRNA were prepared from undifferentiated BAP (Und.) or from BAP having undergone differentiation for 17 days (Diff.) as described in method 1. Two adipogenic markers have been analyzed by qPCR. (C) After 17 days of differentiation, UCP1, lipid droplets and nuclei are visualized by immunofluorescence on BA cultures.

[0229] FIG. 20. Summary of the experiments of comparison. hiPS cells are differentiated using the method 2, the method 3.1, 3.2 and the method 4. For each method tested, BAP (Und.) and BA (Diff.) are also generated from hiPS cells according to the method 1. mRNA were prepared from the different conditions and the expression of the brown adipogenic UCP1 has been analyzed by qPCR.

TABLE-US-00002 TABLE1 Sequencesoftheinvention Bank Reference Proteins number SEQID Sequences hRspondin3 NP-116173.2 SEQID MHLRLISWLFIILNFMEYIGSQNASRGRRQ (CAI20141.1) NO:1 RRMHPNVSQGCQGGCATCSDYNGCLSCKPR or LFFALERIGMKQIGVCLSSCPSGYYGTRYP Q9BXY4-1 DINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVSIVHCEV SEWNPWSPCTKKGKTCGFKRGTETRVREII QHPSAKGNLCPPTNETRKCTVQRKKCQKGE RGKKGRERKRKKPNKGESKEAIPDSKSLES SKEIPEQRENKQQQKKRKVQDKQKSVSVSTVH mRspondin3 NP-082627.3 SEQID MHLRLISCFFIILNFMEYIGSQNASRGRRQ NO:2 RRMHPNVSQGCQGGCATCSDYNGCLSCKPR LFFVLERIGMKQIGVCLSSCPSGYYGTRYP DINKCTKCKVDCDTCFNKNFCTKCKSGFYL HLGKCLDSCPEGLEANNHTMECVSIVHCEA SEWSPWSPCMKKGKTCGFKRGTETRVRDIL QHPSAKGNLCPPTSETRTCIVQRKKCSKGE RGKKGRERKRKKLNKEERKETSSSSDSKGL ESSIETPDQQENKERQQQQKRRARDKQQKS VSVSTVH hRspondin2 NP-848660.3 SEQID MQFRLFSFALIILNCMDYSHCQGNRWRRSK or NO:3 RASYVSNPICKGCLSCSKDNGCSRCQQKLF Q6UXX9-1 FFLRREGMRQYGECLHSCPSGYYGHRAPDM NRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLEETMECVEGCEVGHW SEWGTCSRNNRTCGFKWGLETRTRQIVKKP VKDTILCPTIAESRRCKMTMRHCPGGKRTP KAKEKRNKKKKRKLIERAQEQHSVFLATDR ANQ mRspondin2 NP-766403.1 SEQID MRFCLFSFALIILNCMDYSQCQGNRWRRNK NO:4 RASYVSNPICKGCLSCSKDNGCSRCQQKLF FFLRREGMRQYGECLHSCPSGYYGHRAPDM NRCARCRIENCDSCFSKDFCTKCKVGFYLH RGRCFDECPDGFAPLDETMECVEGCEVGHW SEWGTCSRNNRTCGFKWGLETRTRQIVKKP AKDTIPCPTIAESRRCKMAMRHCPGGKRTP KAKEKRNKKKRRKLIERAQEQHSVFLATDR VNQ hRspondin3 CAI20142.1 SEQID MHLRLISWLFIILNFMEYIGSQNASRGRRQ isoform2 or NO:5 RRMHPNVSQGCQGGCATCSDYNGCLSCKPR Q9BXY4-2 LFFALERIGMKQIGVCLSSCPSGYYGTRYP DINKCTKCKADCDTCFNKNFCTKCKSGFYL HLGKCLDNCPEGLEANNHTMECVSIVHCEV SEWNPWSPCTKKGKTCGFKRGTETRVREII QHPSAKGNLCPPTNETRKCTVQRKKCQKGE RGKKGRERKRKKPNKGESKEAIPDSKSLES SKEIPEQRENKQQQKKRKVQDKQKSGIEVT LAEGLTSVSQRTQPTPCRRRYL hRspondin2 Q6UXX9.2 SEQID MRQYGECLHSCPSGYYGHRAPDMNRCARCR isoform2 NO:6 IENCDSCFSKDFCTKCKVGFYLHRGRCFDE CPDGFAPLEETMECVEGCEVGHWSEWGTCS RNNRTCGFKWGLETRTRQIVKKPVKDTILC PTIAESRRCKMTMRHCPGGKRTPKAKEKRN KKKKRKLIERAQEQHSVFLATDRANQ hRspondin2 Q6UXX9-3 SEQID FRLFSFALIILNCMDYSHCQGNRWRRSK isoform3 NO:7 RGCRIENCDSCFSKDFCTKCKVGFYLHRGR CFDECPDGFAPLEETMECVGCEVGHWSEWG TCSRNNRTCGFKWGLETRTRQIVKKPVKDT ILCPTIAESRRCKMTMRHCPGGKRTPKAKE KRNKKKKRKLIERAQEQHSVFLATDRANQ mMsgn1 NM.019544.1 SEQID ATGGACAACCTGGGTGAGACCTTCCTCAGC NO:8 CTGGAGGATGGCCTGGACTCTTCTGACACC GCTGGTCTGCTGGCCTCCTGGGACTGGAAA AGCAGAGCCAGGCCCTTGGAGCTGGTCCAG GAGTCCCCCACTCAAAGCCTCTCCCCAGCT CCTTCTCTGGAGTCCTACTCTGAGGTCGCA CTGCCCTGCGGGCACAGTGGGGCCAGCACA GGAGGCAGCGATGGCTACGGCAGTCACGAG GCTGCCGGCTTAGTCGAGCTGGATTACAGC ATGTTGGCTTTTCAACCTCCCTATCTACAC ACTGCTGGTGGCCTCAAAGGCCAGAAAGGC AGCAAAGTCAAGATGTCTGTCCAGCGGAGA CGGAAGGCCAGCGAGAGAGAGAAACTCAGG ATGCGGACCTTAGCCGATGCCCTCCACACG CTCCGGAATTACCTGCCGCCTGTCTACAGC CAGAGAGGCCAACCGCTCACCAAGATCCAG ACACTCAAGTACACCATCAAGTACATCGGG GAACTCACAGACCTCCTCAACAGCAGCGGG AGAGAGCCCAGGCCACAGAGTGTGTGA hMsgn1 NM001105569.1 SEQID ATGGACAACCTGCGCGAGACTTTCCTCAGC NO:9 CTCGAGGATGGCTTGGGCTCCTCTGACAGC CCTGGCCTGCTGTCTTCCTGGGACTGGAAG GACAGGGCAGGGCCCTTTGAGCTGAATCAG GCCTCCCCCTCTCAGAGCCTTTCCCCGGCT CCATCGCTGGAATCCTATTCTTCTTCTCCC TGTCCAGCTGTGGCTGGGCTGCCCTGTGAG CACGGCGGGGCCAGCAGTGGGGGCAGCGAA GGCTGCAGTGTCGGTGGGGCCAGTGGCCTG GTAGAGGTGGACTACAATATGTTAGCTTTC CAGCCCACCCACCTTCAGGGCGGTGGTGGC CCCAAGGCCCAGAAGGGCACCAAAGTCAGG ATGTCTGTCCAGCGGAGGCGGAAAGCCAGC GAGAGGGAGAAGCTCAGGATGAGGACCTTG GCAGATGCCCTGCACACCCTCCGGAATTAC CTGCCACCTGTCTACAGCCAGAGAGGCCAG CCTCTCACCAAGATCCAGACACTCAAGTAC ACCATCAAGTACATCGGGGAACTCACAGAC CTCCTTAACCGCGGCAGAGAGCCCAGAGCC CAGAGCGCGTGA mNoggin NP_032737 SEQID MERCPSLGVTLYALVVVLGLRAAPAGGQHY NO:10 LHIRPAPSDNLPLVDLIEHPDPIFDPKEKD LNETLLRSLLGGHYDPGFMATSPPEDRPGG GGGPAGGAEDLAELDQLLRQRPSGAMPSEI KGLEFSEGLAQGKKQRLSKKLRRKLQMWLW SQTFCPVLYAWNDLGSRFWPRYVKVGSCFS KRSCSVPEGMVCKPSKSVHLTVLRWRCQRR GGQRCGWIPIQYPIISECKCSC hNoggin EAW94528 SEQID MERCPSLGVTLYALVVVLGLRATPAGGQHY NO:11 LHIRPAPSDNLPLVDLIEHPDPIFDPKEKD LNETLLRSLLGGHYDPGFMATSPPEDRPGG GGGAAGGAEDLAELDQLLRQRPSGAMPSEI KGLEFSEGLAQGKKQRLSKKLRRKLQMWLW SQTFCPVLYAWNDLGSRFWPRYVKVGSCFS KRSCSVPEGMVCKPSKSVHLTVLRWRCQRR GGQRCGWIPIQYPIISECKCSC hEGF Q6QBS2 SEQID NSDSECPLSHDGYCLHDGVCMYIEALDKYA (fragment) NO:12 CNCVVGYIGERCQYRDLKWWELR hUCP1 P25874 SEQID MGGLTASDVHPTLGVQLFSAGIAACLADVI NO:13 TFPLDTAKVRLQVQGECPTSSVIRYKGVLG TITAVVKTEGRMKLYSGLPAGLQRQISSAS LRIGLYDTVQEFLTAGKETAPSLGSKILAG LTTGGVAVFIGQPTEVVKVRLQAQSHLHGI KPRYTGTYNAYRIIATTEGLTGLWKGTTPN LMRSVIINCTELVTYDLMKEAFVKNNILAD DVPCHLVSALIAGFCATAMSSPVDVVKTRF INSPPGQYKSVPNCAMKVFTNEGPTAFFKG LVPSFLRLGSWNVIMFVCFEQLKRELSKSR QTMDCAT

