Hamster-derived induced pluripotent stem cells

Abstract

Herein is reported a method for the production of one or more hamster-derived induced pluripotent stem cells comprising the steps of transducing a hamster cell with a Sendai virus comprising coding sequences for reprogramming factors, cultivating the transduced cell in the presence of at least a WNT pathway activator for about 12 days, replating the cells, adding a medium comprising one or more proteinogenic growth factors and cultivating the replated cells in the presence of the at least one WNT pathway activator, and selecting one or more cells that are positive for alkaline phosphatase and SSEA1 and that has stem cell morphology, wherein the at least one WNT pathway activator is in a small molecule cocktail comprising CHIR99021 (CHIR), 3-deazaneplanocin A (DZNep), forskolin, RepSox, tranylcypromine, aprotinoid acid (TTNPB), and valproic acid (VPA).

Description

[0001] Hamster-derived induced pluripotent stem cells

[0002] The current invention is in the field of cell line generation. More specifically, herein is reported a method for the generation of induced pluripotent stem cells from hamster cells or cell lines.

[0003] Somatic mammalian cells can be effectively reprogrammed to induced pluripotent stem cells (iPSCs) by specific introduction of the Yamanaka transcription factors Sox-2, Oct-4, Klf-4 and c- Myc into the target cells. This technology allowed for a source of pluripotent stem cell models beside ethically critical embryonic stem cell sources. By this, these iPSCs can be used for the generation of specific disease models, e.g. from patient derived cancer cells and rare allele species as well as new tools for “animal-free” drug development.

[0004] Current protocols were optimized for the generation of iPSCs from human and mouse progenitor primary cells and cell lines. The reprogramming of hamster cells into iPSCs, however, were shown only by one report (Pei et al., Stem Cell Research, Volume 21, May 2017, pp. 132-136) and by the use of an integrating Lentiviral system. However, integrating systems bear the risk of inducing genomic instability and disruption of critical, vital coding and non-coding genetic loci, e.g. by reprogramming factor and vector sequences that were integrated into the reprogrammed iPSC genome. Furthermore, the viral transgenes will / could be present in the reprogrammed cellular systems much longer, which requires laborious safety considerations (e.g. S2 in Germany). Nonintegrating systems for cell reprogramming, however, are safe, convenient to use in normal cell culture labs once viral transcripts cannot be detected anymore and does not interfere directly with the genomic background of the cells.

[0005] Use of iPSC-derived hamster cells, e.g. primary hamster fibroblasts and Chinese Hamster Ovary (CHO) cells, are of high interest for the biopharmaceutical research, development and manufacturing sector, since most biopharmaceuticals are produced by hamster-derived cellular expression systems and a holistic use of similar cellular models during drug development could simplify development activities and reduce unintended different experimental outcome.

[0006] An alternative approach for generation of murine chemically induced iPSCs (miPSCs) using a combination of seven small-molecule compounds has been shown previously (Huo et al., Science, Volume 341, Aug 2013, Issue 6146, pp. 651-654). However, the efficiency of chiPSC generation from mouse somatic cells using small molecule cocktails was only low at a frequency up to 0.2%, yet comparable to alternative reprogramming approaches.

[0007] It is important to note, that the cellular and molecular mechanisms in cell reprogramming by using small molecule compounds remains still unclear and is discussed controversially in the stem cell field. However, despite the missing mechanistic understanding, combination of small molecule cocktails with other reprogramming approaches are proposed to boost the efficiency of reprogramming (Malik and Rao, Meth. Mol. Biol., 2013, Issue 997: pp. 23-33).

[0008] So far, the advantageous use and combination of small molecule cocktails with other reprogramming methods, such as Sendai viral transduction of reprogramming factors to generate “hard-to-reprogram” cells and cell lines, such as primary Chinese hamster fibroblast cells has not been reported.

[0009] Herein is reported for the first time the need of small molecule BIO, CHIR99021, CD437, TTNPB, Valproic Acid, SAHA, BIXO 1294, Cyclic Pifithrin-a, G66983, SB431542, SP600125, Thiazovivin either alone or in combination for the generation of hamster iPSCs. It has further been found that maintenance and proliferation of Chinese hamster iPSCs requires specific media composition, i.e. presence of the proteinogenic media factor bFGF and the GSK3P inhibitor CHIR99021.

[0010] Herein is reported a method for the generation and production of hamster-derived induced pluripotent stem cells.

[0011] It has been found that for generating Chinese hamster iPSCs the findings made for the generation of murine iPSCs are not transferable and that findings from the prior art point in a non-working direction.

[0012] The current invention is based at least in part on the finding that specific factors, i.e. additives, and a specific culturing protocol are required for the successful generation and maintenance of hamster derived induced pluripotent stem cells. The required conditions were identified by a nonintegrating and an inducible integrating reprogramming approach using novel combinations of small molecules and primary Chinese hamster fibroblasts and an immortalized hamster cell line.

[0013] The current invention is based, at least in part, on the finding that of the different compounds of the small molecule cocktail SM51, which has been found to be essential for hamster iPSC generation with Sendai virus mediated expression of Sox-2, Oct-4, Klf-4 and c-Myc, the presence of CHIR99021 is mandatory to maintain hamster iPSC colony morphology and proliferation.

[0014] The current invention encompasses at least the following independent and dependent embodiments:

[0015] 1. A method for the production of one or more hamster-derived induced pluripotent stem cells comprising the following steps: a) transducing a hamster cell with a Sendai virus comprising coding sequences for reprogramming factors, b) cultivating the transduced cell in the presence of at least one specific WNT pathway activator for at least 7 days and less than 18 days, c) optionally replating the cells, adding a medium comprising one or more proteinogenic growth factors and cultivating the replated cells in the presence of the at least one specific WNT pathway activator, d) selecting one or more cells that are positive for alkaline phosphatase and SSEA1 and that has pluripotent stem cell morphology, or a) transfecting a hamster cell with one or more nucleic acids comprising expression cassettes for one or more reprogramming factors, wherein the expression cassettes comprise an inducible promoter, b) cultivating the transfected cell in the presence of at least one specific WNT pathway activator for at least 7 days and less than 18 days and inducing the promoter of the reprogramming factors after 0-1 days, c) optionally replating the cells, adding a medium comprising one or more proteinogenic growth factors and cultivating the replated cells in the presence of the at least one specific WNT pathway activator, d) selecting one or more cells that are positive for SSEA1, thereby producing one or more hamster-derived induced pluripotent stem cells. . The method according to embodiment 1 , wherein the cultivating in step b) is in the presence of a small molecule cocktail comprising at least one specific WNT pathway activator. a. The method according to any one of embodiments 1 to 2, wherein the at least one specific WNT pathway activator inhibits GSK3b. b. The method according to any one of embodiments 1 to 2a, wherein the at least one specific WNT pathway activator inhibits GSK3b and is selected from the group consisting of LiCl, CHIR99021, SB-216736, BIO, SB-415286, TWS-119, AR-A014418, LY-2090314, PF- 04802367, L807mts and Tideglusib. 2c. The method according to embodiment 1, wherein the cultivating in step b) or / and c) is in the presence of a small molecule cocktail comprising at least one of the specific WNT pathway activators CHIR99021 and BIO.

[0016] 3. The method according to any one of embodiments 1 to 2c, wherein the cultivating in steps b) or / and c) is in the presence of a small molecule cocktail comprising CHIR99021.

[0017] 4. The method according to any one of embodiments 2 to 3, wherein the small molecule cocktail is completely removed at the time of replating and replaced by CHIR99021 only.

[0018] 5. The method according to any one of embodiments 1 to 4, wherein the replating is on feeder cells or on a matrigel-coated surface.

[0019] 6. The method according to embodiment 5, wherein the feeder cells are y-irradiated murine embryonic fibroblast (MEF) cells.

[0020] 6a. The method according embodiments 1 to 4, wherein the replating is on extracellular matrix proteins.

[0021] 6b. The method according to embodiment 6a, wherein the extracellular matrix proteins are at least one of laminin, vitronectin, gelatine, and collagen.

[0022] 7. The method according to any one of embodiments 1 to 6b, wherein the Sendai virus is a non-integrating Sendai virus.

[0023] 8. The method according to any one of embodiments 1 to 7, wherein the reprogramming factors are Sox-2, Oct-4, Klf-4 and c-Myc.

