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WO2022219523A1 - Procédé amélioré de reprogrammation cellulaire - Google Patents

Procédé amélioré de reprogrammation cellulaire Download PDF

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WO2022219523A1
WO2022219523A1 PCT/IB2022/053418 IB2022053418W WO2022219523A1 WO 2022219523 A1 WO2022219523 A1 WO 2022219523A1 IB 2022053418 W IB2022053418 W IB 2022053418W WO 2022219523 A1 WO2022219523 A1 WO 2022219523A1
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reprogramming
medium
day
sll6r
cells
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Onelia GAGLIANO
Camilla Luni
Nicola Elvassore
Davide Cacchiarelli
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Onyel Biotech Srl
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Onyel Biotech Srl
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2501/20—Cytokines; Chemokines
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts

Definitions

  • the present invention relates to an improved method of reprogramming human adult cells into hiPSCs, the method comprising supplementing the reprogramming medium or the reprogramming medium and the expansion medium with one or more biological molecules selected from Hepatocyte Growth Factor (HGF), Interleukin 6 (IL6) together with soluble Interleukin 6 Receptor (slL6R), and Neuregulin 1 (NRG1).
  • HGF Hepatocyte Growth Factor
  • IL6 Interleukin 6
  • slL6R soluble Interleukin 6 Receptor
  • NGF Neuregulin 1
  • hiPSCs Human induced pluripotent stem cells
  • hiPSCs are pluripotent stem cells derived from human adult cells that hold great promise especially for in vitro studying embryogenesis and for the derivation of in vitro models of human tissues, and in general as a source of patient-specific human cells of potentially any tissue. They are characterized by key molecular signatures at genetic network and signaling pathway levels.
  • NANOG, POU5F1 (encoding OCT4 protein) and SOX2 genes constitute the essential pluripotency master transcriptional network that in a feed-forward manner supports secondary transcription factors (e.g., MYC family members, ZFX) and stabilizes pluripotency.
  • the pluripotency in somatic cells is normally obtained through molecular changes induced by forced expression of four transcription factors: OCT4, SOX2, KLF4 and cMYC.
  • OCT4, SOX2, KLF4 and cMYC transcription factors or other factor sets suitable for cell reprogramming.
  • these transcription factors perturbs the transcriptional network of the somatic cells.
  • the cells integrate intrinsic and extrinsic cues to remodel chromatin and reach a new epigenetic state.
  • TGF-b pathway activation could be a negative regulator during acquisition of epithelial phenotype, whereas has a positive effect on reprogramming during the very first steps and at later stages, when it acts as a promoter of pluripotency.
  • hiPSCs human induced pluripotent stem cells
  • the authors of the present invention have provided an improved reprogramming protocol from human somatic cells to hiPSCs in which said reprogramming is carried out in conventional cell culturing devices (i.e. non-microfluidic systems or devices, such as petri dishes, multi well plates, cell culture flasks and the like).
  • conventional cell culturing devices i.e. non-microfluidic systems or devices, such as petri dishes, multi well plates, cell culture flasks and the like.
  • HGF hepatocyte growth factor
  • IL6 interleukin 6
  • slL6R soluble interleukin 6 receptor
  • NGF-1 neuregulin 1
  • supplementing the reprogramming medium with HGF and/or a combination of IL6 and slL6R during all the early phase of the reprogramming and supplementing the reprogramming medium and the expansion medium with NRG-1 during all the late phase of the reprogramming improves the process providing a 3-fold increase of hiPSCs when compared to the number of hiPSCs obtained with the same protocol without said addition (see figure 2).
  • Object of the invention is, therefore, a method of reprogramming human somatic adult cells into human induced pluripotent stem cells (hi-PSCs), comprising supplementing the reprogramming medium or the reprogramming medium and the expansion medium with at least one of: hepatocyte growth factor (HGF); a combination of interleukin 6 (IL6) and interleukin 6 soluble receptor (slL6R); neuregulin 1 (NRG-1).
  • HGF hepatocyte growth factor
  • IL6 interleukin 6
  • slL6R interleukin 6 soluble receptor
  • NGF-1 neuregulin 1
  • reprogramming indicates the process that is used to de-differentiate adult, differentiated cells to cells that have an immature and pluripotent phenotype, such as induced pluripotent stem cells (iPSC).
