METHODS OF PREPARING NAÏVE HUMAN PLURIPOTENT STEM CELLS

Abstract

Methods of preparing naive human pluripotent stem cells are described. The methods include the use of xeno-free media and do not include the use of feeder cells.

Claims

1. A method for preparing a nave human pluripotent stem cell, the method comprising: providing a human pluripotent stem cell (HPSC); and culturing the HPSC in the absence of feeder cells and in the presence of vitronectin; thereby producing a nave HPSC.

2. The method of claim 1, wherein providing a HPSC comprises providing a primed HPSC.

3. The method of claim 1, wherein the vitronectin comprises full-length vitronectin.

4. The method of claim 1, wherein the vitronectin comprises vitronectin coated on a surface.

5. The method of claim 1, wherein culturing the HPSC in the absence of feeder cells comprises culturing the HPSC in a medium comprising at least one of insulin, fibroblast growth factor (FGF), transforming growth factor beta (TGF), and Activin.

6. The method of claim 1, the method further comprising culturing the HPSC in the absence of fibroblast growth factor (FGF) and transforming growth factor beta (TGF).

7. The method of claim 6, wherein the cells are cultured under hypoxic conditions.

8. The method of claim 1, the method further comprising culturing the HPSC in a medium comprising at least one of insulin, fibroblast growth factor (FGF), transforming growth factor beta (TGF), and Activin prior to culturing the HPSC in the presence of vitronectin.

9. The method of claim 1, wherein culturing the HPSC comprises culturing the HPSC in a xeno-free medium.

10. A method for preparing a nave human pluripotent stem cell, the method comprising: providing a human pluripotent stem cell (HPSC); culturing the primed HPSC in the absence of feeder cells and in the presence of vitronectin coated on a surface, wherein the vitronectin comprises full-length vitronectin, and wherein the HPSC are cultured in a xeno-free medium comprising at least one of insulin, fibroblast growth factor (FGF), transforming growth factor beta (TGF), and Activin; and then culturing the HPSC in a xeno-free medium in the absence of fibroblast growth factor (FGF) and transforming growth factor beta (TGF), wherein the cells are cultured under hypoxic conditions; thereby producing a nave HPSC.

11. The method of claim 10, the method further comprising culturing the HPSC in a xeno-free medium comprising at least one of insulin, fibroblast growth factor (FGF), transforming growth factor beta (TGF), and Activin prior to culturing the primed HPSC in the presence of vitronectin.

12. The method of claim 10, wherein providing an HPSC comprises providing a primed HPSC.

13. The method of claim 1, wherein the nave HPSC exhibits a normal karyotype.

14. The method of claim 1, wherein the nave HPSC does not differentiate in the presence of an ERK inhibitor.

15. The method of claim 10, wherein the nave HPSC exhibits a normal karyotype.

16. The method of claim 10, wherein the nave HPSC does not differentiate in the presence of an ERK inhibitor.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0022] FIG. 1 shows that nave pluripotent stem cells derived on vitronectin according to the methods described in Example 1 are distinct from primed pluripotent stem cells. (A) Morphology of a primed HPSC colony. (B) Morphology of a transitioned nave HPSC colony (Passage 10). ERK inhibitor PD0325901 induces differentiation in primed HPSC (C), as evidenced by morphological changes, but not in nave HPSC (D). Expression of NANOG, a transcription factor involved with self-renewal of undifferentiated embryonic stem cells, in primed (E) and transitioned (F) nave HPSCs. Expression of stage-specific embryonic antigen-3 (SSEA3), a marker of stem cell-like characteristics, in primed (G) and transitioned (H) nave HPSCs. Expression of stage-specific embryonic antigen-4 (SSEA4), a marker of stem cell-like characteristics, in primed (I) and transitioned (J) nave HPSCs.