[0024] 9. The method according to any one of embodiments 1 to 8, wherein the reprogramming factors are Sox-2, Oct-4, Klf-4, c-Myc, Esrrb, Nanog, Lin28, and Sall4.

[0025] 10. The method according to any one of embodiments 1 to 9, wherein the at least one WNT pathway activator or CHIR99021 or the small molecule cocktail is added with or directly after the Sendai virus.

[0026] 11. The method according to any one of embodiments 1 to 10, wherein the cultivating in steps b) and c) is for at least 11 days.

[0027] 12. The method according to any one of embodiments 1 to 10, wherein the cultivating in steps b) and c) is for at least 14 days. 13. The method according to any one of embodiments 1 to 10, wherein the cultivating in steps b) and c) is for at least 21 days.

[0028] 14. The method according to any one of embodiments 1 to 10, wherein the cultivating in steps b) and c) is for at least 35 days.

[0029] 15. The method according to any one of embodiments 1 to 14, wherein the cultivating in step b) is for 7 days and in step c) for at least for 4 days.

[0030] 16. The method according to any one of embodiments 1 to 15, wherein the cultivating in step b) is for 7 days and in step c) for at least for 7 days.

[0031] 17. The method according to any one of embodiments 1 to 16, wherein the cultivating in step b) is for 12 days and in step c) for at least for 4 days.

[0032] 18. The method according to any one of embodiments 1 to 17, wherein the cultivating in step b) is for 12 days and in step c) for at least for 7 days.

[0033] 19. The method according to any one of embodiments 1 to 18, wherein the cultivating in step b) is for 14 days and in step c) for at least for 4 days.

[0034] 20. The method according to any one of embodiments 1 to 19, wherein the cultivating in step b) is for 14 days and in step c) for at least for 7 days.

[0035] 21. The method according to any one of embodiments 1 to 20, wherein the replating is after 10 to 14 days.

[0036] 22. The method according to any one of embodiments 1 to 21, wherein the replating is after about 12 days.

[0037] 23. The method according to any one of embodiments 1 to 22, wherein the proteinogenic growth factor is bFGF.

[0038] 23a. The method according to any one of embodiments 1 to 23, wherein the proteinogenic growth factor is bFGF at a concentration of about 10 ng / mL.

[0039] 24. The method according to any one of embodiments 1 to 23a, wherein the transduction of step a) and the cultivation of step b) are in fibroblast medium.

[0040] 25. The method according to any one of embodiments 1 to 24, wherein the method comprises prior to step a) the following step:

[0041] 0) cultivating a hamster cell in a fibroblast cultivation medium for about 7 days. The method according to any one of embodiments 1 to 25, wherein the cultivating in step c) is in embryonic stem cell (ESC) medium. The method according to any one of embodiments 1 to 25, wherein the cultivation medium in step c) is changed to embryonic stem cell (ESC) medium on the replating day or one day later. The method according to any one of embodiments 1 to 27, wherein pluripotent stem cell morphology denotes cobblestone-like colonies with sharp delimited colony boundaries. The method according to any one of embodiments 1 to 28, wherein the pluripotent stem cell morphology is determined by bright field microscopy. The method according to any one of embodiments 1 to 29, wherein the alkaline phosphatase is determined by epifluorescence microscopy. The method according to any one of embodiments 1 to 30, wherein each expression cassette has an inducible promoter. The method according to any one of embodiments 1 to 31, wherein each expression cassette has a different inducible promoter. The method according to any one of embodiments 31 to 32, wherein the inducible promoter is a Tet-inducible promoter. The method according to any one of embodiments 1 to 33, wherein the inducer of the inducible promoter is added in step b) simultaneously with the at least one WNT pathway activator or CHIR99021 or the small molecule cocktail comprising CHIR99021. The method according to any one of embodiments 1 to 34, wherein positive denotes the existence of at least one iPSC marker, at least SSEA1 or at least AP or SSEA1 and AP, present in pluripotent hamster stem cells. The method according to any one of embodiments 1 to 35, wherein the hamster cell is a primary hamster fibroblast cell or immortalized hamster cell. The method according to any one of embodiments 1 to 36, wherein the hamster cell is a Chinese hamster ovary cell. The method according to any one of embodiments 1 to 37, wherein CHIR99021 is added at a concentration of about 200 mM. 39. The method according to any one of embodiments 1 to 38, wherein the concentration of CHIR99021 in the cultivation is 20 pM.

[0042] 40. The method according to any one of embodiments 1 to 39, wherein the small molecule cocktail comprises CHIR99021 (CHIR), 3-deazaneplanocin A (DZNep), forskolin, RepSox, tranylcypromine, aprotinoid acid (TTNPB), and valproic acid (VP A).

[0043] 41. The method according to any one of embodiments 1 to 40, wherein the concentration of the compounds of the small molecule cocktail in step b) or / and c) are about 10 pM CHIR99021 (CHIR), about 0.05 pM 3-deazaneplanocin A (DZNep), about 10 pm forskolin, about 5 pM RepSox, about 5pM tranylcypromine, about 1 pM aprotinoid acid (TTNPB) and about 500 pM valproic acid (VP A)..

[0044] 42. The method according to any one of embodiments 1 to 41, wherein the small molecule cocktail is added at a concentration of about 200 mM - 1 mM.

[0045] 43. The method according to any one of embodiments 1 to 42, wherein the concentration of the small molecule cocktail in the cultivation is 500 pM-5 pM.

[0046] 44. The method according to any one of embodiments 1 to 43, wherein for the induction of reprogramming an inhibition of GSK3beta pathway, an activation of retinoid pathway, an inhibition of histone deacetylase pathway, an inhibition of histone methyltransferase pathway, an inhibition of p53 pathway, an inhibition of PKC pathway, an inhibition of TGFbeta pathway, and an inhibition of JNK pathway has to be effected.

[0047] 45. The method according to any one of embodiments 1 to 43, wherein for the induction of reprogramming the GSK3beta pathway has to be inhibited, the retinoid pathway has to be activated, the histone deacetylase pathway has to be inhibited, the histone methyltransferase pathway has to be inhibited, the p53 pathway has to be inhibited, the PKC pathway has to be inhibited, the TGFbeta pathway has to be inhibited and the JNK pathway has to be inhibited.

[0048] 46. The method according to any one of embodiments 1 to 45, wherein the cultivation of step b) is in a medium comprising BIO, CHIR99021 (CHIR), CD437, TTNPB, valproic acid, SAHA, BIX01294, cyclic Pifithrin-a, G66983, SB431542, SP600125, and Thiazovivin.

[0049] ***

[0050] In addition to the various embodiments depicted and claimed, the disclosed subject matter is also directed to other embodiments having other combinations of the features disclosed or claimed herein. As such, the particular features presented herein can be combined with each other in other manners within the scope of the disclosed subject matter such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to those embodiments disclosed.

[0051] Figure 1 General scheme for the generation of hamster derived induced pluripotent stem cells according to the current invention. Primary Chinese hamster fibroblasts (CHAF) and an immortalized hamster ovary (CHO) cell line were used in combination with non-integrating Sendai virus and stable integration of inducible reprogramming factors, respectively. Advantageous replating timing, growth factor requirement and small molecule factors were identified by combining a phenotypic and Design-of-Experiment (DoE) approach.

[0052] Figure 2 Hamster fibroblasts cannot be reprogrammed by Sendai virus transduction of Yamanaka factors.

[0053] A) General protocol for Sendai virus iPSC generation.

[0054] B) Bright field images of murine embryonic fibroblasts (MEF) (positive control) and primary Chinese hamster fibroblasts (CHAF); row 1 and 2: 9 days and 14 days, respectively, after Sendai virus transduction for analysis of cell morphology; row 3: epifluorescence imaging of MEFs and CHAFs, respectively, 18 days after Sendai virus transduction for alkaline phosphatase detection. Circle and black arrowhead indicate iPSC colonies.

[0055] Figure 3 Chinese hamster fibroblasts cannot be reprogrammed by Sendai virus transduction. Different combinatorial tests of cell culture media and matrix conditions do not enable hamster iPSC generation. Bright field images of Sendai virus transduced CHAFs were replated 14 days after transduction and cultured in the presence of combinations of different cell culture surface matrixes and cell culture media. mESC: murine embryonic stem cell media; yMEF: gamma irradiated murine embryonic fibroblasts; yCHAF: gamma irradiated Chinese hamster fibroblasts; Lam: laminin; Vtn: vitronectin; Gel: gelatine.

