Patent Application
20240384232
The present disclosure relates to methods for generating both patient-specific and universal donor banked HLA-defined therapeutic progenitors in clinical grade, current good manufacturing practice (cGMP)-compliant conditions where both the patient-specific and the universal donor banked HLA-defined therapeutic progenitors are derived from a new class of tankyrase/PARP inhibitor-regulated naïve human induced pluripotent stem cells (TIRN-hiPSCs).
Although human pluripotent stem cell (hPSC)-derived progenitors have wide impact for regenerative medicine, their broad clinical application via patient-specific approaches faces important challenges and limitations. For example, the current paradigm of autologous human induced pluripotent stem cell (hiPSC) therapeutics poses logistical and financial challenges. Specifically, the labor involved in screening individual hiPSC lines for high-quality clones and the costs associated with such screens makes patient-specific therapies inaccessible in health care systems with limited resources. Moreover, the costs of validating genomic integrity and functionality for individualized autologous hiPSC is not sustainable in the long term in a cost-conscious health care system. Even if such labor and cost challenges could be addressed, personalized patient-specific hiPSC lines are not readily available for numerous acute disorders requiring immediate therapeutic intervention (e.g., myocardial infarction, cerebrovascular stroke).
As an alternative to patient-specific cellular therapies, global efforts have begun to develop HLA-defined iPSC banks, including from inventories of clinical-grade, HLA-typed cord blood (CB). Clinical bone marrow transplantation (BMT) provides important paradigms to facilitate such hiPSC bank therapies. For example, the existing infrastructure of BMT routinely leverages partially HLA-matched, or haplo-identical HLA-matched hematopoietic stem cells.
Computational models have predicted that a small number of 80-200 HLA-defined hiPSC lines derived from existing HLA-typed cord or peripheral blood banks could generate allogeneic matches to serve the transplantation needs of the majorities of the populations in Japan, UK, and the USA. To prioritize immune-compatibility, HLA A, B, and DR loci (and O-negative blood group)-genotyped individuals from 2-10homozygous HLA haplotype donors could serve the needs of ˜5-30% of the population. Such ‘haplo’ banks of clinical-grade hiPSC lines could significantly expand therapeutic feasibility and reduce graft rejection, but are still likely to generate immune responses to hiPSC-derived tissues in non-autologous recipients. Moreover, the broad genetic diversity of the USA may limit the feasibility of HLA-matched hiPSC, even with the establishment of haplo banks.
In view of the foregoing, there remains a need in the field for cost effective methods for generating both patient-specific and universal donor banked HLA-defined therapeutic progenitors, particularly with respect to methods involving clinical grade, current good manufacturing practice (cGMP)-compliant conditions.
In certain embodiments, the presently disclosed subject matter provides methods for generating a population of therapeutic progenitor cells under clinical grade, current good manufacturing practice (cGMP)-compliant conditions, comprising contacting a population of human induced pluripotent stem cells (hiPSCs) with a tankyrase/PARP-inhibitor-containing composition comprising (i) leukemia inhibitory factor (LIF), (ii) a Glycogen Synthase Kinase 3-β (GSK3β) signaling pathway inhibitor, (iii) a mitogen-activated protein kinase (MEK) signaling pathway inhibitor, (iv) a tankyrase/PARP inhibitor, and (v) a protein kinase C (PKC) inhibitor; the method further comprises performing feeder-free (FF) and xeno-free (XF) culture of the hiPSCs to generate a population of these therapeutic progenitor cells under clinical grade, cGMP-compliant conditions.