EXAMPLES

Example 1

[0230] Methods

[0231] The full names and manufacturer of all compounds used herein is detailed in the Appendix

[0232] Primary Differentiation and Derivation of Human Brown Adipocyte Progenitors (hBAP) from hiPS Cells:

[0233] Undifferentiated hiPS cells are dissociated to single cells using trypsin and they are seeded at a density of 5.5.10.sup.4 cells/cm.sup.2 on matrigel-coated dishes in mTESR-1 medium supplemented with Rock-1 inhibitor (10 M). One day after, medium is changed to fresh mTESR-1 without Rock-1 inhibitor. When the cells form small aggregates, determined at the day 0 of differentiation, they are changed to a sequence of differentiation media.

[0234] At day 0, medium is changed to medium composed of DMEM supplemented with ITS (1%), CHIR99021 (3 M) and LDN-193189 (500 nM). The medium is refreshed daily until day 6.

[0235] At day 6, medium is changed to medium composed of DMEM supplemented with KSR (15%), LDN-193189 (500 nM), HGF (10 ng/ml) and IGF-1 (2 ng/ml). The medium is changed daily until day 8.

[0236] At day 8, medium is changed to medium composed of DMEM supplemented with KSR (15%) and IGF-1 (2 ng/ml). The medium is changed daily until day 12.

[0237] From day 12 to day 22, medium is changed to medium composed of DMEM supplemented with KSR (15%), HGF (10 ng/ml) and IGF-1 (2 ng/ml). The medium is changed every 2 days (FIGS. 1 and 2).

[0238] At the selected day to derive hBAP (between day 12 and day 22), medium is changed to a medium composed of DMEM supplemented with FBS (10%). The medium is refreshed every 2 days. After a week, the cells are passaged using trypsin and they're seeded on tissue culture grade plate. This is fixed passage number 0. Cells are maintained in the previous medium supplemented with FGF-2 (5 ng/ml).

[0239] When the cells reach confluence, they are passaged and seeded at a density of 5.10.sup.4 cells/cm.sup.2.

[0240] Passages are repeated (usually 4 to 9 times) until a cell population with homogeneous morphology is obtained (FIG. 2).