[0056] Figure 4 Exemplary results of the method according to the current invention showing that a hybrid reprogramming approach of Sendai virus transduction of Yamanaka factors in combination with the specific small molecule cocktail SM51 enables hamster iPSC generation:

[0057] A) Protocol for hybrid approach using Sendai virus and small molecule cocktails for hamster iPSC generation according to the current invention;

[0058] B) Comparative data - bright field images of murine embryonic fibroblasts (MEF) and two primary hamster fibroblasts (CHAF) lines, CHAF-0 and CHAF-2, 3 days and 11 days, respectively, after exclusively Sendai virus transduction for analysis of cell morphology and identification of iPSC colonies. Murine iPSC colony originated from MEFs is highlighted by circle and black arrowhead;

[0059] C) Bright field images of hamster fibroblast (CHAF) line CHAF-0, 3 days and 11 days, respectively, after combined Sendai virus transduction and cultivation in the presence of a small molecule (SM) cocktail, SM40, SM41, SM43, SM44, SM46 and SM51, for analysis of cell morphology and identification of hamster iPSC colonies. Hamster iPSCs colony originated from CHAFs by Sendai virus transduction and small molecule cocktail SM51 treatment is highlighted by a circle and black arrowhead.

[0060] Figure 5 Mean CT (cycle threshold) values of a qRT-PCR analysis for analysis of reprogramming efficiency. Analysis of Yamanaka factor mRNA expression by qPCR in MEFs (M), two different Chinese hamster fibroblasts: CHAF-0 (CO), CHAF-2 (C2), and of CHAF-2 with SM40, SM41, SM43, SM44, SM46, and SM5 1. Numbers represent values of qPCR crossing point analysis. Probes for SeV : Sendai virus; cMYC: cMYC; KOS: KLF4, Oct4, Sox2; KLF4: Kruppel-like factor 4; Oct4: Octamer binding transcription factor 4, Sox2: sex determining region Y- box 2. “x”, gene transcript not detectable.

[0061] Figure 6 Example for the method according to the current invention showing that maintenance of hamster iPSC status and morphology is dependent on small molecule cocktail, such as SM51. Bright field images of a hamster fibroblast (CHAF) line at day 13 after combined Sendai virus and small molecule cocktail SM51 treatment (A), and MEFs and images of two cell culture sections after day 17 with removal of SM51 at day 14 (B). SM46 was used as a negative control. Hamster iPSCs colonies originated from CHAFs by Sendai virus transduction and small molecule cocktail, i.e. SM51, treatment and requirement on small molecule cocktail, i.e. SM51, is highlighted by circles and black arrowheads.

[0062] Figure 7 Example of the method according to the current invention showing that maintenance of hamster iPSC status and morphology is dependent on small molecule CHIR99021. Bright field images (A) and epifluorescence AP-life staining (B) of cell line CHAF-0, at day 21 after combined Sendai virus and small molecule cocktail, i.e. SM51, treatment, then removal of individual components of the small molecule cocktail, i.e. SM51, or reduction to half the concentration of SM51 at day 14. Hamster iPSCs colonies originated from CHAFs by Sendai virus transduction and small molecule cocktail, i.e. SM51, treatment, and requirement on small molecule cocktail, specifically SM51, component CHIR99021 is highlighted by black and white arrowheads.

[0063] Figure 8 Example of the method according to the current invention showing that maintenance of iPSC status and morphology is dependent on the culture media composition. Brightfield images of iPSC colonies originated from cell line CHAF- 0 and MEF at day 21 after combined Sendai virus and small molecule cocktail, i.e. SM51, treatment for CHAFs and exclusively Sendai virus treatment for MEFs. Representative cell culture sections of CHAF derived hamster iPSC colonies cultured in ESC (embryonic stem cell) media with 0.5xSM51 or ESC / mTeSR (1 : 1) blend with 0.5xSM51 and MEF derived iPSC colonies with ESC media are shown.

[0064] Figure 9 Overview of used small molecule cocktails, concentration of compounds and mode of action.

[0065] A) List of tested small molecule cocktails used for reprogramming experiments.

[0066] B) Small molecule compounds of SM51 with respective target pathway, concentration ranges according to the current invention, and tested concentrations, Cl and C2.

[0067] Figure 10 Example of the method according to the current invention showing that CHIR99021 and bFGF and replating after 12 days are mandatory factors for colony formation during reprogramming of CHAFs.

[0068] A) List of 12 different media supplemented with known pluripotency inducers. CHAFs were transduced with Sendai virus and either replated after 7 days, 12 days or 18 days on irradiated MEFs in the respective medium.

[0069] B) The diagram shows the number of S SEA 1 -positive (SSEA1+) colonies after 35 days in the different media and replated on different days.

[0070] C) Exemplary AP staining of CHAF-derived reprogrammed hamster fibroblast induced pluripotent stem cells (chiPSCs) by Sendai virus and medium condition 8 (see A) at passage 12. The pictures show exemplary chiPSC colonies in 4x and lOx magnification.

[0071] Figure 11 Example of the method according to the current invention showing that Inducible expression of reprogramming factors and small molecule safeguard factors are mandatory for CHO SSEA1 formation. An exemplary Chinese hamster ovary (CHO) cell line was transfected with different combinations of reprogramming factors either under constitutive or Tetracycline-inducible (TetOn) expression. For the generation of stable pools, cells were cultivated for 14 days with selective pressure containing medium.

[0072] A) Only TetOn pools 9, 10 and 11 recovered, indicating a toxic and / or cytostatic effect by constitutive expression of reprogramming factors (red: no pool recovery; green: pool recovery).

[0073] B) SSEA1 presence of pool 9 and 10 were detected exclusively by simultaneously reprogramming factor expression by tetracycline and small molecule supplementation to the cell culture medium (red: no SSEA1 detection; green: SSEA1 detection).

[0074] C) Exemplary SSEA1 FACS staining of stable CHO pool 9 with small molecule DoE experiment media cocktail at day 4 after induction of reprogramming factors compared to induced pool 9 without small molecules. The amount of SSEA1 positive cells is shown in the gated area.

[0075] Figure 12 Exemplary approaches for design of experiment-based (DoE-based) approaches to identify suitable process conditions. For example, optimization of three factors are normally varied between a minimum (-1), medium (0), and maximum level (+1) for optimization DoEs. (Picture adopted from http: / / www.gmpua.com and modified).

[0076] Figure 13 Exemplary method according to the current invention showing the identification of small molecule safeguard factors required for hamster SSEA1 expression.

[0077] A) Actual vs. predicted plot of Mean Fluorescence Intensity (MFI) of SSEA1 for all 96 single experiments of the DoE approach D-optimal.

[0078] B) Sorted parameter estimates for the p-value guided stepwise reduced relevant factors for model building.

[0079] Figure 14 Used vector constructs in this study for constitutive and inducible overexpression of reprogramming factors in CHO cells.

[0080] A) Reprogramming factor organizations on “Front” (in list called “F Plasmid”) and “Back” (in list called “B Plasmid”) vector with respective factor localization, plasmid assignment, and controls: eGFP and a tri-fluorescence expression cassette is shown.

[0081] B) Design of the tri-fluorescence expression cassette, with marker gene specific artificial promoters coupled to fluorescence proteins eGFP, eBFP, and mRUBY. Fluorescence proteins are tagged with a PEST sequence from mouse ornithine decarboxylase (MODC) for reduction of fluorescence in vivo half-life. An upstream located selection marker is used for stable CHO pool generation (note, all “Back” plasmids contain this marker to allow for selection).

[0082] The current invention is based on the finding that for generating Chinese hamster induced pluripotent stem cells (chiPSCs) a hybrid approach of non-integrating Sendai virus and stable integration of inducible reprogramming factors in combination with a small molecule cocktail has to be used.

[0083] The current invention is based on the finding that for generating Chinese hamster iPSCs the activation of the WNT pathway is essential. By using small molecule cocktails, generation of Chinese hamster iPSCs is supported by inhibiting GSK3B, histone deacetylase, histone methyltransferase, p53, PKC, and TGFP and activating the retinoid pathway either alone or in combination.