  • iPSC induced pluripotent stem cells
  • pluripotent stem cells stands for human-induced pluripotent stem cells, which are a type of pluripotent stem cell that can be generated directly from adult cells.
  • “Early” phase of reprogramming in the present description and claims is a phase commonly known by the skilled person as the phase that ends with the appearance of the epithelial cell clusters after the first days of reprogramming. It refers averagely to a time period starting from day 1 or from day 2 after somatic cell seeding and of said up to averagely ending from day 3 to day 6 of said reprogramming.
  • the early phase ends with the appearance of the epithelial cell clusters, i.e. the last day of the early phase is the day before the appearance of the epithelial cell clusters.
  • “Late” phase of reprogramming in the present description and claims is a phase commonly known by the skilled person and refers to a time period starting averagely from day 6, or in any case from the day in which epithelial cell clusters are visible, and ends on the last day of said reprogramming. As the beginning of the late phase coincides with the appearance of the epithelial cell clusters therefore, when needed, the last day of the early phase and the first day of the late phase can be easily adjusted in each experiment by the skilled person.
  • the early phase can be defined as a time period starting from day 1 or day 2 and ending the day before the appearance of epithelial cell clusters
  • the late phase can be defined as a time period starting on the day when epithelial cell clusters appear and ending on the last day of said reprogramming.
  • days of the reprogramming protocol it is to be understood that before starting a reprogramming method/protocol, seeding of the cells to reprogram is needed. In the present specification said seeding period is referred as day 0.
  • the progressive counting of the days in the reprogramming protocol begins on the same day in which the somatic cells are exposed for the first time to the transcription factors (transcription factors).
  • Reprogramming medium is any kind of medium available to the skilled person suitable for cells submitted to a reprogramming protocol.
  • expansion medium is intended in the present specification with the meaning commonly used in the art and refers to medium for the expansion of reset naive human induced pluripotent stem (hiPSC) cells, this medium is used after the appearance of epithelial cell clusters during reprogramming instead of the previously used reprogramming medium.
  • hiPSC reset naive human induced pluripotent stem
  • Reprogramming medium is any kind of medium available to the skilled person suitable for cells submitted to a reprogramming protocol.
  • the present invention provides an improved method of reprogramming human somatic adult cells into human induced pluripotent stem cells (hi-PSCs), comprising supplementing the reprogramming medium or the reprogramming medium and the expansion medium with at least one of:
  • HGF a combination of IL6 and slL6R;
  • reprogramming indicates the process to de-differentiate differentiated cells to cells that are immature and pluripotent, also known as induced pluripotent stem cells (iPSc).
  • iPSc induced pluripotent stem cells
  • the reprogramming method of the invention can be any conventional reprogramming method in which adult somatic cells are exposed to suitable reprogramming transcription factors in conventional reprogramming medium for a suitable number of days; the method further comprising supplementing the reprogramming medium or the reprogramming medium and the expansion medium with at least one of:
  • HGF a combination of IL6 and slL6R;
  • Suitable known reprogramming methods according to the invention include the method disclosed by Takahashi K. and Yamanaka S. in 2006 (Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors Cell 2006, 07:024), by Takahashi K. et al in 2007 (Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors Cell 2007, 11: 19) or any modification thereof.
  • any of said methods will further comprise supplementing the reprogramming medium or the reprogramming medium and the expansion medium with at least one of:
  • HGF a combination of IL6 and slL6R;
  • suitable known reprogramming methods are methods in which adult somatic cells are reprogrammed to be “induced pluripotent stem cells (iPS cells)”, through retro-viral introduction or transient expression suitable transcription factors such as Oct4, Sox2, Klf4 and c-Myc or Oct4, Sox2, Nanog, Lin-28 and optionally other known suitable transcription factors.
  • the transcription factors can be provided to the cells by transient expression e.g. by submitting the cells to reprogramming by delivering into said cells mRNAs or mmRNAs encoding said transcription factors and/or inducing into the cells the transient expression of said factors, with suitable inducible vectors.
  • the reprogramming is carried out by transient expression of the selected transcription factors.
  • suitable transcription factors are at least Oct4, Sox2, Klf4 and c-Myc.
  • suitable transcription factors are Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28 (OSKMNL).