[0241] Differentiation of Human Brown Adipocyte Progenitors (hBAP):

[0242] The hBAP are plated at a high density (i.e. 5.10.sup.5 cells/cm.sup.2) and maintained in derivation medium. When the cells reach confluence, determined at the day 0 of differentiation, the medium is changed to differentiation medium composed of DMEM with low glucose supplemented with FBS (10%), rosiglitazone (1 M), insulin (10 pg/ml), T3 (0.2 nM), SB431542 (5 M), ascorbic acid (25.5 pg/ml), EGF (10 ng/ml), hydrocortisone (4 g/ml), dexamethasone (1 M) and IBMX (500 M). Dexamethasone and IBMX are discarded after day 3 (FIG. 3). The differentiation medium is then changed twice a week.

[0243] Results

[0244] Experimental Results

[0245] Workflow to get BA.

[0246] This protocol is made of sequential differentiation sessions to obtain BA from the initial batch of hiPS cells (FIG. 1). After induction of the iPAM cells, a putative subpopulation of BAP is enriched by sequential replating from passage 5 up to passage 9. BAP were picked at several end points ranging from day 12 to day 22 before starting the second part of the protocol. The enriched BAP population is then differentiated for 2 weeks in a culture media containing key adipogenic factors (FIGS. 1 and 2).

[0247] BAP Can Be Enriched Over Passages

[0248] After 12 to 22 days of differentiation (day 16 showed), we passaged the cells and plated them in a serum+FGF2 containing culture media to enrich the BAP population. Passaging the cells from P0 to P4, enabled to eliminate aggregates and contaminating cells (FIG. 3). Cell reached an homogeneous and 100% confluent population of fibroblast-like BAP as illustrated by phase-contrast microscopy. After passage 4, the cellular shape of the BAP did not change anymore until P9 (data not shown), allowing the differentiation process indifferently from P5 to P9.

[0249] BAP-Derived BA Express Relevant Markers of Differentiated Brown Adipocytes

[0250] BAP or the BAP-derived BA (from P5 to P9) were differentiated in DMEM containing 10% Fcetal bovine serum+rosiglitazone (1 M) +insulin (10 g/ml) +T3 hormone (200 M) +SB431542 (5 m)+Ascorbic acid (25.5 g/ml)+EGF (10 ng/ml)+hydrocortisone (4 g/ml)+dexamethasone (1 M)+IBMX (500 M). After 3 days, medium was replaced by fresh one lacking the two last compounds. The levels of expression of transcripts by RT-qPCR were assessed on BA and BAP cells (FIG. 4). The pan-adipocyte markers FABP4 (fatty acid binding protein 4), PLIN1 (perilipin 1), PPAR (peroxisome proliferator-activated receptor gamma) increased into BA cells compared to BAP, reflecting the commitment to adipocyte pathway. On the other hand, brown adipocyte markers: UCP1 (uncoupling protein 1), CIDE-A (cell death-inducing DFFA-like Effector A) and PGC1- (peroxisome proliferator-activated receptor gamma coactivator alpha) were more expressed in BA than in BAP. This effect was observed from passages 5 to 9 (FIG. 4).

[0251] After 17 days of maturation, BA cultures were next characterized by immunofluorescence with an antibody against UCP1 and with a neutral lipid probe identifying the intracellular lipid droplets. As shown, the BA harboured a strong and homogeneous staining for UCP1 (FIG. 6). The quantification of the number of cells expressing UCP1+ lipid droplets showed a range of 39,5% (for P9 cells) to 79% (for P6 cells) positive cells.

Example 1.a

[0252] Methods

[0253] The full names and manufacturers of all compounds used herein are detailed in the Appendix

[0254] Primary Differentiation and Derivation of Human Brown Adipocyte Progenitors (hBAP) From hiPS Cells (FIGS. 6 and 7):

[0255] Step a) Undifferentiated hiPS cells are dissociated to single cells using trypsin and seeded at a density ranging from 3.10.sup.4 to 9.10.sup.4 cells/cm.sup.2. This example was carried out with 5.5.10.sup.4 cells/cm.sup.2 on matrigel-coated dishes in mTESR-1 medium supplemented with Rock-1 inhibitor (10 M). One day after, medium is changed to fresh mTESR-1 without Rock-1 inhibitor. When the cells form small aggregates, determined at the day 0 of differentiation, they are changed to a sequence of primary differentiation media.

[0256] At day 0, medium is changed to medium composed of DMEM supplemented with ITS (1%), CHIR99021 (3 M) and LDN-193189 (500 nM), supplemented or not with FGF-2 (20 ng/ml) from day 3. The medium is refreshed daily until day 6. This corresponds to the induction of the paraxial mesoderm lineage.

[0257] Step b) At day 6, medium is changed to medium composed of DMEM supplemented with KSR (15%), LDN-193189 (500 nM), HGF (10 ng/ml) and IGF-1 (2 ng/ml), supplemented or not with FGF-2 (20 ng/ml). The medium is changed daily until day 8.

[0258] At day 8, medium is changed to medium composed of DMEM supplemented with KSR (15%) and IGF-1 (2 ng/ml). The medium is changed daily until day 12.

[0259] Starting at day 12 and until the selected timepoint is reached (at the latest on day 22), medium is changed to a medium composed of DMEM supplemented with KSR (15%), HGF (10 ng/ml) and IGF-1 (2 ng/ml). The medium is changed every 2 days. This step corresponds to the initiation of the derivation of cells into hBAP.

[0260] Step c) On the day selected to derive hBAP (between day 12 and day 22 of the primary differentiation or after 6 to 15 days after initiation of step c)), medium is changed a medium composed of DMEM supplemented or not with FBS (10%), with or without FGF-2 (5 ng/ml).This example was carried out with DMEM supplemented with 10% FBS without FGF-2. The medium is refreshed every 2 days.

[0261] Step d) After 7 days, the cells are passaged using trypsin and seeded on tissue culture grade plate. This is passage number 0 (P0). Cells are maintained in a medium composed of DMEM and FBS (10%) supplemented with FGF-2 (5 ng/ml).

[0262] When the cells reach confluence, they are passaged and seeded at a density ranging from 3.10.sup.4 to 9.10.sup.4 cells/cm.sup.2 on tissue culture grade plate. This experiment has been carried out using 5.10.sup.4 cells/cm.sup.2.