[0084] The current invention is based on the finding that for generating Chinese hamster iPSCs that the iPSC cells have to be replated at day 12.

[0085] The current invention is based on the finding that for generating hamster iPSCs that LIF, which is a mandatory factor for mouse iPSC generation, is not needed for generation of hamster iPSCs.

[0086] DEFINITIONS

[0087] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular, and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0088] Useful methods and techniques for carrying out the current invention are described in e.g. Ausubel, F.M. (ed.), Current Protocols in Molecular Biology, Volumes I to III (1997); Glover, N.D., and Hames, B.D., ed., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, R.I. (ed.), Animal Cell Culture - a practical approach, IRL Press Limited (1986); Watson, J.D., et al., Recombinant DNA, Second Edition, CHSL Press (1992); Winnacker, E.L., From Genes to Clones; N.Y., VCHPublishers (1987); Celis, J., ed., Cell Biology, Second Edition, Academic Press (1998); Freshney, R.I., Culture of Animal Cells: A Manual of Basic Technique, second edition, Alan R. Liss, Inc., N.Y. (1987). The content of which is incorporated herein by reference, The use of recombinant DNA technology enables the generation of derivatives of a nucleic acid. Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion. The modification or derivatization can, for example, be carried out by means of site directed mutagenesis. Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, England).

[0089] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the terms "comprising", "including", and "having" can be used interchangeably.

[0090] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s)” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms or words that do not preclude the possibility of additional acts or structures. The term “comprising” also encompasses the term “consisting of’. The present disclosure also contemplates other embodiments “comprising”, “consisting of’ and “consisting essentially of’ the embodiments or elements presented herein, whether explicitly set forth or not.

[0091] The term “about” as used herein in connection with a specific value (e.g. temperature, concentration, time and others) shall refer to a variation of + / - 1 % of the specific value that the term “about” refers to.

[0092] SPECIFIC EMBODIMENTS OF THE INVENTION

[0093] It has been found that for generating Chinese hamster iPSCs the findings made for the generation of murine iPSCs are not transferable and that findings from the prior art point in a non-working direction.

[0094] The current invention is based at least in part on the finding that specific factors, i.e. additives, and a specific culturing protocol are required for the successful generation and maintenance of hamster derived induced pluripotent stem cells. The required conditions were identified by a nonintegrating and an inducible integrating reprogramming approach using novel combinations of small molecules and primary Chinese hamster fibroblasts and an immortalized hamster cell line. The current invention is exemplified in the following using Chinese hamster cells. These cells are used merely to exemplify the invention and shall not be construed as limiting the scope of the invention that is set forth in the appended claims, i.e. the method according to the current invention is applicable to any hamster cell.

[0095] In Figure 1 a way for the identification of mandatory components to be used in the method according to the current invention is exemplified for Chinese hamster cell derived iPSC generation. In more detail, primary Chinese hamster fibroblasts (CHAF) and an immortalized Chinese hamster ovary (CHO) cell line were used in combination with non-integrating Sendai virus and stable integration of inducible reprogramming factors, respectively. Advantageous replating timing, growth factor requirement and essential small molecule factors could be identified.

[0096] Primary Chinese hamster fibroblasts (CHAF) were generated from skin biopsies of Chinese hamsters (Cytogen Research and Development, Inc.) by enzymatic and mechanical separation of cells and culturing with appropriate cell culture media. To generate hamster iPSCs, a state of the art Sendai virus transduction protocol (see Figure 2A) using a commercially available kit (CytoTune™-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher) to introduce the Yamanaka factors Sox-2, Oct-4, Klf-4 and c-Myc was used. As reference and positive control for Sendai virus mediated generation of iPSCs, murine embryonic fibroblasts (MEFs) (MEF (C57BL / 6) [MEF- BL / 6-1], ATCC) were employed. Briefly, cells were thawed and plated to cell culture dishes at day “-7” and cultured in fibroblast medium for 7 days to increase cell number and reduce stress of cells from the thawing event. At day “0”, cells were transduced with Sendai virus particles and cultured another 7 days in fibroblast cell culture media. At day “7”, cells were replated onto y- irradiated MEF feeder layer and medium was changed to embryonic stem cell (ESC) medium the day after. After additional 7 days in culture, cells were expanded and replated on y-irradiated MEFs or different combinations of cell culture surface matrixes and cell culture media.

[0097] First iPSC colonies from MEF were identified 9 days after Sendai virus transduction by characteristic cobblestone-like colonies with sharp delimited colony boundaries, which increased in size during time in culture (see Figure 2B). The colonies showed a bright fluorescence live cell staining of the characteristic stem cell maker alkaline phosphatase at day 18. Unexpectedly, in the period of the experiment, no iPSC colonies could be detected originating from CHAFs.

[0098] Thus, despite knowledge from the art it is shown in Figure 2 that Chinese hamster cells, especially fibroblasts, cannot be reprogrammed by Sendai virus transduction of Yamanaka factors alone.

[0099] In Figure 3 it is shown that certain combinations of cell culture media and matrix conditions do not enable Chinese hamster derived iPSC generation. In more detail, bright field images of Sendai virus transduced CHAFs replated at 14 days and cultured by combinations of different cell culture surface matrixes and cell culture media were taken. That is, testing Sendai virus transduced CHAF by different combinations of cell culture surface matrixes and cell culture media did also not rescue the formation of iPSC colonies.

[0100] In a subsequent experiment, the protocol was adapted to a combination of Sendai virus mediated expression of Yamanaka factors Sox-2, Oct-4, Klf-4 and c-Myc with small molecule cocktails that have been described to induce or boost iPSC generation in other cellular systems as hamster cells. Briefly, the respective small molecule cocktail (see Figure 9A for the respective compositions) was added at day “0” parallel to the Sendai virus transduction and removed from the cell culture media at day 14.

[0101] Again, first iPSC colonies derived from MEFs were detected at day 11, and no colonies were formed in two different CHAF cell lines in absence of small molecule cocktails (see Figure 4B).

[0102] The test of six different small molecule cocktails SM40, SM41, SM43, SM44, SM46, and SM51 added to the Sendai virus transduced CHAF-2 cultures revealed no colonies at day 3, same as for the MEF control experiment.

[0103] Surprisingly, at day 11 iPSC-line colonies were detected in the CHAF culture that was treated with the small molecule cocktail SM51 (see Figure 4C) but not with the other small molecule cocktails.

[0104] In Figure 4 it is shown that the hybrid reprogramming approach according to the current invention employing Sendai virus transduction of Yamanaka factors in combination with a specific small molecule cocktail, i.e. SM51, enables Chinese hamster iPSC generation. In more detail, in Figure 4 comparative bright field images of murine embryonic fibroblasts (MEF) and two primary CHAF lines, CHAF-0 and CHAF-2, after day 3 and 11 of exclusively Sendai virus transduction are shown. These were used for analysis of cell morphology and identification of iPSC colonies. A murine iPSCs colony that originated from MEFs is highlighted by a circle and black arrowhead. Additionally, bright field images of CHAF line CHAF-0, after day 3 and 11 of combined Sendai virus transduction and small molecule (SM) cocktails SM40, SM41, SM43, SM44, SM46, and SM51 treatment are shown. These were likewise used for analysis of cell morphology and identification of iPSC colonies. It can be seen that in contrast to the exclusive Sendai virus transfection, Chinese hamster iPSCs colony originating from CHAFs by Sendai virus transduction and small molecule cocktail, i.e. SM51, treatment could be identified (highlighted by circle and black arrowhead).

[0105] To rule out that different background expression of Yamanaka factors Sox-2, Oct-4, Klf-4 and c- Myc caused this observation, all cultures were tested for the mRNA expression of Sox-2, Oct-4, Klf-4 and c-Myc by qPCR. By this it could be shown that all cultures transduced with Sendai virus had similar high levels of reprogramming factor mRNA compared to negative controls (see Figure 5), indicating that SM51 enables CHAF cells to reprogram while the other conditions do not. In more detail, in Figure 5 the efficiency of Sendai virus induced expression of the Yamanaka factor in MEFs and CHAFs is shown. Yamanaka factor mRNA expression has been analyzed by qPCR in MEFs (M), CHAF-0 (CO), CHAF-2 (C2), and of CHAF-2 with SM40, SM41, SM43, SM44, SM46, and SM51. Numbers represent values of qPCR crossing point analysis.