  • the reprogramming method can be as follows: a. replacing the culture medium of somatic cells previously seeded and grown for about 16 to 24 hours with a suitable reprogramming medium; b. preparing a transfection solution comprising mRNAs coding for the transcription factors for reprogramming Oct4, Sox2, Klf4 and c-Myc; preferably comprising the mRNAs of the transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28 (OSKMNL); c. adding said transfection solution to a new batch of said standard reprogramming medium; d. replacing the medium used in a. with the reprogramming medium with said transfection solution prepared in c.; e.
  • the reprogramming medium, or the reprogramming medium and the expansion medium are further supplemented with at least one of: HGF; a combination of IL6 and slL6R;
  • cells at Day one, should be put in reprogramming medium at least 9 h before starting transfection, and should have reached -50-60% confluence before the first transfection.
  • cell seeding on day 0, i.e. before starting the reprogramming should be done at a density of 40-80 cells/mm 2 , preferably, about 60 cell/mm 2 .
  • All human somatic cells are suitable cells for the reprogramming method of the invention, In a preferred embodiment, said human somatic cells are cells from skin or blood.
  • said cells are adult cells.
  • Suitable media for cell reprogramming can be any commercial reprogramming medium such as Pluriton reprogramming medium (e.g. Stemgent, Nordic Diagnostica AB), Nutristem hPSC XF reprogramming medium (Biological Industries) ReproTeSR reprogramming medium (STEMCELL Technologies), TeSR-E7 reprogramming medium (STEMCELL Technologies), Essential 6 reprogramming medium (Thermo Fisher Scientific), Essential 8 reprogramming medium (Thermo Fisher Scientific) and the like. Where needed or advisable, FGF2 supplementation can be at a final concentration of 20 ng/ml.
  • Pluriton reprogramming medium e.g. Stemgent, Nordic Diagnostica AB
  • Nutristem hPSC XF reprogramming medium Biological Industries
  • ReproTeSR reprogramming medium Biological Industries
  • TeSR-E7 reprogramming medium STEMCELL Technologies
  • Essential 6 reprogramming medium Thermo Fisher Scientific
  • Suitable antibiotics such as penicillin and/or streptomycin can be also used.
  • kits for the preparation of the transfection solution as well as mRNAs of the suitable transcription factors are commercially available.
  • information on media, transfection solution and suitable mRNAs for reprogramming protocols are published on a wide number of scientific papers and are now common knowledge for the skilled person.
  • the method described above is average of 12 days and can be divided in an early phase which is averagely from day 1 or day 2 until day 5, and in late phase, which is averagely from day 6 to day 12.
  • the beginning of the late phase coincides with the appearance of the epithelial cell clusters therefore, when needed, the last day of the early phase and the first day of the late phase can be easily adjusted in each experiment.
  • the method of the invention can comprise any commonly used protocol for reprogramming human somatic cells into hiPSCs known in the art, to which the steps of supplementing of the reprogramming medium with at least one of: hepatocyte growth factor (HGF); a combination of interleukin 6 (IL6) and interleukin 6 soluble receptor (slL6R); neuregulin 1 (NRG-1); are added.
  • HGF hepatocyte growth factor
  • IL6 interleukin 6
  • slL6R interleukin 6 soluble receptor
  • NSG-1 neuregulin 1
  • HGF hepatocyte growth factor
  • human HGF e.g. as identified by the UniProt identifier P14210-1.
  • recombinant human HGF is used for the method of the invention.
  • Both human and mouse HGF are commercially available (e.g. Abeam, Gibco, MitenyiBiotec, Sinobiologic, Peprotech).
  • IL-6 is a pleiotropic cytokine well known in the art. Any IL-6 can be used, preferably recombinant human IL-6 is used for the method of the invention. Various recombinant human IL-6 are commercially available (e.g. Peprotech, Abeam, MiltenyiBiotech, Gibco, Invivogen etc.).
  • IL-6 receptor has several isoforms, among them there is the soluble form of IL6 receptor (slL6R) that acts as an agonist of IL6 activity.
  • slL6R soluble form of IL6 receptor
  • Human soluble IL-6 receptor as is preferred for the method of the invention.
  • Human slL6R is commercially available (e.g. Peprotech, Abeam, MiltenyiBiotech, Gibco, Invivogen etc.).
  • Supplementing with the combination IL6 and slL6R according to the invention can be carried out by supplementing the reprogramming medium with separated aliquots of each of IL6 and slL6R, or by supplementing the reprogramming medium with a mixture of IL6 and slL6R.