[0023] FIG. 2A and FIG. 2(B-J) show nave pluripotent stem cells derived on vitronectin have a normal karyotype and pluripotent differentiation potential. FIG. 2A shows a representative karyotype of nave HPSCs after 10 passages in RSet media. FIG. 2(B-J) shows exemplary differentiated nave cells express the endoderm markers FOXA2 (B) and SOX17 (C), mesoderm markers CXCR4 (E) and Brachyury (F), and the ectoderm markers PAX6 (H) and Nestin (I). Corresponding DAPI staining is shown for the endoderm markers (D), the mesoderm markers (G), and the ectoderm markers (J).

[0024] FIG. 3 shows expression of pluripotent markers in primed and nave HPSCs. Expression of the pluripotency markers CDH1 and OCT4 is changed less than two-fold in nave HPSCs compared to primed HPSCs while expression of NANOG is increased in nave HPSCs compared to primed HPSCs. P18: primed HPSCs passage 18; N7: nave HPSCs passage 7; N10: nave HPSCs passage 10; N12: nave HPSCs passage 12.

[0025] FIG. 4 shows exemplary morphology of nave pluripotent stem cells derived in feeder-free conditions. (A) Morphology of a primed HPSC colony cultured on vitronectin. Colony morphology was followed during transition in RSet media after passage 1 (B), passage 7 (C) and passage 10 (D). (E) Morphology of a primed HPSC colony cultured on Matrigel. Colony morphology was followed during transition in RSet media after passage 1 (F), passage 7 (G), and passage 10 (H).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0026] This disclosure describes methods for the derivation of a nave human pluripotent stem cell (HPSC) from a primed HPSC using defined conditions.

[0027] Human pluripotent stem cells (HPSCs) cultured in conditions that maintain pluripotency via fibroblast growth factor (FGF) and transforming growth factor beta (TGF) signaling are described as being in a primed state. These cells have been shown to exhibit characteristics more closely related to mouse epiblast-derived stem cells than to so-called nave mouse pluripotent stem cells said to possess a more ground state pluripotency that mimics the early mouse embryo inner cell mass (Tesar et al. (2007) Nature, 448: 196-199; Hanna et al. (2010) PNAS, 107: 9222-9227).

[0028] At the time of the invention, culture conditions favorable for generation of nave HPSCs from primed HPSCs required the use of mouse embryonic fibroblasts as a feeder layer or a mixture of truncated vitronectin mixed with gelatin to support the transition from primed HPSCs to a nave HPSCs (Theunissen et al. (2014) Cell Stem Cell, 15: 471-487; Gafni et al. (2013) Nature, 504: 282-286). This disclosure describes a protocol for producing nave HPSCs from primed HPSCs in defined, xeno-free conditions. The methods described herein further describe maintenance of nave HPSCs in defined, xeno-free conditions. These methods are expected to allow stem cell researchers to enhance the study and clinical translation of nave HPSCs.

[0029] In one aspect, this disclosure describes a method for preparing a nave human pluripotent stem cell. The method includes providing a HPSC and culturing the HPSC in the absence of feeder cells and in the presence of vitronectin. In some embodiments, culturing the HPSC in the absence of feeder cells preferably includes culturing the HPSC in the absence of gelatin.

[0030] As used herein, a feeder cell is a cell on which stem cells, particularly a HPSC, may be plated and/or which provide a milieu conducive to the growth and maintenance of the stem cells in a pluripotent state. In some embodiments, culturing the HPSC in the absence of feeder cells includes culturing the HPSC in the absence of a conditioned medium. A conditioned medium is a medium taken from a culture of a feeder cell to maintain the HPSC in a pluripotent state without direct contact with the feeder cells.

[0031] In some embodiments, the HPSC provided is a primed HPSC. As used herein, a primed HPSC is a cell characterized by a flattened morphology (in a colony of cells), intolerance to passaging as single cells, and a dependence on bFGF and TGF/Activin signaling rather than LIF/Stat3 (Hanna et al. (2010) PNAS, 107: 9222-9227).