[0106] According to state of the art protocol, a small molecule cocktail should be removed from the cultures on cultivation day 14 after transduction. This was done accordingly. Three days after the removal, i.e. at cultivation day 17, the cell cultures were re-inspected by bright field microscopy. Unexpectedly, iPSC colonies could not be detected anymore. This unexpectedly showed that the presence of SM51 is required in order to maintain the state of hamster iPSC colonies in culture (see Figure 6).

[0107] In Figure 6 it is shown that maintenance of iPSC status and morphology is dependent on the small molecule cocktail used for reprogramming, i.e. on the small molecule cocktail SM51 as one preferred small molecule cocktail of the method according to the current invention. In more detail, in Figure 6 bright field images of a CHAF line at day 13 after combined Sendai virus and small molecule cocktail SM51 treatment and of MEFs are shown. Further images of two cell culture sections after day 17 with removal of the small molecule cocktail, i.e. SM51, on day 14 are shown. Small molecule cocktail SM46 was used as a negative control. Chinese hamster iPSCs colonies originated from CHAFs by Sendai virus transduction and small molecule cocktail, i.e. SM51, treatment and the dependency on the small molecule cocktail, i.e. SM51, is highlighted by circles and black arrowheads.

[0108] The relevance of the different compounds of the small molecule cocktail SM51, which has been found to be essential for hamster iPSC generation with Sendai virus mediated expression of Sox- 2, Oct-4, Klf-4 and c-Myc, was evaluated by withdrawing at day 14 after transduction single compounds of SM51, i.e. CHIR99021 (CHIR), 3-deazaneplanocin A (DZNep), forskolin, RepSox, tranylcypromine, aprotinoid acid (TTNPB), or valproic acid (VP A), from the small molecule cocktail added to the cultures (see Figure 9C). The cell cultures were examined at day 21, i.e. 7 days after the removal, for cell colony morphology by bright field microscopy and life cell epifluorescence microscopy for alkaline phosphatase (AP).

[0109] Unexpectedly, the requirement for the presence of CHIR was found, which was found to be mandatory to maintain hamster iPSC colony morphology and proliferation (see Figure 7A, 7B). IPSC colonies in all other tested conditions indicated the start of cell differentiation as evidenced by the presence / occurrence of long elongated cellular outgrowths from the colonies and disruption of the sharp colony boundaries.

[0110] Summarizing this, in Figure 7 it is shown that maintenance of iPSC status and morphology is dependent on the small molecule additive CHIR (CHIR99021). This is evidenced by bright field images and epifluorescence AP-life staining of CHAF line CHAF-0, at day 21 after combined Sendai virus and small molecule cocktail, i.e. SM51, treatment for 14 days post transduction, with removal of individual components of the small molecule cocktail, i.e. SM51, at day 14, i.e. after cultivation for 14 days with the complete small molecule cocktail SM51, and for an additional 7 days with individual components of the small molecule cocktail SM51 removed. Chinese hamster iPSCs colonies originated from CHAFs by Sendai virus transduction and small molecule cocktail, i.e. SM51, treatment and dependency on the small molecule cocktail component CHIR is highlighted by black and white arrowhead.

[0111] In a subsequent experiment, the relevance of different cell culture media and the proteinogenic factor bFGF (basic fibroblast growth factor) on hamster iPSC maintenance were tested. For this, cells were cultivated with fresh ESC (embryonic stem cell) media, containing LIF (leukemia inhibitory factor) or with ESC media, which contains LIF, blended with mTeSR medium, which contains bFGF, both with 50% reduced SM51 compound concentrations, which according to the current invention supports and maintains the hamster iPSC colony morphology until day 14. Unexpectedly, the use of the ESC / mTeSR (1 : 1) blend fully recovered the hamster iPSC colony phenotype and restarted cell proliferation (see Figure 8).

[0112] In more detail, in Figure 8 it is shown that iPSC status and morphology is dependent on the specific culture medium composition. Bright field images of iPSC colonies originated from cell lines CHAF-0 and MEF at day 21 after combined Sendai virus and small molecule cocktail, i.e. SM51, treatment for CHAFs and exclusively Sendai virus treatment for MEFs as well as images of representative cell culture sections of CHAF derived iPSC colonies cultured in ESC media with 0.5x SM51 or ESC / mTeSR (1 : 1) blend with 0.5x SM51 and MEF derived iPSC colonies with ESC media are shown.

[0113] In Figure 9 an overview of the herein used small molecule cocktails, concentration of compounds and proposed mode of action is provided. The composition of small molecule cocktail SM51 as one preferred small molecule cocktail for use in the method according to the current invention with the respective target pathway of the compounds, proposed concentration ranges, and tested concentrations, Cl and C2 is given in detail.

[0114] In subsequent studies the effect of bFGF alone and / or LIF and bFGF in combination for long-term cultivation of hamster iPSCs was determined. Different replating days after Sendai virus transduction and different media with different combinations of SM51, LIF, bFGF, CHIR and mTeSR were tested (see Figure 10A). According to state of the art methods, Sendai-Virus transduced cells are normally replated after 7 days on irradiated MEFs, followed by a switch to stem cell medium on the next day (i.e. on day 8 (d8) after transduction). Later replating days (dl2 and dl 8) were examined and the effect of CHIR99021, bFGF, LIF in different combinations in ESC medium was evaluated (see Figure 10A). After 35 days, samples from all conditions were fixed and stained for the pluripotency marker SSEA1. Although SSEA1+ colonies were visible in all replating days-conditions (replated after 7d, 12d and 18d), unexpectedly the highest amount of SSEA1+ colonies was detected in the condition replated at day 12 after transfection, i.e. maintained 5 days longer than the state of the art replating at day 7 (see Figure 10B).

[0115] Furthermore, a clear effect of the supplemented small molecules was found. This is shown in Figure 10B. Medium 8 (see Figure 10 A), which is supplemented with CHIR99021 and bFGF, showed in all three replating-day conditions the highest number of SSEA1+ colonies, supporting our previous findings, that CHIR99021 and bFGF are supporting and enhancing factors for the generation and cultivation of hamster iPSC. This is one preferred embodiment of the method according to the current invention.

[0116] Further is shown in Figure 10C that an exemplary alkaline phosphatase (AP) staining of CHAF- derived reprogrammed Chinese hamster fibroblast induced pluripotent stem cells (chiPSC) by Sendai virus transduction in the presence of SM51 and Medium 8 at passage day 12. The pictures show example chiPSC colonies in 4x and lOx magnification.

[0117] Thus, from Figure 10 it can be seen that CHIR99021 and bFGF as well as a replating after 12 days are advantageous factors for colony formation during reprogramming of CHAFs. These are all individually and in combination preferred embodiments of the current invention.

[0118] An immortalized Chinese Hamster Ovary (CHO) cell line was used to evaluate the relevance of different reprogramming factors with and without tetracycline-inducible expression and with and without addition of small molecule factors to the cultivation medium. In total, eight different reprogramming factors, namely Sox-2, Oct-4, Klf-4, c-Myc, Esrrb, Nanog, Lin28, and Sall4 were cloned and combined for stable CHO pool generation by eight variations (see Figure 11 A). As controls, a single fluorescence cassette or a tri-fluorescence cassette with endogenous promoters were used. The fluorescence cassettes contained fluorescence sensor protein expression regulated by differentiation status active synthetic promoters to allow for live cell imaging and sorting of respective cells (Fig. 14C). For the generation of stable CHO pools, transfected CHO cells were cultivated in selection pressure Puromycin containing cell culture medium. Unexpectedly, none of the pools with constitutive expression of the reprogramming factors recovered, which indicated a toxic and / or cytostatic effect of constitutive reprogramming factor expression.