[0263] Passages are repeated (usually 4 to 9 times) until a cell population with homogeneous morphology as determined by a person skilled in the art is obtained (FIG. 8), thus generating a population of hBAP.

[0264] Secondary Differentiation of Human Brown Adipocyte Progenitors (hBAP) Into Human Brown Adipocytes (hBA):

[0265] Step e) The hBAP are plated at a density ranging from 3.10.sup.4 to 9.10.sup.4 cells/cm.sup.2) and maintained in derivation medium i.e. a medium composed of DMEM supplemented with FBS (10%) and FGF-2 (5 ng/ml). this experiment has been carried out using 5.10.sup.4 cells/cm.sup.2. When the cells reach confluence, determined at the day 0 of secondary differentiation, the medium is changed to differentiation medium composed of DMEM with low glucose (1 g/l) supplemented with FBS (10%), rosiglitazone (1 M), insulin (10 g/ml), T3 (0.2 nM), SB431542 (5 M), ascorbic acid (25.5 g/ml), EGF (10 ng/ml), hydrocortisone (4 g/ml), dexamethasone (1 M) and IBMX (500 M). Dexamethasone and IBMX are discarded after day 3 The differentiation medium is then changed twice a week during 8 to 30 days (usually about 2 weeks).

[0266] Quantitative RT-PCR:

[0267] Total RNA was extracted from cell cultures using the nucleo spin RNA plus kit (Macherey-Nagel). RT-PCR was performed on 500 ng total RNA using iScript gDNA clear cDNA synthesis Kit (Biorad), appropriate primers and run on a LightCycler 48011 (Roche). TBP was used as the internal control.

[0268] Immunocytochemistry:

[0269] Cell cultures were fixed with PFA 4%. Cells were incubated for 30 minutes with a blocking solution composed of 5%NGS, 1% fetal bovine serum and 0.2%Triton in Phosphate Buffered Saline (PBS). Primary antibodies incubation was performed during 1 h30 at room temperature and antibodies working dilutions were as follow: anti-UCP1 (R&D) was 1:250, anti-Desmin (Santa Cruz) was 1:800. After PBS washing, cells were incubated with AlexaFluor488-conjugated secondary antibodies (Invitrogen) at 1:1000 for 30 minutes, and counterstained with Dapi. HCS lipidtox neutral lipid staining was done according to standard protocol.

[0270] Lipolysis:

[0271] The BAP were differentiated for 17 days. Then the cells were maintained for 24 hours in DMEM 1g/Iglucose supplemented with BSA 0,2%. Lipolysis was stimulated with forskolin (10 M) for 24 hours.

[0272] Experimental Results

[0273] Workflow to Obtain BA as Described in the Present Invention.

[0274] This protocol is made of sequential differentiation sessions to obtain BA from the initial batch of hiPS cells (FIG. 6). After induction of the iPAM cells (step a)) and subsequent exposure to a myogenic medium (step b)), a putative subpopulation of BAP is derived at different timepoints ranging from day 12 to day 22 to be enriched by sequential replating (usually 4 to 9 passages) (steps c) and d)).. This enriched BAP population is then differentiated for about 2 weeks in a culture media containing key adipogenic factors (step e)) (FIGS. 6 and 7).

[0275] Phenotypic Characterization of BAP

[0276] After 12 to 22 days of differentiation (day 16 showed) according to the method described in this example, cells were plated in a culture media containing FBS+FGF2 to enrich the BAP population. Passaging the cells several times (usually 4) allowed the elimination of aggregates and contaminating cells (FIG. 8). The cell population reached a 100% confluent state of fibroblast-like BAP, homogeneous to a person skilled in the art, as illustrated by phase-contrast microscopy. Additional passages once homogenization is obtained did not change the cellular shape of the BAP (data not shown).

[0277] Characterization of BAP-Derived BA Over Passages

[0278] BAP (from P1 to P9) were differentiated into BA in DMEM containing FBS (10%)+rosiglitazone (1 M)+insulin (10 g/ml)+T3 hormone (200 M)+SB431542 (5 m)+Ascorbic acid (25.5 g/ml)+EGF (10 ng/ml)+hydrocortisone (4 g/ml)+dexamethasone (1 M)+IBMX (500 M). Dexamethasone and IBMX were discarded after day 3. The levels of expression of certain transcripts typical of the adipocyte lineage were assessed by RT-qPCR both on BAP and BA (FIGS. 9 and 11). The pan-adipocyte markers FABP4 (fatty acid binding protein 4), PLIN1 (perilipin 1), PPAR (peroxisome proliferator-activated receptor gamma) greatly increased in BA cells compared to BAP (respectively up to 1.10.sup.5; 3,5.10.sup.4 and 8 times higher expressed), reflecting the commitment to adipocyte pathway. In addition, brown adipocyte markers: UCP1 (uncoupling protein 1), CIDE-A (cell death-inducing DFFA-like Effector A) and PGC1-a (peroxisome proliferator-activated receptor gamma coactivator alpha) were significantly more expressed in BA than in BAP (respectively up to 1,2.10.sup.5, 300 and 150 times higher expressed).

[0279] BA cultures at day 17 of differentiation were next characterized by immunofluorescence with an antibody against UCP1 and with a neutral lipid probe identifying the intracellular lipid droplets. The BA harboured a strong and homogeneous staining for UCP1 (FIGS. 10 and 12). The quantification of the number of cells expressing UCP1+ lipid droplets showed a range of 40% (for P2 cells) to 79% (for P6 cells) positive cells for this experiment. The yield of differentiation obtained using the present invention reached 85% of cells expressing UCP1 and presenting lipid droplets (FIG. 19).

[0280] Importantly, to complete the characterization, BAP derived at day 12 or 16 and after 5 passages were differentiated for 17 days and then treated with forskolin (10 M). After 24 hours, we observed an increase of the free glycerol released (by about 60%) for the treated cells compared to untreated cells (FIG. 13.D). The BA were able able to respond to forskolin to increase the lipolysis, thus showing the functionality of these cells.

[0281] These results show without doubt that the method of the invention generates not only adipocytes, but specifically brown adipose progenitors and adipocytes which are functional, with very high yield (up to 85% purity) that is unprecedented (see Example 2), allowing to obtain a population of BA with a high purity amenable to industrial applications.