In certain embodiments, the tankyrase/PARP inhibitor is selected from: XAV939, IWR-1, G007-LK, JW55, AZ1366, JW 74, NVP-TNKS656 and combinations thereof. In certain embodiments, the GSK3β signaling pathway inhibitor is selected from: 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR 99021), 5-Ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]isoquinoline-1,3(2H)-dione (3F8), 1-(7-Methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea (A 1070722), N6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine (CHIR 98014), lithium chloride (LiCl), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 5-iodo-indirubin-3′-monoxime (I3′M), N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) and combinations thereof. In certain embodiments, the MEK signaling pathway inhibitor is selected from: PD032590, CI-1040 (PD184352), cobimetinib (GDC-0973, XL518), Selumctinib (AZD6244), MEK162, AZD8330, TAK-733, GDC-0623, Refametinib (RDEA119; BAY 869766), Pimasertib (AS703026), RO4987655 (CH4987655), RO5126766, WX-554, HL-085 and combinations thereof. In certain embodiments, the PKC inhibitor is Go6983.
In certain embodiments, the hiPSCs are derived from primed hiPSCs. In certain embodiments, the priming of hiPSCs comprises contacting the hiPSCs with a serum replacer-containing media. In certain embodiments, priming the hiPSCs comprises contacting the hiPSCs with a ROCK inhibitor. In certain embodiments, the ROCK inhibitor is Y-27632.
In certain embodiments, the population of therapeutic progenitor cells are generated from a population of HLA-defined hiPSCs. In certain embodiments, the population of HLA-defined hiPSCs comprise human CD34+ cord blood cells.
In certain embodiments, the FF and XF culture of the hiPSCs in contact with the tankyrasc/PARP inhibitor-continuing composition comprises culturing the hiPSCs on vitronectin-coated substrates. In certain embodiments, the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-containing composition is performed for 1to 10 passages. In certain embodiments, passages 1-3 of the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-containing composition comprise selection of dome-shaped colonies for each subsequent passage.
The present disclosure relates to methods for generating both patient-specific and universal donor banked HLA-defined therapeutic progenitors in clinical grade, current good manufacturing practice (cGMP)-compliant conditions. In certain embodiments, the patient-specific and the universal donor banked HLA-defined therapeutic progenitors are derived from a new class of tankyrase/PARP inhibitor-regulated naïve human induced pluripotent stem cells (TIRN-hiPSCs).
In one aspect, the present disclosure is directed to methods and compositions finding use in the production of patient-specific TIRN-hiPSCs. For example, patient-specific TIRN-hiPSCs can be derived from any human somatic cell using the methods described herein. These patient-specific TIRN-hiPSCs represent a new class of human naïve pluripotent stem cells exhibiting high epigenetic plasticity, stable epigenomic imprints, and more efficient multi-lineage functionality than conventional, lineage-primed hiPSC. The methods described herein result in efficient, bulk, and rapid chemical reversion of conventional, lineage-primed, xenofree (XF) feeder-free (FF) hiPSC lines into a stable preimplantation naïve epiblast-like pluripotent state in cGMP-grade conditions. For example, but not limitation, the chemical tankyrase/PARP inhibitor-based methodologies described herein rapidly revert conventional, lineage-primed hiPSC lines to adopt transcriptional, epigenetic, and biochemical features of the human pre-implantation naïve epiblast. Thus, in certain embodiments, the TIRN-hPSCs described herein possess multiple naïve ICM characteristics, including MEK-ERK/bFGF signaling independence, activated phosphorylated JAK/STAT3 signaling, distal OCT4 enhancer usage, global DNA CpG hypomethylation, and/or increased expression of activated beta-catenin. In certain embodiments, TIRN-hPSCs do not require reversion culture back to primed culture conditions prior to differentiation. In certain embodiments, reversion of conventional hiPSCs into TIRN-hiPSCs results in decreased lineage-primed gene expression and marked improvement in directed multi-lineage differentiation of conventional hiPSC lines across a broad repertoire of genetically-independent somatic cell hiPSC donors. In addition, TIRN-hPSCs can be protected against erosion of CpG methylated genomic imprinted regions and, in certain embodiments, maintain DNMT1 expression.