[0119] For inducible expression of reprogramming factors, two genetic gene combination conditions combining either Sox-2, Oct-4, Klf-4, and c-Myc, or Sox-2, Oct-4, Klf-4, c-Myc, Esrrb, Nanog, Lin28, and Sall4 were selected. A tetracycline-repressor regulated CMV promoter was introduced for inducible expression of the reprogramming factors. By this, both respective inducible pools, i.e. Pool 9 and Pool 10, recovered as expected and stable pools could be generated. To evaluate the potential of the stable CHO pools to be reprogrammed, doxycycline (an oxytetracycline derivative of the group of tetracyclines) was added to the cell culture medium of growing stable CHO pools. The activation of the reprogramming factors expression in Pool 9 and 10 alone in the absence of SM51 does not induce reprogramming of the CHO pools and the cultures stopped growing and died after a few days.

[0120] Thus, from Figure 11 it can be seen that inducible expression of reprogramming factors and small molecule cocktail are required for CHO SSEA1 formation. In more detail, a CHO cell line was transfected with different combinations of reprogramming factors either under constitutive or tetracycline-inducible (TetOn) expression promoters. For the generation of stable pools, cells were cultivated for 14 days with selective pressure containing medium. It can be seen that only TetOn Pools 9, 10 and 11 recovered, indicating a toxic and / or cytostatic effect of constitutive reprogramming factor expression (red: no pool recovery; green: pool recovery). SSEA1 presence in Pool 9 and 10 were detected exclusively by simultaneously reprogramming factor expression by tetracycline induction and small molecule cocktail supplementation to the cell culture medium (red: no SSEA1 detection; green: SSEA1 detection). An exemplary SSEA1 FACS staining of stable CHO Pool 9 with small molecule cocktail at day 4 after induction of reprogramming factors compared to induced Pool 9 without small molecules is shown in Figure 11C. The amount of SSEA1 positive cells is shown in the gated area.

[0121] Without being bound by this theory, it is assumed that one or more endogen supportive pathways are dysregulated in hamster cells, yet needed for correct induced stem cell formation initiation, maturation and stabilization.

[0122] Potential dysregulated pathways in hamster cells needed for iPSC generation are not known. To determine the relevance of small molecule factors on CHO cells, a library of compounds reported to support reprogramming events in eukaryotic cells, yet dominantly used in human and mouse iPSC models, was generated (see Table 1). None of the factors has been reported before to support hamster iPSC generation.

[0123] Table 1: Small molecule library tested for the Chinese hamster iPSC generation approaches.

[0124] The type of cellular mechanism and modulated signaling pathway as well as molecular target is listed for each small molecule factor.

[0125] Statistical experiment planning by Design of Experiments (DoE) is a well-known technique especially for biological and chemical reactions where many factors independently influence the final result. For example, DoE was successfully used for cell culture media (Zhang, H., et al., Cytotechnol. 65 (2013) 363-378), fermentation processes (Fu, Z., et al., Biotechnol. Prog. 28 (2012) 1095-1105), protein purification processes (Pezzini, J., et al., J. Chrom. A. 1218 (2011) 8197-8208), and cell culture conditions (Koopaei, N.N., AMB Express. 8 (2018) 135).

[0126] Figure 12 shows the different approaches for DoE that can be used depending on the desired experiment outcome. For example, three factors are normally varied between a minimum (-1), medium (0), and maximum level (+1) for DoEs. (Picture adopted from http: / / www.gmpua.com and modified).

[0127] Fractional factorial designs, often used for systems with a huge number of factors of influence, allow the identification of significant principal modulators in the process. On the other site, experiment designs like Full factorial, Box Behnken, or Central composite not only identify critical modulators but rather allow quantification of discrete optimal concentrations or settings thereof. For that, so-called Prediction profiler tools postulate interactively experiment outcomes when concentrations or settings of critical modulators are in silico varied in a minimum (-1) and maximum (1) state. For calculating optimal concentrations, a predicted optimum level (a value between -1 and +1) has to be determined by multiplication with used set up concentration.

[0128] To test the 37 selected small molecule factors alone and in each possible combination a comprehensive DoE approach was applied to identify relevant small molecule factors advantageous for CHO reprogramming. The DoE approach was planned and analyzed using the statistical software tool JMP (SAS Institute GmbH, Heidelberg, Germany). A custom D-optimal design with 37 small molecule factors was generated, including the test factors LIF and doxycycline, with three levels of concentrations, and four center points. By this, 48 single experiments were defined. The experimental D-optimal design was applied for both stable CHO pools, i.e. Pool 9 and Pool 10, to allow for generalization of results. In sum, 96 single experiments were performed and the expression level of SSEA1 (determined by mean fluorescence intensity (MFI) by flow cytometry) were used as readout. For the cell cultivation experiment, stable CHO pools, Pool 9 and Pool 10, were seeded on Matrigel in mTeSR medium. The respective upper, medium and lower level of each tested small molecule factor in the DoE is listed in Table 2 and the DoE matrix with respective used small molecule factor combinations and concentrations is shown in Table 3. Doxycycline addition to induce reprogramming factor expression was added simultaneously to the other small molecule factors in the respective concentrations for each experiment (see Table 3). The cells were cultivated for 8 days and subsequently analyzed for SSEA1 expression by flow cytometry (FACS). Table 2: Test compounds used in the D-optimal DoE design with applied upper, middle and lower limit concentrations.

[0129] Table 3: D-optimal DoE matrix with respective tested compounds and concentrations. The

[0130] 48 experiments were applied to both stable CHO pools, Pool 9 and Pool 10.

[0131]

[0132]

[0133] The cells did not die in the DoE approach as observed before without any small molecule factor included. The cells showed high viability and SSEA1 levels could be analyzed for each single experiment. The SSEA1 levels and the data were used to fit a multi regression model by the software JMP. A p-value guided stepwise reduction of factors in the model were used to generate the best fit of the data with R2=0.95, RMSE=25.631, and p<0.0001 as plotted by the predicted versus observed SSEA1 MFI data (see Figure 13A). In more detail, Figure 13 shows the identification of small molecule factors advantageous for hamster SSEA1 expression. A plot of the actual versus predicted Mean Fluorescence Intensity (MFI) of SSEA1 for all 96 single experiments of the D-optimal DoE approach is shown. Further are shown sorted parameter estimates for the p-value guided stepwise reduced relevant factors that are needed for model building. Finally, 17 factors were identified with a p-value <0.05 needed to fit the observed data and 12 factors have a positive effect on improved SSEA1 MFI when supplemented to the cell culture medium (see Table 4). The identified advantageous concentration of the 12 factors supporting Chinese hamster iPSC generation is based on positive linear regression, and by this, at least equal or even higher than the tested upper limit in the DoE (see Table 5). Table 4: Identified small molecule factors to enable Chinese hamster iPSC generation.

[0134] Table 5: Identified Chinese hamster iPSC generation enabling small molecule factors with optimal concentrations.

[0135] In sum, these twelve advantageous small molecule factors that target nine different pathways that need to be inhibited or activated to enable stem cell factor SSEA1 formation were identified. Unexpectedly, LIF was not mandatorily needed for the formation of SSEA1 in stable CHO pools.

[0136] Thus, the current invention is based, at least in part, on the finding that the WNT pathway has to be activated, preferably by inhibiting GSK3b. Any WNT pathway activators that inhibit GSK3b can be used. GSK3b inhibitors that can be used in the method according to the current invention to activate the WNT pathway are reported in, e.g., Tran and Zheng (Prot. Sci. 26 (2017) 650-661) as well as Law and Zheng (iScience 25 (2022) 104159). In certain embodiments, the WNT pathway activator is selected from the group consisting of Lithium chloride, preferably at a concentration of about 1-2 mM, CHIR99021, preferably at a concentration of about 0.004-5 mM, SB-216763, preferably at a concentration of about 5.7-12 mM, BIO, preferably about 0.008- 0.48 mM, SB-415286, preferably about 0.078-1 mM, TWS-119, preferably about 0.03-3 mM, AR-A014418, preferably about 0.142-10 mM, LY-2090314, preferably about 0.010-5 mM, PF- 04802367, preferably about 0.009-1 mM, L807mts, preferably about 1-100 mM, and Tideglusib.

[0137] In one preferred embodiment, the small molecule as well as its concentration in the method according to the current invention in step b) or / and c) are at least 0.1 nM BIO, at least 20 pM CHIR99021 (CHIR), at least 6.5 pM CD437, at least 5 pM SAHA, at least 1 pM BIX01294, at least 0.3 pM Cyclic Pifithrin-a, at least 5 pM G66983, at least 2.5 pM SB431542, at least 5.6 pM SP600125, at least 1 pM aprotinoid acid (TTNPB), at least 1 pM Thiazovivin, or at least 275 pM valproic acid (VP A) or any combination thereof.