[0282] Time Interval to Derive BAP

[0283] As described above, before the enrichment of BAP with the consecutive passages, the cells are cultivated in myogenic medium from day 6 to day 22 and then maintained in medium containing serum.

[0284] The BAP derived at different time points were differentiated (as described in example 1) for 17 days (data shown for passage 5). The BA cultures all expressed strongly the UCP1 protein with numerous lipid droplets (FIG. 13, A). The quantification of the number of cells expressing UCP1 and presenting lipid droplets showed a range of 43% (for BAP derived at day 14 to 69% (for BAP derived at day 12). The expression of UCP1 is confirmed by qPCR analysis (FIG. 13, B). Both FABP4 and UCP1 were, as expected, more expressed in BA than in BAP. These data show that BAP can be derived at any timepoint from day 12 to day 22 and lead to the generation of BA with a satisfying efficiency. Furthermore, after stimulation with forskolin, we observed an increase of the lipolysis for BA obtained after differentiation of BAP derived at day 12 and 16, confirming previous results (FIG. 13.D).

[0285] In FIG. 14, the method of the invention was performed without step c) in order to assess the importance of this step. The BAP were derived at day 20 with (1) or without (2) a culturing step in medium containing serum. After differentiation, the rate of UCP1-positive cells is 60% for the BAP derived with this additional step and 23% for those derived without it. These results show that while step c) significantly increases the yield of BA generation, its absence does not prevent the obtention of a BA population, and is therefore optional.

[0286] These results show that after induction of the iPAM cells, BAP and BA can be generated with high yield whatever the timepoint selected for BAP derivation, as long as this timepoint is comprised between 12 and 22 days of primary differentiation, followed by 2 to 9 serial passages. Among these possibilities, the preferred conditions are to derive BAP at day 16 and passage them 5 times before differentiation.

Example 2

[0287] In this example, the method of the invention was compared to other methods identified in the prior art as claiming to generate BAP and/or BA, in order to ascertain the superiority of the method described in the present application. For each method (2 to 5), BAP and BA were also generated in parallel using the method of the invention as described below (method 1), using the same batch of undifferentiated hiPS cells at the same time.

Methods

[0288] The full names and manufacturers of all compounds used herein are detailed in the Appendix

[0289] Method 1: Primary Differentiation and Derivation of hBAP From hiPS Cells and Differentiation of hBAP Into hBA:

[0290] Step a) Undifferentiated hiPS cells are dissociated to single cells using trypsin and seeded at a density of 5.5.10.sup.4 cells/cm.sup.2 on matrigel-coated dishes in mTESR-1 medium supplemented with Rock-1 inhibitor (10 M). One day after, medium is changed to fresh mTESR-1 without Rock-1 inhibitor. When the cells form small aggregates, determined at the day 0 of primary differentiation, they are changed to a sequence of differentiation media.

[0291] At day 0, medium is changed to medium composed of DMEM supplemented with ITS (1%), CHIR99021 (3 M) and LDN-193189 (500 nM). The medium is refreshed daily until day 6.

[0292] Step b) At day 6, medium is changed to medium composed of DMEM supplemented with KSR (15%), LDN-193189 (500 nM), HGF (10 ng/ml) and IGF-1 (2 ng/ml). The medium is changed daily until day 8.

[0293] At day 8, medium is changed to medium composed of DMEM supplemented with KSR (15%) and IGF-1 (2 ng/ml). The medium is changed daily until day 12.

[0294] From day 12 to day 16, medium is changed to medium composed of DMEM supplemented with KSR (15%), HGF (10 ng/ml) and IGF-1 (2 ng/ml). The medium is changed every 2 days (FIGS. 6 and 7).

[0295] Step c) At day 16, medium is changed to a medium composed of DMEM supplemented with FBS (10%). The medium is refreshed every 2 days.

[0296] Step d) After 7 days, the cells are passaged using trypsin and seeded on tissue culture grade plate. (passage number 0). Cells are maintained in the previous medium composed of DMEM supplemented with FBS (10%) and FGF-2 (5 ng/ml).

[0297] When the cells reach confluence, they are passaged and seeded at a density of 5.10.sup.4 cells/cm.sup.2.

[0298] Passages are repeated 5 times until a cell population with homogeneous morphology is obtained .

[0299] Step e) The hBAP are plated at a density of 5.10.sup.4 cells/cm.sup.2 and maintained in derivation medium i.e. DMEM supplemented with FBS (10%) and FGF-2 (5 nh/ml). When the cells reach confluence, determined at the day 0 of secondary differentiation, the medium is changed to differentiation medium composed of DMEM with low glucose (1 g/l) supplemented with FBS (10%), rosiglitazone (1 M), insulin (10 g/ml), T3 (0.2 nM), SB431542 (5 M), ascorbic acid (25.5 g/ml), EGF (10 ng/ml), hydrocortisone (4 g/ml), dexamethasone (1 M) and IBMX (500 M). Dexamethasone and IBMX are discarded after day 3. The differentiation medium is then changed twice a week until day 17 of the secondary differentiation.

[0300] Method 2: Protocol Described in WO2013/030243

[0301] WO2013/030243 claims a method for preparing populations comprising adipocytes by culturing a population of iPAM cells under appropriate conditions for their differentiation into adipocytes, i.e. in the presence of an efficient amount of at least one or more compounds known to induce adipocyte differentiation. In order to determine whether BAP and BA comparable to method 1 can be obtained using the method of WO2013/030243, iPAM cells were generated and subsequently exposed to either a myogenic culture medium (2.a.) or an adipogenic medium (2.b. and 2.c.), adipogenic media being considered potential appropriate conditions for the generation of adipocytes.

[0302] Generation of iPAM Cells

[0303] Undifferentiated hiPS cells are dissociated to single cells using trypsin and seeded on matrigel-coated dishes in mTESR-1 medium supplemented with Rock-1 inhibitor (10 M). One day after, medium is changed to fresh mTESR-1 without Rock-1 inhibitor. When the cells form small aggregates, determined at the day 0 of primary differentiation, they are changed to a sequence of differentiation media.

[0304] At day 0, medium is changed to medium composed of DMEM supplemented with ITS (1%), CHIR99021 (3 M) and LDN-193189 (500 nM).