In another aspect, the present disclosure is directed to methods and compositions for establishing cGMP-grade banks of clinical grade ‘Universal’ donor TIRN-hiPSCs (UTIRN-hiPSCs) from HLA-defined CD34+ hematopoietic progenitors. These hematopoietic progenitor-derived UTIRN-hiPSCs can be utilized, for example, but not limitation, for comprehensive multi-lineage repair of diseased tissues in any recipient via approaches employing drug-mediated allogeneic tissue transplantation tolerance (e.g., with post-transplant cyclophosphamide). In certain embodiments, banks of UTIRN-hiPSC lines can be used to generate downstream, secondary banks of differentiated, HLA-defined, cryo-preserved universal donor cells (e.g., cardiac, vascular, neural progenitors) for “off-the-shelf” cellular therapies. In certain embodiments, UTIRN-hiPSCs will have high impact for regenerative medicine by facilitating allogeneic tissue tolerance induction strategies with a universal supply of cellular therapies in a manner that decreases the costs and broadens availability of hiPSC therapies to a wider number of individuals (e.g., as illustrated in
The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below, or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure and how to make and use them.
As used herein, the use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification can mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”
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 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.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
As used herein, the terms “comprising,” “comprise” or “comprised,” and variations thereof, in reference to defined or described elements of an item, composition, apparatus, method, process, system, etc. are meant to be inclusive or open ended, permitting additional elements, thereby indicating that the defined or described item, composition, apparatus, method, process, system, etc. includes those specified elements—or, as appropriate equivalents thereof—and that other elements can be included and still fall within the scope/definition of the defined item, composition, apparatus, method, process, system, etc.
An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.
As used in this specification and the appended claims, the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.
“Parenteral” administration of an immunogenic composition includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), or intrasternal injection, or infusion techniques.
The terms “patient” or “individual” or “subject” are used interchangeably herein, and refers to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters, and primates.
As defined herein, a “therapeutically effective” amount of a compound or agent (i.e., an effective dosage) means an amount sufficient to produce a therapeutically (e.g., clinically) desirable result. The compositions can be administered from one or more times per day to one or more times per week; including once every other day. The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of the compounds of the invention can include a single treatment or a series of treatments.
As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, are intended to encompass homologous and/or orthologous genes and gene products from other species.
Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4,from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
In one aspect, the subject matter of the present disclosure is directed to defined feeder-free (FF), xenofree (XF) cGMP-compliant culture medium systems for the production of patient-specific TIRN-hiPSCs. For example, the methods described herein are directed, in certain embodiments, to defined FF/XF cGMP-compliant culture medium systems for CB (or PBSC or BM) pluripotency episomal reprogramming. In certain embodiments, the methods described herein employ FF/XF medium systems that revert conventional XF/FF human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESC) to a naïve epiblast-like state, referred alternatively herein as the “LIF-4i FF TIRN system”, the “LIF-4i XF/FF TIRN system”, the “LIF-4i FF method”, the “LIF-4i XF/FF method” and modifications thereof. The LIF-4i FF TIRN system is based on a tankyrasc/PARP inhibitor-based small molecule approach. The method contains a novel recipe of LIF, four proprietary small molecules including a XAV939 (LIF-4i), defined media components, and KnockOut serum replacer (KSOR, e.g., Knockout™ Serum Replacement (Knockout™ SR) from Thermo Fisher), but does not contain growth factors typically required to maintain primed, conventional hiPSC (e.g., bFGF or TGFβ).
The LIF-4i FF TIRN system involves a modified version of the classic murine 2i naïve reversion cocktail to human pluripotent stem cells. For example, but not limitation, the self-renewal of hPSC (which cannot expand in 2i alone) is stabilized in LIF-2i by supplementing this cocktail with the tankyrase/PARP inhibitor XAV939. Although the mechanisms of action of XAV939 in hPSC are likely complex and synergistic with 2i, it likely includes at a minimum, an important stabilization and augmentation of hPSC self-renewal via WNT signaling pathways.