[0138] ***

[0139] All publications, patents, and patent applications cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, and patent application were specifically and individually indicated to be so incorporated by reference. In the event that one or more of the incorporated literature and similar materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls.

[0140] ***

[0141] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0142] That is, although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein and the claimed invention below. The examples are provided to better illustrate the disclosed teachings and are not intended to limit the scope of the teachings presented herein. While the present teachings have been described in terms of these exemplary embodiments, the skilled artisan will readily understand that numerous variations and modifications of these exemplary embodiments are possible without undue experimentation. All such variations and modifications are within the scope of the current teachings.

[0143] General Methods

[0144] Chinese hamster skin fibroblast (CHAF) cell line and cultivation

[0145] Chinese hamsters (Cytogen Research and Development, Inc.) were anesthetized following institutional animal care and use committee (IACUC) guidelines. A small area of the skin was shaved and sterilized with 70 % ethanol. Using sterile surgical instruments, a small skin biopsy (approximately 0.5 cm2) was excised from the dorsal region of the hamster. The biopsy was immediately placed in a sterile 50 mL conical tube containing cold PBS. In a laminar flow hood, the skin biopsy was transferred to a sterile Petri dish containing PBS. The biopsy was minced into small pieces (1-2 mm3) using sterile scissors and forceps. Minced tissue was transferred to a 15 mL conical tube containing 5 mL of DMEM supplemented with 1 mg / mL collagenase type I. The tube was incubated at 37 °C for 1-2 hours with gentle agitation to facilitate enzymatic digestion. After digestion, the tissue suspension was filtered through a 70 pm cell strainer into a new 50 mL conical tube to remove undigested tissue fragments. The filtrate was centrifuged at 300 x g for 5 minutes to pellet the cells. The supernatant was aspirated, and the cell pellet was resuspended in 5 mL DMEM supplemented with 10 % FBS and 1 % Pen-Strep (Penicillin / Streptomycin). The cell suspension was transferred to a 6-well culture plate and incubated at 37 °C in a humidified atmosphere with 5 % CO2. The medium was changed every 2-3 days to remove non-adherent cells and debris. Once fibroblast colonies were observed (typically within 5-7 days), cells were allowed to reach 70-80 % confluence. Confluent fibroblast cultures were washed with PBS and detached using 0.05 % Trypsin-EDTA. Cells were collected in a 15 mL conical tube, centrifuged at 300 x g for 5 minutes, and resuspended in fresh DMEM with 10 % FBS and 1 % Pen-Strep. Cells were seeded into new culture plates at a 1 :3 or 1 :4 split ratio and maintained under standard culture conditions. Fibroblast identity was confirmed by morphological assessment under a phase-contrast microscope. Immunocytochemistry or flow cytometry for fibroblast-specific markers (e.g., vimentin) was performed to further validate the cell type.

[0146] Murine embryonic fibroblasts (MEFs) cultivation

[0147] Mouse embryonic fibroblasts (MEF (C57BL / 6) [MEF-BL / 6-1], ATCC SCRC-1008™) were cultured in DMEM supplemented with 10 % FBS and 1 % Pen-Strep.

[0148] Chinese hamster ovary cell line

[0149] A modified Chinese hamster ovary (CHO) cell line (see WO 2019 / 126634), adapted to suspension, was cultivated in a chemically defined, protein-free DMEM / F12-like cell culture media. Exemplarily, used CHO cell lines and pools can be cultivated in DMEM media (e.g. DMEM, high glucose, no glutamine, no methionine, no cystine Cat.# 21013, Thermo Fisher Scientific - supplemented with glutamine, methionine, reducing agent and glucose). The cells were incubated at 37 °C in a humidified atmosphere with 5 % CO2. The medium was changed and cells were split every 3-4 days.

[0150] Inactivation of murine and Chinese hamster iPSCs:

[0151] 6-well culture plates were coated with 0.1 % gelatin solution and incubated at 37 °C for at least 30 minutes.

[0152] MEFs were mitotically inactivated by treatment with mitomycin C (10 pg / mL) for 2-3 hours or by y-irradiation (60 Gy). Inactivated MEFs or CHAFs were seeded onto gelatin-coated 6-well culture plates at a density of 2E5 cells / well and allowed to adhere overnight.

[0153] Alkaline

[0154] Murine iPSCs and Chinese hamster pluripotent stem cells (chiPSCs) were cultured in 6-well plates using standard stem cell culture medium. Cells were maintained at 37 °C in a humidified atmosphere with 5 % CO2. The culture medium was aspirated, and cells were gently washed twice with PBS. For Alkaline Phosphatase Live Staining (Thermo Fisher Scientific, Cat. No. A14353), the reagent was diluted 1 :500 in PBS according to the manufacturer's instructions. 1 mL of the diluted stain was added to each well, and the plates were incubated at 37 °C for 20 minutes. After incubation, the stain was aspirated, and cells were washed twice with PBS to remove excess stain. Stained cells were analyzed under an inverted fluorescence microscope (Zoe Fluorescence Imager, BioRad).

[0155] For enzymatic Alkaline Phosphatase Staining (abeam Cat. No. ab242286) the culture media from each well was aspirated and rinsed with PBS. The rinse solution was removed and the cells were fixed with 4 % PFA at room temperature for 5 min. After fixation, the PFA solution was removed and the fixed cells rinsed with PBS. The staining was conducted according to the manufacturer's protocol using staining solutions. In short, FRV was mixed with Naphtol and water in a ratio 2: 1 : 1. The solution was added to the cells and incubated for 15 min. at RT in the dark. Subsequently, the staining solution was removed by aspiration and the cells were washed two times with PBS. Images were captured to determine the presence and distribution of alkaline phosphatase activity, indicative of pluripotent stem cells with positive AP staining.

[0156] SSEA1 FACS:

[0157] Murine iPSCs and Chinese hamster pluripotent stem cells (chiPSCs) were dissociated into single cells using 0.05 % Trypsin-EDTA (Thermo Fisher, Cat. No. 15400054). Cells were collected in 15 mL conical tubes and centrifuged at 300 x g for 5 minutes. The cell pellet was resuspended in PBS with 2 % FBS to a concentration of 1E6 cells / mL. 100 pL of the cell suspension (1E5 cells) was transferred to individual flow cytometry tubes. Cells were incubated with 5 pL of anti-SSEAl FITC antibody (BioLegend, Cat. No. 125610) 30 min. at 4 °C in the dark. An isotype control antibody was used to stain a separate aliquot of cells under the same conditions. After incubation, cells were washed twice with PBS with 2 % FBS and resuspended in 500 pL of PBS with 2 % FBS. Stained cells were analyzed using flow cytometry (BD Celesta). Data were collected and analyzed to determine the percentage of cells expressing the SSEA1 marker, indicative for mouse pluripotent stem cells.

[0158] Sendai Virus Reprogramming Approach

[0159] Primary Chinese hamster fibroblasts (CHAF) were generated from skin biopsies of Chinese hamsters (Cytogen Research and Development, Inc.) by enzymatic and mechanical separation of cells and culturing with appropriate cell culture media.

[0160] A classical Sendai virus transduction protocol (Figure 2A) using a commercially available kit (CytoTune™-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher) to introduce the Yamanaka factors Sox-2, Oct-4, Klf-4 and c-Myc was used. As reference cell model and positive control for Sendai virus mediated generation of iPSCs, murine embryonic fibroblasts (MEFs) (MEF (C57BL / 6) [MEF-BL / 6-1], ATCC) were integrated in the experimental design.

[0161] Murine embryonic fibroblasts were cultured in fibroblast culture medium in 6-well plates until they reached 70-80 % confluence. The culture medium was aspirated, and cells were washed once with PBS. Commercially available kit (CytoTune™-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher, Cat. No. A16517) for Sendai virus vectors encoding the reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) was used according to the manufacturer's instructions. The virus mixture was added to the fibroblasts in a fresh fibroblast culture medium, and the plates were incubated at 37 °C for 24 hours. After 24 hours, the virus-containing medium was replaced with fresh fibroblast culture medium. Cells were cultured for an additional 5-7 days, with medium changes every other day. Transduced fibroblasts were harvested using 0.05 % Trypsin-EDTA and seeded onto gammairradiated MEFs plates in stem cell culture medium. Medium was changed daily, and colonies with iPSC morphology began to appear around day 10-14 post-transduction. Individual iPSC colonies were manually picked and transferred to new Matrigel-coated plates for expansion. Established iPSC lines were maintained in stem cell culture medium and characterized for pluripotency markers.