  • Neuregulin-1 is a member of the neuregulin growth factor family. According to the present invention any NRG-1 can be used. In particular recombinant human NRG-1 is preferred for the method of the invention. Recombinant human NRG-1 is commercially available (e.g. R&D, Abeam, Sinobiological).
  • HGF a combination of IL6 and slL6R;
  • NRG-1 can be carried out starting from day 1 or from day 2 of said reprogramming until the end of the reprogramming.
  • supplementing is carried out starting from day 1 or from day 2 of said reprogramming until the end of the reprogramming, both the reprogramming medium and the expansion medium are supplemented.
  • this can be made with HGF, or with a combination IL6 and slL6R, or with NRG-1. As shown in figure 1, said embodiment results in a two-fold increase of the hi-PSCs obtained.
  • the supplementing from day 1 or from day 2 of said reprogramming until the end of the reprogramming can be made with HGF and a combination IL6 and slL6R; with HGF and NRG-1 or with a combination IL6 and slL6R and NRG-1.
  • the supplementing from day 1 or from day 2 of said reprogramming until the end of the reprogramming is made with HGF, a combination IL6 and slL6R, and NRG-1.
  • said embodiment results in a five-fold increase of the hi-PSCs obtained.
  • New supplementing is carried out each time the medium is changed.
  • the supplementing can be carried up to the last day of the early phase of the reprogramming. In other terms, it is carried up until the appearance of the epithelial cell clusters, which is averagely at day 6 of reprogramming.
  • the supplementing can be carried out from day 1 or 2 to day 5 of the reprogramming. In one embodiment said supplementing from day 1 or 2 up to the last day of the early phase of the reprogramming can be carried out with HGF; and/or with a combination of IL6 and slL6R.
  • the method of the invention may further comprise supplementing the reprogramming medium and the expansion medium with NRG-1 for the entire late phase of said reprogramming (i.e. from the appearance of the epithelial cell clusters, which is averagely at day 6, to the end of reprogramming).
  • the supplementing can be carried out from day 6 to the end of the reprogramming.
  • the reprogramming method can comprise the following steps: a. replacing the culture medium of somatic cells previously seeded and grown for about 16 to 24 hours with a suitable reprogramming medium supplemented with HGF, or with a combination IL6 and slL6R, or with NRG-1; b. preparing a transfection solution comprising mRNAs coding for the transcription factors for reprogramming Oct4, Sox2, Klf4 and c-Myc; preferably comprising the mRNAs of the transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28 (OSKMNL); c.
  • transfection solution to a new batch of said standard reprogramming medium supplemented with HGF, or with a combination IL6 and slL6R, or with NRG-1; d. replacing the medium used in a. with the reprogramming medium with said transfection solution prepared in c.; e. Incubating the cells overnight at 37 °C; f. replacing the medium used in d. with a new batch of said suitable reprogramming medium supplemented with HGF, or with a combination of IL6 and slL6R, or with NRG- 1 used in a.; g. repeating steps b. to f. for each day the first day of the late phase, i.e.
  • each supplementing in a. c. f. h. and i. of the method described above can be made with HGF and a combination of IL6 and slL6R; or with HGF and NRG-1 or with a combination of IL6 and slL6R and NRG-1.
  • each supplementing in a. c. f. h. and i. of the method described above can be made with HGF; a combination of IL6 and slL6R; and NRG-1.
  • the reprogramming method can comprise the following steps: a. replacing the culture medium of somatic cells previously seeded and grown for about 16 to 24 hours with a suitable reprogramming medium supplemented with HGF and/or with a combination of IL6 and slL6R; b.
  • a transfection solution comprising mRNAs coding for the transcription factors for reprogramming Oct4, Sox2, Klf4 and c-Myc; preferably comprising the mRNAs of the transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog and Lin-28 (OSKMNL); c. Adding said transfection solution to a new batch of said standard reprogramming medium supplemented with HGFand/or with a combination of IL6 and slL6R; d. replacing the medium used in a. with the reprogramming medium with said transfection solution prepared in c.; e. Incubating the cells overnight at 37 °C; f. replacing the medium used in d.
  • the final concentration of HGF is from 80 to 120 ng/mL; preferably, about 100 ng/mL.