Furthermore, although the promiscuous PARP inhibitor XAV939 has been widely employed as a small molecule WNT inhibitor when used alone, another method, the LIF-3i method, accentuated several other studies that also demonstrated that when used simultaneously with GSK-3b inhibition, XAV939 induced a stabilization of AXIN isoforms that enhanced self-renewal of both conventional human pluripotent stem cells and murine EpiSC. The mechanism of action was shown to be XAV939 synergizing with GSK-3b inhibition, to paradoxically augment canonical WNT signaling by reinforcing the stability of the active isoform of β-catenin in both cytoplasmic and nuclear subcellular compartments. However, unlike previous studies, Zimmerlin et al., Development 2016; 143(22): 4368-4380, exploited dual use of GSK-3b/tankyrase/PARP co-inhibition along with simultaneous (PD0325901) MEK inhibition to stably revert a wide repertoire of conventional hPSC to a naïve epiblast-like state without requirement for exogeneous FGF2. In that study, initial culture in LIF-5i (including forskolin and purmorphamine) followed by LIF-3i culture reverted hPSC (TIRN-hPSC) re-activated naïve epiblast-like STAT3 signaling, and possessed naïve epiblast-like globally hypomethylated genomes, but without hypomethylated genomic imprinting aberrations. TIRN-hPSC were functionally competent for multi-lineage differentiation without need for an additional ‘capacitation’ step or re-culture back to primed state (i.e., ‘re-priming’). Moreover, these studies not only validated the functional pluripotency of reverted TIRN-hPSC in multi-lineage directed differentiation assays, but also revealed that TIRN-hPSC possessed significantly improved differentiation efficiencies relative to their isogenic primed, conventional hPSC counterparts.
The LIF-4i FF TIRN reversion method described herein is reproducible in a broad variety of independent FF hESC and transgene-free, non-integrated FF hiPSC lines. The method requires minimal training with basic cell culture skill and has been employed to revert >20 independent hESC and hiPSC lines from a broad array of donors. Furthermore, the LIF-4i FF TIRN system supports robust bulk clonal expansion efficiencies throughout all the steps between lineage-primed conventional hPSC culture all the way to completed naïve-like hPSC reversion (i.e., adaptation, transition and expansion for 7-10 passages in LIF-4i FF alone). The stability of this culture system does not depend on the presence of feeders and allows complete FF/XF expansion of TIRN-hPSC.
TABLE 1, below, outlines a two-step LIF-4i XF/FF TIRN method developed following extensive media analysis of the LIF-3i/MEF protocol. The LIF-4i FF chemical naïve reversion method employs the classical leukemia inhibitory factor (LIF), GSK3B, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only the tankyrase/PARP inhibitor XAV939 in a novel completely FF protocol. To stabilize TIRN-hPSC without feeders, the substrate and media composition were modified from the original LIF-3i/MEF method, and a 4th inhibitor (i.e., Go6983) targeting protein kinase C (LIF-4i FF medium) was introduced, which had been shown to reinforce pluripotency in human primed and rodent ESC. This mechanism is independent from STAT3 activation and MEK/GSK3β inhibition and antagonizes lineage commitment. This inhibitor has previously been introduced into other human naive culture systems to minimize spontaneous differentiation.
In certain embodiments, the present disclosure is directed to methods for generating a population of therapeutic progenitor cells under clinical grade, cGMP-compliant conditions, comprising: (a) contacting a population of hiPSCs with a tankyrasc/PARP-inhibitor-containing composition comprising: (i) leukemia inhibitory factor; (ii) a GSK3β signaling pathway inhibitor; (iii) a MEK signaling pathway inhibitor; (iv) a non-specific tankyrase/PARP inhibitor; and (v) a PKC inhibitor; (b) performing FF and XF culture of the hiPSCs to generate a population of therapeutic progenitor cells under clinical grade, cGMP-compliant conditions. In certain embodiments, the tankyrase/PARP inhibitor is selected from: XAV939, IWR-1, G007-LK, JW55, AZ1366, JW 74, NVP-TNKS656 and combinations thereof. In certain embodiments, the GSK3β signaling pathway inhibitor is selected from: 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR 99021), 5-Ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]isoquinoline-1,3(2H)-dione (3F8), 1-(7-Methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea (A 1070722), N6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine (CHIR 98014), lithium chloride (LiCl), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 5-iodo-indirubin-3′-monoxime (I3′M), N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) and combinations thereof. In certain embodiments, the MEK signaling pathway inhibitor is selected from: PD032590, CI-1040 (PD184352), cobimetinib (GDC-0973, XL518), Selumctinib (AZD6244), MEK162, AZD8330, TAK-733, GDC-0623, Refametinib (RDEA119; BAY 869766), Pimasertib (AS703026), RO4987655 (CH4987655), RO5126766, WX-554, HL-085 and combinations thereof. In certain embodiments, the PKC inhibitor is Go6983.