[0162] Combined Sendai Virus Reprogramming and Small Molecule Cocktail Approach

[0163] Primary Chinese hamster fibroblasts (CHAF) were generated from skin biopsies of Chinese hamsters (Cytogen Research and Development, Inc.) by enzymatic and mechanical separation of cells and culturing with appropriate cell culture media.

[0164] A combined Sendai virus transduction using a commercially available kit (CytoTune™-iPS 2.0 Sendai Reprogramming Kit, Thermo Fisher) to introduce the Yamanaka factors Sox-2, Oct-4, Klf- 4 and c-Myc and cultivation with a small molecule cocktail was used. As reference cell model and positive control for Sendai virus mediated generation of iPSCs, murine embryonic fibroblasts (MEFs) (MEF (C57BL / 6) [MEF-BL / 6-1], ATCC SCRC-1008™) were integrated in the experimental design.

[0165] Briefly, cells were thawed and plated to cell culture dishes at day “-7” and cultured in fibroblast media (DMEM (PAN Biotech, P04-03609), 15 % FBS, 2 mM L-glutamine, 10 pM 2- mercaptoethanol, 10 U / mL Penicillin-Streptomycin) for 7 days to increase cell number and reduce stress of cells from the thawing event. On day “0”, cells were transduced with Sendai virus particles and the small molecule cocktails (Figure 9A) were added parallel to the Sendai virus transduction. The transduced cells were cultured another 7 days in fibroblast cell culture media. At day “7”, cells were replated onto y-irradiated MEF feeder cell layer and media was changed to embryonic stem cell (ESC) media the day after. After additional 7 days in culture (dl4), cells were expanded and replated on y-irradiated MEFs or different combinations of cell culture surface matrixes (Vitronectin (Stem Cell Technologies Cat. No. 07180), Geltrex (Thermo Fisher Cat. No. A1569601), Matrigel (Corning, Cat. No. 354277) or Laminin 521 (Stem Cell Technologies, Cat. No. 77003) and cell culture media in the presence or absence of the respective small molecule cocktail or individual components thereof.

[0166] Examnle 3 - method according to the invention

[0167] Combined Inducible Overexpression of Reprogramming Factors and Small Molecule Cocktail Approach

[0168] A CHO cell line, adapted for growing in suspension was transfected with various plasmid combinations to allow for constitutive or tetracycline inducible-expression of reprogramming factors Sox-2, Oct-4, Klf-4, and c-Myc, or Sox-2, Oct-4, Klf-4, c-Myc, Esrrb, Nanog, Lin28, and Sall4. For the inducible expression cassettes, a tetracycline-repressor regulated CMV promoter (Tet) was introduced in the expression cassettes of the respective reprogramming factors. Expression was regulated by addition of doxycycline (an oxytetracycline derivative of the group of tetracyclines) to the cell culture respective the cell culture medium.

[0169] In more detail, for plasmid generation, the coding sequences of reprogramming factors, namely Sox-2, Oct-4, Klf-4, c-Myc, Esrrb, Nanog, Lin28, and Sall4, or fluorescence sensors as control were used (see Figure 14).

[0170] All plasmid constructs were generated by standard cloning procedures, with the respective sequences synthesized (Twist Biosciences). The factors were combined for stable CHO pool generation as described in Figure 11 A using a CHO host cell line with a defined integration locus (see WO 2019 / 126634) either with or without Tet-inducible expression. CHO cells were cultured in a proprietary Dulbecco's Modified Eagle's Medium / F 12-based medium in 125-500 mL shake flask vessels at 150 rpm, 37 °C, 80 % rH and 5 % CO2. Cells were passaged at a seeding density of 3-6E5 cells / ml every 3-4 days. Pools of cells that stably express reprogramming factors either without or with Tetracycline induction were generated as described by Carver et al. (Biotechnol. Prog. 36 (2020) e2967). Briefly, expression plasmids were transfected into CHO cells by MaxCyte STX electroporation (MaxCyte, Inc.).

[0171] For the identification of mandatory activated or / and inhibited pathways in CHO cells, a DoE with small molecules was performed (see Table 2). Here, recombinant CHO cell pools 9 and 10 were seeded on matrigel-coated plates (Coming, Cat. No. 354277) in the presence of defined small molecule combinations as well as doxycycline. FACS analysis for pluripotency marker SSEA1 was performed every other day to evaluate reprogramming potential and efficiency.

Claims

- 33 -Patent Claims1. A method for the production of one or more hamster-derived induced pluripotent stem cells comprising the following steps: a) transducing a hamster cell with a Sendai virus comprising coding sequences for reprogramming factors, b) cultivating the transduced cell in the presence of at least a WNT pathway activator for at least 7 days and less than 18 days, c) optionally replating the cells, adding a medium comprising one or more proteinogenic growth factors and cultivating the replated cells in the presence of the at least a WNT pathway activator, d) selecting one or more cells that are positive for alkaline phosphatase and SSEA1 and that has stem cell morphology, or a) transfecting a hamster cell with one or more nucleic acids comprising expression cassettes for one or more reprogramming factors, wherein the expression cassettes comprise an inducible promoter, b) cultivating the transfected cell in the presence of at least a WNT pathway activator for at least 7 days and less than 18 days and inducing the promoter of the reprogramming factors after 0-1 days, c) optionally replating the cells, adding a medium comprising one or more proteinogenic growth factors and cultivating the replated cells in the presence of the at least a WNT pathway activator, d) selecting one or more cells that are positive for SSEA1, thereby producing one or more hamster-derived induced pluripotent stem cells.

2. The method according to claim 1, wherein the cultivating in step b) or / and c) is in the presence of a small molecule cocktail comprising a WNT pathway activator.

3. The method according to claim 2, wherein the small molecule cocktail is completely removed at the time of replating and replaced by a WNT pathway activator only.- 34 -4. The method according to any one of claims 1 to 3, wherein the feeder cells are y-irradiated murine embryonic fibroblast (MEF) cells.

5. The method according to any one of claims 1 to 4, wherein the reprogramming factors are Sox-2, Oct-4, Klf-4 and c-Myc.

6. The method according to any one of claims 1 to 5, wherein the WNT pathway activator or the small molecule cocktail is added with or directly after the Sendai virus.

7. The method according to any one of claims 1 to 6, wherein the cultivating in steps b) and c) is for at least 14 days.

8. The method according to any one of claims 1 to 7, wherein the cultivating in step b) is for 12 days.

9. The method according to any one of claims 1 to 8, wherein the proteinogenic growth factor is bFGF.

10. The method according to any one of claims 1 to 9, wherein the transduction of step a) and the cultivation of step b) are in fibroblast medium.

11. The method according to any one of claims 1 to 10, wherein the method comprises prior to step a) the following step:0) cultivating a hamster cell in a fibroblast cultivation medium for about 7 days.

12. The method according to any one of claims 1 to 11, wherein the cultivating in step c) is in embryonic stem cell (ESC) medium.

13. The method according to any one of claims 1 to 12, wherein the cultivating in step b) or / and c) is in the presence of a small molecule cocktail comprising at least one of the specific WNT pathway activators CHIR99021 and BIO.

14. The method according to any one of claims 2 to 13, wherein the small molecule cocktail comprises CHIR99021 (CHIR), 3-deazaneplanocin A (DZNep), forskolin, RepSox, tranylcypromine, aprotinoid acid (TTNPB), and valproic acid (VP A).

15. The method according to any one of claims 2 to 14, wherein the concentration of the compounds of the small molecule cocktail in step b) or / and c) are about 10 pM CHIR99021 (CHIR), about 0.05 pM 3-deazaneplanocin A (DZNep), about 10 pm forskolin, about 5 pM RepSox, about 5pM tranylcypromine, about 1 pM aprotinoid acid (TTNPB) and about 500 pM valproic acid (VP A).

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