[0305] After 3 days, the previous medium is supplemented with FGF-2 (20ng/ml). The medium is refreshed daily until day 6.

[0306] Method 2.a: Generation of iPAM Cells and Culturing Step in Myogenic Medium:

[0307] At day 6, medium is changed to medium composed of DMEM supplemented with KSR (15%), LDN-193189 (500 nM), HGF (10 ng/ml), FGF-2 (20 ng/ml) and IGF-1 (2 ng/ml). The medium is changed daily until day 8.

[0308] At day 8, the medium is changed to medium composed of DMEM supplemented with KSR (15%) and IGF-1 (2 ng/ml).

[0309] At day 12, the medium is changed to medium composed of DMEM supplemented with KSR (15%), HGF (10ng/ml) and IGF-1 (2 ng/ml). Medium is refreshed every 2-3 days.

[0310] Method 2. b and c: Generation of iPAM Cells and Culturing Step in Adipogenic Medium

[0311] iPAM cells are generated as described above.

[0312] At day 6, medium is changed to a medium composed of DMEM supplemented with KSR (15%), LDN-193189 (500 nM), HGF (10 ng/ml), FGF-2 (20 ng/ml) and IGF-1 (2 ng/ml). The medium is changed daily until day 8. At day 8, medium is then changed to one of two adipocyte differentiation media:

[0313] b. An adipocyte differentiation medium composed of DMEM-based medium containing 15% KSR and supplemented with dexamethasone (1 M), IBMX (500 M), insulin (10 g/ml), T3 (0,2 nM) and rosiglitazone (1 M). This medium would be considered a standard adipogenic medium by a person skilled in the art.

[0314] c. The adipocyte differentiation medium described in step e) of the present application. The medium is composed of DMEM-based medium containing 15% KSR, dexamethasone (1 M), IBMX (500 M), insulin (10 g/ml), T3 (0,2 nM), SB431542 (5 M), ascorbic acid (25,5 g/ml), EGF (10 ng/ml) and hydrocortisone (4 g/ml).

[0315] At day 11, dexamethasone and IBMX are removed for both adipogenic media. The medium is refreshed every 2-3 days.

[0316] Method 3: Protocol Described in WO17223457

[0317] WO17223457 claims an in vitro method of generating induced Brown Adipose Tissue (iBAT) cells that express UCP1 by providing a population of iPAM cells followed by culturing this population under 2 different sets of conditions: HIFL or PRA-Adipomix. In order to determine whether BAP and BA comparable to method 1 can be obtained using method 3, iPAM cells were generated and subsequently exposed each of the methods described in WO17223457.

[0318] Method 3.1. Method HIFL:

[0319] Day 0 to day 6: see previously described in Method 2, Generation of iPAM cells

[0320] At day 6, medium is changed to a medium composed of DMEM supplemented with KSR (15%), LDN-193189 (100 nM), HGF (10 ng/ml), FGF-2 (20 ng/ml) and IGF-1 (2 ng/ml). The medium is changed daily until day 8.

[0321] At day 8, the medium is only supplemented with HGF (10 ng/ml) and IGF-I (2 ng/ml). The medium is changed every 2-3 days.

[0322] Method 3.2. Method PRA-Adipomix:

[0323] Day 0 to day 6: see previously described in Method 2, Generation of iPAM cells

[0324] At day 6, medium is changed to DMEM-based medium containing PD173074 (250 nM and retinoic acid (100 nM).

[0325] At day 8, cultures are changed to two adipocyte differentiation media:

[0326] a. The Adipomix medium described into WO17223457. The medium is composed of DMEM based medium containing 15% KSR, 1X insulin-transferrin-selenium (ITS), 500 M IBMX, 125 nM indomethacin, 1 nM T3, 5 M dexamethasone and 1 M rosiglitazone. The medium is refreshed every 2-3 days.

[0327] b. The adipocyte differentiation medium described in the present application. The medium is composed of DMEM based medium containing 15% KSR, dexamethasone (1 M), IBMX (500 M), insulin (10 g/ml), T3 (0,2 nM), rosiglitazone (1 M), SB431542 (5 M), ascorbic acid (25,5 g/ml), EGF (10 ng/ml) and hydrocortisone (4 g/ml). At day 11, dexamethasone and IBMX are removed. The medium is refreshed every 2-3 days.

[0328] Method 4: Protocol Described in Hafner et al., 2016

[0329] Hafner et al. discloses a method to generate BAP and BA cells from hiPS cells by forming embryoid bodies (EBs), culturing them in a medium containing DMEM, serum and FGF-2, followed by serial passaging and culture in the adipogenic medium of step e). Methods 1 and 4 share common steps to derive the BAP and differentiate them into BA, but the differ by the first steps of differentiation of the pluripotent cells with the formation of embryoid bodies for method 4 or the generation of iPAM cells followed by exposure to a myogenic medium for method 1.

[0330] In order to determine whether BAP and BA can be obtained using method 4 with a yield comparable to that of method 1, both methods were conducted in parallel.

[0331] EBs were formed by floating culture in DMEM/F12 medium supplemented with 20% Knock-out Serum Replacement. Ten days after EBs formation, EBs are plated on gelatin-coated culture plates and maintained in DMEM/F12 medium supplemented with 20% KSR for 8 days. At day 18, medium is changed to a medium composed of DMEM supplemented with 10% FBS. After a week, the cells are passaged and seeded on tissue culture grade plates. Passages are repeated until a cell population with homogeneous morphology is obtained, after passage 4 or 5.

[0332] The cells are plated at a density of 5.10.sup.4 cells/cm.sup.2 and maintained in derivation medium i.e. DMEM supplemented with FBS (10%) and FGF-2 (5 nh/ml). When the cells reach confluence, determined at the day 0 of differentiation, the medium is changed to differentiation medium composed of DMEM with low glucose (1 g/l) supplemented with FBS (10%), rosiglitazone (1 M), insulin (10 g/ml), T3 (0.2 nM), SB431542 (5 M), ascorbic acid (25.5 g/ml), EGF (10 ng/ml), hydrocortisone (4 g/ml), dexamethasone (1 M) and IBMX (500 M). Dexamethasone and IBMX are discarded after day 3. The differentiation medium is then changed twice a week until day 17 of the differentiation.