[0032] As used herein, a nave HPSC is a cell that can be cloned with high efficiency, can (in a colony of cells) grow in a packed dome colony, and is stabilized by LIF/Stat3 and destabilized by bFGF and TGF/Activin signaling. In some embodiments, a nave HPSC is characterized by having a tightly packed cell morphology that (in a colony of cells) forms a rounded, three-dimensional colony with distinct phase bright edges. In some embodiments, a nave HPSC can be passaged routinely using Tryp1E mediated single cell dissociation.

[0033] For example, the representative images in FIG. 1 of primed HPSCs show flattened cell colonies that are made up of numerous flattened individual cells; in contrast, the representative images in FIG. 1 of nave HPSCs show more rounded cells that contribute to a more rounded, dome-like colony.

[0034] The primed HPSC can include any suitable primed HPSC. In some embodiments, the primed HPSC is a primed human induced pluripotent stem cell (hiPSC). In some embodiments, a hiPSC can include a cell from the PCBC16ipS cell line (Ye et al. (2013) PLoS ONE 8 (1):e53764).

[0035] In some embodiments the vitronectin includes a full-length vitronectin. In some embodiments the vitronectin is a human vitronectin. In some embodiments, the vitronectin may include the amino acid sequence of SEQ ID NO:1. In some embodiments, the vitronectin may be recombinant. In some embodiments, the vitronectin is full-length vitronectin from PeproTech, Rocky Hill, N.J.

[0036] In some embodiments the vitronectin may preferably be coated on a surface. The surface may include a cell culture surface including, for example, a plate. In some embodiments, the vitronectin may be coated at a concentration of at least 1 microgram per milliliter (g/mL), at least 2 g/mL, at least 3 g/mL, at least 4 g/mL, or at least 5 g/mL. In some embodiments, the vitronectin may be coated at a concentration of up to 2 g/mL, up to 3 g/mL, up to 4 g/mL, up to 5 g/mL, up to 6 g/mL, up to 8 g/mL, or up to 10 g/mL. For example, in some embodiments, a 12-well tissue culture treated plate may preferably be coated with 5 g/mL full-length vitronectin.

[0037] Without wishing to be bound by theory, the culture matrix including vitronectin may be the limiting factor defining a successful transition to a nave pluripotent state in the feeder-free conditions described herein.

[0038] In some embodiments, culturing the HPSC in the absence of feeder cells may include culturing the HPSC in a medium including insulin, FGF, TGF, and/or Activin A. In some embodiments, the medium may preferably include mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada). In some embodiments, the medium may include a FGF/TGF1/Activin A-containing media described by Gafni et al. (2013) Nature, 504: 282-286 and/or by WIS-NHSM Human Nave Stem Cell Platform Approaches and Protocols, available on the world wide web at hannalabweb.weizmann.ac.il/wp-content/uploads/2015/08/Hanna-Lab-Detailed-and-Extended-WIS-NHSM-Formulations.pdf.

[0039] In some embodiments, the method further includes culturing the HPSC in the absence of FGF and TGF. In some embodiments, culturing the HPSC in the absence of FGF and TGF includes culturing the HPSC in the absence of Activin. In some embodiments, the HPSC may preferably be cultured in a medium including RSet media (STEMCELL Technologies, Vancouver, Canada). In some embodiments, the HPSC may be cultured in a medium including a FGF/TGF1/Activin A-free media described by Gafni et al. (2013) Nature, 504: 282-286 and/or by WIS-NHSM Human Nave Stem Cell Platform Approaches and Protocols, available on the world wide web at hannalabweb.weizmann.ac.il/wp-content/uploads/2015/08/Hanna-Lab-Detailed-and-Extended-WIS-NHSM-Formulations.pdf.

[0040] For example, the HPSC may be cultured in the absence of feeder cells and in the presence of vitronectin, FGF, TGF, and/or Activin, and then the HPSC may be cultured in the absence of FGF and TGF. In some embodiments, the HPSC may be cultured in the absence of FGF and TGF under hypoxic conditions. As used herein, hypoxic conditions may be defined as conditions having an oxygen level in a range from 1% to 15%. In some embodiments, hypoxic conditions may be defined as conditions having an oxygen level of 5%.