  • IL6 final concentration when the medium is supplemented with the combination of IL6 and slL6R, IL6 final concentration can be from 40 to 60 ng/mL final concentration can be from 8 to12 ng/mL; preferably, IL6 final concentration can be about 50 ng/mL and slL6R final concentration can be about 10 ng/mL.
  • the final concentration can be from 80 to 120 ng/mL; preferably about 100 ng/mL.
  • the method can be performed under hypoxia conditions (5%02).
  • Table 1 shows the reprogramming efficiency when the medium is supplied with only one of the factors (HGF, IL6, slL6R and NRG1)
  • Table 2 displays the efficiency data when the factors are used in different combinations.
  • Reprogramming of human fibroblasts to hiPSC colonies hiPSCs from human foreskin BJ fibroblasts were generated.
  • a total of 6 mRNA transfections were performed using StemRNA-NM reprogramming kit (Stemgent, 00- 0076) and StemMACS mRNA transfection kit (Miltenyi, 130-104-463), in reprogramming medium (NutriStem® hPSC XF Medium SKU: 06-5100-01-1 A, supplemented with FGF2 (Peprotech, cat. no.
  • medium was supplemented with HGF 100 ng/mL (Peprotech, 100-39), IL6 50 ng/mL (Peprotech, 200-06), slL6R 10 ng/mL (Peprotech, 200-06R), NRG1 100 ng/mL (R&D, 396-HB), during the whole process duration, according to the specified perturbation conditions.
  • Reprogramming efficiency was quantified after immunostaining with TRA-1-60 and NANOG markers and calculated as the ratio of the number of TRA-1-60+ and NANOG+ colonies and initial cells seeded.
  • Human somatic cells from skin seeded at a density 60 cell/mm 2 were used.
  • CRITICAL STEP Cells should be put in reprogramming medium at least 9 h before transfection, and should have reached -50-60% confluence before the first transfection.
  • CRITICAL STEP Use RNase-free tubes and tips, and freshly thawed aliquot of RNA each day.
  • transfection solution to the medium of reprogramming (Nutristem+FGF2 or Pluriton medium and optionally penicillin-streptomycin), prewarmed to room temperature, and gently mix.
  • medium of reprogramming Nutristem+FGF2 or Pluriton medium and optionally penicillin-streptomycin
  • hiPSC expansion medium E8 Voden, cat. no. 05991, or StemMACS iPSBREW XF Miltenyi Biotech, cat. no. 130.107.086, and optionally penicillin-streptomycin.
  • HGF HGF 100 ng/mL (Peprotech, 100-39), IL-6 50 ng/mL (Peprotech, 200-06), IL-6r 10 ng/mL (Peprotech, 200-06R), NRG-1 100 ng/mL (R&D, 396-HB)
  • supplementing was made daily from Day1 or Day2 until Day12, when alone or all together.
  • HGF and IL6 and N6-r or only HGF were added daily from Day1 or Day2 until Day5 (early phase) and NRG1 from Day6 to Day12 (late phase). The beginning of the late phase coincided with the appearance of the epithelial cell clusters. Final concentrations were HGF 100 ng/mL (Peprotech, 100-39), IL-6 50 ng/mL (Peprotech, 200-06), IL-6r 10 ng/mL (Peprotech, 200-06R), NRG-1 100 ng/mL (R&D, 396- HB)
  • TR and TB are component of the StemMACS mRNA transfection kit.

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Abstract

La présente invention concerne un procédé amélioré de reprogrammation de cellules adultes humaines en hiPSC, le procédé comprenant la supplémentation du milieu de reprogrammation ou du milieu de reprogrammation et du milieu de multiplication avec une ou plusieurs molécules biologiques choisies parmi le facteur de croissance des hépatocytes (HGF), l'interleukine 6 (IL6) ainsi que le récepteur soluble de l'interleukine 6 (slL6R), et la neuréguline 1 (NRG1). Le procédé de l'invention augmente considérablement l'efficacité de la reprogrammation.
PCT/IB2022/053418 2021-04-13 2022-04-12 Procédé amélioré de reprogrammation cellulaire Ceased WO2022219523A1 (fr)

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WO2011084747A2 (fr) * 2009-12-21 2011-07-14 The Johns Hopkins University Compositions et procédés pour des cellules souches pluripotentes induites de tissu somatique, provenant de l'endoderme
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