[0333] Method 5: Protocol Described in WO2012/147853

[0334] WO2012/147853 claims a method for the high-efficiency (>90%) production of brown adipocytes from hiPS cells with a 2 step-process: first, cells aggregates are produced by floating culture from pluripotent stem cells in the presence of a hematopoietic cytokine in a serum-free environment. Then, BA are generated by cell adhesion of the cell aggregates in the presence of a hematopoietic cytokine. Methods 1 and 5 were conducted in parallel in order to compare the yield of differentiation of BA obtainable by each method.

[0335] According to WO2012/147853, the differentiation of hiPS cells is initiated by the formation of embryoid bodies (EBs) by floating culture in IMDM/F12 medium (containing 5 mg/mL BSA, 1% by volume synthetic lipid solution, 1% by volume of 100 ITS, 450 mM MTG, 2 mM L-glutamine, 5% by volume of PFHII, 50 mg/mL of ascorbic acid, 20 ng/mL of BMP4, 5 ng/mL of VEGF, 20 ng/mL of SCF, 2.5 ng/mL of Flt3L, 2.5 ng/mL of IL6, and 5 ng/mL of IGF2). Medium is changed every 3 days.

[0336] After 8 days, EBs are plated on gelatin-coated culture plates in IMDM/F12 medium (containing 5 mg/mL BSA, 1% by volume of a synthetic lipid solution, 1% by volume of 100 ITS, 450 mM MTG, 2 mM L-glutamine, 5% by volume of PFHII, 50 mg/mL of ascorbic acid, 10 ng/mL of BMP7, 5 ng/mL of VEGF, 20 ng/mL of SCF, 2.5 ng/mL of Flt3L, 2.5 ng/mL of IL6, and 5 ng/mL of IGF2) for one week. Medium is changed every 3 days.

[0337] Quantitative RT-PCR:

[0338] Total RNA was extracted from cell cultures using the nucleo spin RNA plus kit (Macherey-Nagel). RT-PCR was performed on 500 ng total RNA using iScript gDNA clear cDNA synthesis Kit (Biorad), appropriate primers and run on a LightCycler 48011 (Roche). TBP was used as the internal control.

[0339] Immunocytochemistry:

[0340] Cell cultures were fixed with PFA 4%. Cells were incubated for 30 minutes with a blocking solution composed of 5%NGS, 1% fetal bovine serum and 0.2%Triton in Phosphate Buffered Saline (PBS). Primary antibodies incubation was performed during 1h30 at room temperature and antibodies working dilutions were as follow: anti-UCP1 (R&D) was 1:250, anti-Desmin (Santa Cruz) was 1:800. After PBS washing, cells were incubated with AlexaFluor488-conjugated secondary antibodies (Invitrogen) at 1:1000 for 30 minutes, and counterstained with Dapi. HCS lipidtox neutral lipid staining was done according to standard protocol.

[0341] Experimental Results

[0342] Comparison of the Method 1 to the Method 2:

[0343] The ability of iPAMs cells to generate brown adipocytes (FIG. 15) was evaluated, with and without applying the culturing steps described in the method of the present invention. Starting from a single batch of hiPS cells at the same time, iPAM cells were generated. Then, the cells were maintained in myogenic medium (method 2.a),cultivated in adipogenic medium (methods 2.b and c), or differentiated according to method 1.

[0344] Expression of adipocyte markers analysed by qPCR: For this comparison, the expression of the genes at day 20, day 30 and for the BAP and BA of the method 1) were normalized against a sample taken at day 8 (i.e. before any commitment to adipocyte lineage). After 20 or 30 days of differentiation the cells of method 2.a. weakly expressed FABP4 (pan-adipogenic marker) but didn't express UCP1 (brown adipocyte specific marker). Expression of UCP1 was however detected at low levels in cells of methods 2.b. and 2.c. at day 20 or 30. Expression of UCP1 was 900 times higher in the control cells generated from method 1.

[0345] These data show that with appropriate induction using adipogenic media, iPAM cells can differentiate into brown adipocytes. However, the expression of UCP1 was significantly lower in cells generated with methods 2.b. or 2.c than with in cells produced with method 1, demonstrating the importance of the additional steps described in the present invention. The combination of culturing steps constituting the present invention generate a surprisingly high yield of BA production compared to any other combination of those steps as would be suggested in prior art.

[0346] Comparison of the Method 1 to the Method 3:

[0347] The ability to generate BA cells according to WO17223457 was evaluated. Starting from a single batch of hiPS cells at the same time, iPAM cells were generated. Then, the cells were exposed to the culturing steps of methods 1, 3.1., 3.2.a. and 3.2.b. (detailed in the section methods).

[0348] Expression of Adipocyte Markers Analysed by qPCR:

[0349] For this comparison, the expression of the genes at day 20, day 30 and for the BAP and BA of the method 1) were normalized against a sample taken at day 8 (i.e. before any commitment to adipocyte lineage).

[0350] After 20 or 30 days of differentiation the cells of method 3.1. named HIFL didn't express any adipocyte markers (FIG. 16) suggesting that the cells did not differentiate into brown adipocytes. After 20 or 30 days, cells of methods 3.2. expressed low levels of FABP4 and UCP1 markers (FIG. 17). Expression of UCP1 was at least 1000 times higher in the control cells generated from method 1.

[0351] These data again show that with appropriate induction, iPAM cells can differentiate into brown adipocytes. However, the expression of UCP1 was significantly lower in cells generated with methods 3 compared to method 1. While the methods described in WO17223457 may generate BA cells with low yield, these results clearly demonstrate the superiority of the method of the present invention.

[0352] Comparison of the Method 1 to the Method 4:

[0353] The ability to generate BA cells according to Hafner et al. was evaluated. hiPS cells were differentiated to generate brown adipocytes according to the method 1 or the method 4. BAP cultures before secondary differentiation and BA cultures after 17 days of maturation in adipogenic medium were analysed by qPCR and immunofluorescence at passage 4 (data not shown) and 5 (FIG. 18).

[0354] Expression of adipocyte markers analysed by qPCR: For this comparison, the expression of the genes in the differentiated cells are normalized by the expression in the undifferentiated cells. BA cells obtained with both methods showed expression of FABP4 and UCP1 (FIG. 18.B.), but cells from method 1 expressed UCP1 a levels 100 times higher.