[0041] In some embodiments, the method further includes culturing the HPSC in a medium including insulin, FGF, TGF, and/or Activin. In some embodiments, the medium may preferably include mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada). In some embodiments, the medium may include a FGF/TGF1/Activin A-containing media described by Gafni et al. (2013) Nature, 504: 282-286 and/or by WIS-NHSM Human Nave Stem Cell Platform Approaches and Protocols, available on the world wide web at hannalabweb.weizmann.ac.il/wp-content/uploads/2015/08/Hanna-Lab-Detailed-and-Extended-WIS-NHSM-Formulations.pdf. In some embodiments, the HPSC may be cultured in a medium including insulin, FGF, TGF, and/or Activin prior to culturing the HPSC in the absence of feeder cells and in the presence of vitronectin.

[0042] In some embodiments, culturing the HPSC includes culturing the HPSC in a xeno-free medium during part of the culturing or during each part of the culturing. As used herein, a xeno-free medium is a medium that does not include a component derived from a different organism than the cell being cultured. In some embodiments, a xeno-free medium but may contain a component derived from the same organism as the cell being cultured. In some embodiments, a xeno-free medium is gelatin-free.

[0043] The methods described herein are intended to produce a nave HPSC. In some embodiments, the nave HPSC preferably exhibits a normal karyotype. In some embodiments, the nave HPSC preferably does not differentiate in the presence of an ERK inhibitor including, for example, PD0325901.

[0044] In some embodiments, the nave HPSC expresses markers characteristic of HPSCs including, for example, CDH1 and/or OCT4. In some embodiments, the nave HPSC has a level of expression of CDH1 and/or OCT4 within two-fold of the gene expression of a primed HPSC. In some embodiments, the nave HPSC has increased NANOG protein expression or NANOG gene expression relative to the protein or gene expression of a primed HPSC. In some embodiments, the nave HPSC have at least 1.1 fold, at least 1.3 fold, or at least 1.5 fold increased NANOG gene expression relative to the NANOG gene expression of a primed HPSC.

[0045] In some embodiments, the nave HPSC exhibits an ability to differentiate into three germ layer derivatives. In some embodiments, differentiation into three germ layer derivatives can be determined in vivo including, for example, by injection of cells into mice followed by pathological examination of hte presence of tissue from the three germ layers. In some embodiments, differentiation into three germ layer derivatives can be determined in vitro. In some embodiments, differentiation into three germ layers can be measured using STEMdiff Trilineage Differentiation Kit (05230, STEMCELL Technologies, Vancouver, Canada).

[0046] In some embodiments, the nave HPSC can be passaged. In some embodiments, the nave HPSC can be stably maintained after being passaged at least 10 times, at least 15 times, at least 20 times, at least 25 times, or at least 30 times. In some embodiments, the nave HPSC can be passaged using a TrypLE reagent (Thermo Fisher Scientific, Waltham, Mass.)-mediated single cell dissociation.

[0047] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

EXAMPLE

Example 1

[0048] This Example describes an exemplary protocol for transitioning primed HPSCs to a nave state using commercial RSet media (STEMCELL Technologies, Vancouver, Canada) and xeno-free recombinant full-length vitronectin.

[0049] Primed HPSCs maintained in defined conditions on a recombinant vitronectin substrate have a stereotypical morphology characterized by small cells with a large nuclear:cytoplasmic ratio that form flat monolayer colonies (FIG. 1A). After transition to the nave state, the HPSCs assumed a tightly packed cell morphology forming rounded, three-dimensional colonies with distinct phase bright edges (FIG. 1B). The nave cells can be passaged using TrypLE-mediated single cell dissociation. In contrast to other published protocols for feeder-free nave HPSC derivation, the cells transitioned to the nave state using the protocol of this Example exhibit a normal karyotype (FIG. 2A). When cultured in media including 10 nanomolar (nM) of an ERK inhibitor (PD0325901), primed HPSCs were not able to maintain pluripotency and displayed immediate differentiation (FIG. 1C). However, ERK signaling has been reported as dispensable for nave HPSCs to maintain a pluripotent state, and nave cells transitioned from the primed state maintain their undifferentiated state in RSet media supplemented with an additional 10 nM PD325901 (FIG. 1D). The transitioned nave HPSCs maintained expression of markers characteristic of HPSCs and could differentiate into three germ layer derivatives in vitro (FIG. 2). Expression of the pluripotent markers CDH1 and OCT4 were changed less than 2-fold in nave HPSCs from primed HPSCs while expression of NANOG was increased (FIG. 3). The cells were stably maintained in the nave state culture conditions for more than 30 passages.

[0050] The use of a full-length recombinant vitronectin in the manner described herein during transition supports the successful transition between pluripotent states; whereas, the use of Matrigel did not support a successful transition (FIG. 4). Although the HPSCs transitioned to the nave state on Matrigel had many of the hallmarks of nave pluripotent stem cells, karyotype analysis revealed clonal abnormalities in all transitioned cell lines.

[0051] The derivation of nave HPSCs from primed HPSCs on vitronectin was repeated with multiple primed HPSC lines from various genetic backgrounds.

Materials and Methods

Feeder Free Nave Pluripotent Stem Cell Derivation

[0052] Primed induced pluripotent stem cell (PCBC16iPS) (Ye et al. (2013) PLoS ONE 8 (1):e53764) to be used for nave cell derivation were cultured for at least two passages in mTeSR1 medium (STEMCELL Technologies, Vancouver, Canada) in either normoxic (20% O.sub.2, 5% CO.sub.2) or hypoxic conditions (5% O.sub.2, 5% CO.sub.2). Primed iPSCs were treated with Gentle Cell Dissociation Reagent (STEMCELL Technologies, Vancouver, Canada) for 5 minutes at room temperature and removed from the plate with a 5 milliliter (mL) pipette in 1 mL TeSR1 medium. Aggregates of 250 primed iPSCs were plated in 1 mL mTeSR1 (STEMCELL Technologies, Vancouver, Canada) per well of a 12-well tissue culture treated plate (Corning, Inc., Corning, N.Y.) coated with 5 micrograms per milliliter (g/mL) full-length vitronectin (PeproTech, Rocky Hill, N.J.) (Parr et al. (2016) Methods in Molecular Biology 1357:221-9). The plate had been previously coated with vitronectin at 37 C. for 2 hours per manufacturer's protocol. The plated cells were incubated overnight at 37 C., to allow adherence to the vitronectin coated plate. The cells may be incubated in either normoxic or hypoxic conditions, and typically were incubated under the same conditions used to maintain the primed HPSC culture. The next day, cells were transferred to hypoxic culture conditions and switched to RSeT medium (STEMCELL Technologies, Vancouver, Canada). Cells were monitored for compaction and defined colony edges. When nave iPSC colonies reached 250 millimeters (mm) in diameter, the cells in the well were passaged. For passaging, cells were washed one time with phosphate buffered saline (PBS) without Ca.sup.2+ or Mg.sup.2+, followed by addition of 250 L TrypLE Express (Thermo Fisher Scientific, Waltham, Mass.) for 3 minutes at 37 C. in hypoxic conditions. TrypLE was neutralized with 750 L RSet supplemented with 10 M ROCK inhibitor Y-27632 (BD Biosciences, San Jose, Calif.). Cells were agitated by pipetting 3 times using a P1000 pipet tip to achieve 10-12 cell clumps. After collection, cells were spun at 300g for 5 minutes. Cells were resuspended in 120 L of RSeT medium supplemented with 10 M Y-27632 by pipetting 6 times with a P200 pipet tip to break cells into clumps of 2-3 cells. Cells were plated in dilutions of 1:3, 1:4, 1:8, and 1:10 to ensure optimal plating density.