[0355] Expression of adipocyte markers analysed by IF: The population of cells expressing UCP1 and presenting lipid droplets was significantly higher with the method 1 than with the method 4 (about 47% for the method 1 and 25% for the method 4) (FIG. 18.A.)

[0356] Thus, these results teach that surprisingly, the combination of the induction of paraxial mesoderm lineage and the enrichment of BAP by passaging as described in the method 1 significantly improves the yield of differentiation.

[0357] Comparison of the Method 1 to the Method 5:

[0358] We could not reproduce the results of method 5. Despite several attempts, after the first step of the formation of the cell aggregates, a high cellular mortality was observed after 2 or 3 days (FIG. 19). Cell cultures were completely lost after 8 days.

[0359] Control cells differentiated according to method 1 using the same batch of undifferentiated hiPS cells however showed expected results whether by qPCR (FABP4 and UCP1 markers were more expressed in BA than in BAP) or by immunofluorescence (after 17 days of differentiation, the cell population expressing UCP1 and presenting lipid droplets reached 85%), suggesting that the cellular mortality observed during the reproduction of the method 5 was not due to the undifferentiated hiPS cells used.

[0360] These results suggest that the method of WO2012/147853 is either not reproducible or highly dependent on external factors, which make this method unsuitable for industrial applications, ascertain the superiority of method 1 and suggest that the cells which can theoretically be obtained from method 5 are not comparable to the cells obtained by the method of the present invention.

[0361] Summary of the Comparisons:

[0362] To demonstrate the technical advantages of the present invention over existing protocols, we have compared it to other protocols disclosed in the prior art which either claim the production of: [0363] adipocytes or brown adipocytes from iPAM cells, or [0364] brown adipocytes from hiPS cells.

[0365] The analysis by qPCR (FIG. 20) showed that the BA generated with method 1 expressed the brown adipocyte specific marker UCP1 at higher level than any of the other protocols (e.g. from 100 times more than method 4 and up to 1000 times more than method 3) testifying to a significantly higher yield of differentiation. These results clearly demonstrate that the method of the present invention is superior to generate BAP and BA compared to existing protocols in terms of yield and/or duration but also of robustness with the absence of EBs. The present application therefore discloses a surprising novel way to generate BAP and BA.

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Abbreviations/Molecules List

[0401] Ascorbic acid: (also known as vitamin C) is an essential nutrient in human diets. Ascorbic acid is a potent reducing and antioxidant agent. Besides anti-oxidant activity, ascorbic acid (ASC) acts as a cofactor of the hydroxylating enzyme of proline and lysine residues in procollagen. Manufacturer: Sigma Aldrich [0402] CHIR99021: is an aminopyrimidine derivative that is an extremely potent inhibitor of GSK3 and functions as a WNT activator. Manufacturer: axon medchem [0403] Dexamethasone: is a synthetic glucocorticoid hormone. Manufacturer: Sigma Aldrich [0404] DMEM: Dulbecco's Modified Eagle Medium (basal culture medium). Manufacturer: Gibco [0405] EGF: epidermal growth factor (EGF) stimulates cell growth and differentiation by binding to its receptor, EGFR. Manufacturer: Miltenyi biotech [0406] FBS: Fcetal bovine serum . Manufacturer: PanSera; Dutscher [0407] FGF-2 (also called bFGF): fibroblast growth factor-2. Manufacturer: Miltenyi biotech [0408] HGF: hepatocyte growth factor. Manufacturer: R&D systems [0409] Hydrocortisone: is a glucocorticoid secreted by the adrenal cortex. Manufacturer: Sigma aldrich [0410] IBMX: (3-isobutyl-1-methylxanthine) is non-specific inhibitor of cyclic AMP and cyclic GMP phosphodiesterases (PDEs). By inhibiting PDEs, IBMX increases cellular cAMP and cGMP levels, activating cyclic-nucleotide-regulated protein kinases. Manufacturer: Stemcell technologies [0411] IGF-1: Insulin growth factor type 1. Manufacturer: Miltenyi biotech [0412] ITS: Insulin-Transferrin-Selenium. It's a cell supplement. Insulin promotes glucose and amino acid absorption, lipogenesis, intracellular transport, and protein and nucleic acid synthesis. Transferrin is a iron-binding glycoprotein that controls the level of free-iron (can also help to reduce the level of oxygen and peroxide free radicals). Selenium is a cofactor for glutathione peroxidase and other proteins, and is used as an antioxydant in culture media. Manufacturer: Gibco [0413] KSR: Knockout serum replacement. It's a more defined, FBS-free medium supplement that supports the growth of pluripotent stem cells (PSCs). Manufacturer: Gibco [0414] LDN-193489: LDN-193189 is a cell permeable small molecule inhibitor of bone morphogenetic protein (BMP) type I receptors ALK2 and ALK3. LDN-193189 was derived from structure-activity relationship studies of Dorsomorphin and functions primarily through prevention of Smad1, Smad5, and Smad8 phosphorylation. Manufacturer: Miltenyi biotech [0415] mTESR-1: Standardized Medium for the Feeder-Independent Maintenance of hESCs & hiPSCs. Manufacturer: Stemcell technologies [0416] Y-27632 (usually called: Rock-1 inhibitor): inhibitor of Rho-associated, coiled-coil containing protein kinase (ROCK). Manufacturer: Tocris bioscience. [0417] Rosiglitazone: Rosiglitazone is an anti-diabetic drug from the thiazolidinedione class. Like other thiazolidinediones, its mechanism of action is by activation of the intracellular receptor class of the peroxisome proliferator-activated receptors (PPARs), specifically PPAR-gamma. Manufacturer: Prestwick [0418] SB431542: is a small molecule inhibitor of the TGF-6/Activin/NODAL pathway that inhibits ALK5, ALK4, and ALK7, but does not inhibit the BMP type I receptors ALK2, ALK3, and ALK6. Manufacturer: Stemcell technologies. [0419] T3: triiodothyronine. T3 is a thyroid hormone resulting of deiodination of thyroxine. Manufacturer: Sigma aldrich.