ERK Inhibitor Assay

[0053] One day after passaging, 10 nM ERK inhibitor PD0325901 (PZ0162, Sigma-Aldrich, St. Louis, Mo.) was added to nave or primed PSCs. Cells were imaged each day for 4 days to follow morphology changes.

Immunocytochemistry

[0054] Cultured cells were fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature (20 C.-25 C.) and then made permeable with 0.2% Triton X-100 in PBS for 30 minutes. Cells were blocked with 3% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 2 hours, then incubated overnight at 4 C. with primary antibodies diluted in 3% BSA. The antibodies used were SSEA-3 (1 g/mL; MAB4303, Millipore, Billerica, Mass.), and SSEA-4 (1 g/mL; MAB4304, Millipore, Billerica, MA). Cells were washed in PBS 3 times (for 5 minutes each wash) and incubated with 0.5 g/mL respective secondary antibody (A21042, Alexa Fluor anti-goat 488; A21432, Alexa Fluor anti-goat 555; A21434, Alexa Fluor anti-rat 555; A31572, Alexa Fluor anti-rabbit 555; A11001, Alexa Fluor anti-goat 488, all from Life Technologies, Thermo Fisher Scientific, Waltham, Mass.) for 1 hour at room temperature and washed with PBS. The cells were stained for 10 minutes at room temperature with 4,6-diamidino-2-phenylindole (DAPI, 1 g/mL; Invitrogen Corporation, Carlsbad, Calif.) diluted in PBS. Images were processed using Adobe Photoshop to optimize brightness and contrast, with all control and experimental images being treated identically.

[0055] To detect NANOG expression, wells were fixed for 10 minutes at room temperature using formalin. The cells were permeabilized with mixture of 1% BSA and 0.2% TritonX1000 in PBS for 10 minutes. The cells were blocked with Block (1% BSA+0.1% Tween) for 30 minutes at room temperature. The cells were incubated overnight at 4 C. with the primary antibody against NANOG (5 g/mL; AF1997, R&D Systems, Minneapolis, Minn.) diluted in Block. The primary antibody was washed off using Block, and the secondary antibody (0.5 g/mL, A21432, Alexa Fluor anti-goat 555) was added for 1 hour at room temperature. The well was washed 3 times with PBS, and DAPI (1 g/mL; Invitrogen Corporation, Carlsbad, Calif.) diluted in PBS was added for 5 minutes at room temperature. The imaging was performed as described above.

Pluripotency Assay

[0056] The differentiation potential of nave HPSCs into each of the three germ layers was performed using STEMdiff Trilineage Differentiation Kit (05230, STEMCELL Technologies, Vancouver, Canada). Briefly, cells were plated onto Matrigel (Corning, Inc., Corning, N.Y.) and treated with STEMdiff Trilineage Endoderm Medium or STEMdiff Trilineage Mesoderm Medium for 5 days or STEMdiff Trilineage Ectoderm Medium for 7 days. Cells were then fixed, stained, and imaged as described above.

Karyotype

[0057] Nave HPSCs were examined by high-resolution G banding after 10 passages in RSet medium.

RT-qPCR

[0058] RNA was isolated from primed HPSCs at passage 18 and nave HPSCs at passage 7, passage 10, and passage 12 using the RNeasy RNA Isolation Kit (Qiagen Company, Hilden, Germany), and cDNA was prepared using Superscript IV (Invitrogen Corporation, Carlsbad, Calif.). RT-qPCR was run on the Mastercycler epgradient S (Eppendorf, Hamburg, Germany) and analyzed using ep realplex software (Eppendorf, Hamburg, Germany). PrimeTime assays for CDH1 (Hs.PT.58.3324071), POU5F1 (Hs.PT.58.14648152.g), NANOG (Hs.PT.58.21480849), and GAPDH (Hs.PT.39a.22214836) were obtained from Integrated DNA Technologies (Coralville, Iowa).

[0059] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.