In certain embodiments, the present disclosure is directed to methods for generating a population of therapeutic progenitor cells under clinical grade, cGMP-compliant conditions, wherein the hiPSCs used to generate the therapeutic progenitor cells are derived from primed hiPSCs. In certain embodiments, the priming of hiPSCs comprises contacting the hiPSCs with a serum replacer-containing media. In certain embodiments, the priming hiPSCs comprises contacting the hiPSCs with a ROCK inhibitor. In certain embodiments, the ROCK inhibitor is Y-27632.
In certain embodiments, the present disclosure is directed to methods for generating a population of therapeutic progenitor cells under clinical grade, cGMP-compliant conditions, wherein the population of therapeutic progenitor cells are generated from a population of HLA-defined hiPSCs. In certain embodiments, the population of HLA-defined hiPSCs comprise human CD34+ cord blood cells.
In certain embodiments, the present disclosure is directed to methods for generating a population of therapeutic progenitor cells under clinical grade, cGMP-compliant conditions comprising a FF and XF culture of the hiPSCs to generate a population of therapeutic progenitor cells under clinical grade, cGMP-compliant conditions, wherein the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-continuing composition is comprises culture of the hiPSCs on vitronectin-coated substrates. In certain embodiments, the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-containing composition for 1 to 10 passages. In certain embodiments, passages 1-3 of the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-containing composition comprise selection of dome-shaped colonies for each subsequent passage.
In certain embodiments, the modified LIF-4i method provides a completely defined XF/FF culture system for the derivation and long-term cryopreservation and storage (TABLE 2) of clinical-grade TIRN-hiPSCs, e.g., from HLA-typed CB or PBSC donors.
In another aspect, the present disclosure relates to the production of cGMP-grade banks of clinical grade “universal” UTIRN-hiPSCs. UTIRN-hiPSC reversion can be used to revert either established or commercially available transgene-free FF hiPSC. With this method, established conventional, primed hESC/hiPSC (or CB-hiPSC derived entirely with FF/XF episomal CB reprogramming) that are cultured in FF E8 medium can be rapidly reverted in bulk with LIF-4i to an FF naïve-like state. FF TIRN-hiPSC or UTIRN-hiPSC are cultured on vitronectin-coated vessels in physiological (5%) O2, and can be cryopreserved, or used directly for differentiation without further manipulation. In addition to enhanced functionality, UTIRN-hPSC possess multiple advantages over conventional hPSC culture, and over other LIF-3i/MEF naïve reversion systems. These advantages include, in certain embodiments, ease of single cell passaging in a defined, reproducible FF system that uses pre-screened, high-quality commercially-available components. As outlined herein, episomally-reprogrammed FF/XF cGMP-compliant HLA-defined CB-derived UTIRN-hiPSC lines from start to finish have been established. The same methods can be employed with clinical grade CD34+ PBSC or BM, among other cell types.
Multiple other culture systems have also been reported to promote conventional hPSC to similar naïve-like pluripotent states. Although these hPSC culture systems have also relied on utilization of classical mouse naïve 2i conditions, in most cases these single-cell passaging methods also required additional chemical modulation for stabilizing an inherently unstable/metastable human naïve state. Unlike the TIRN system, most of these other methods demonstrated impaired functional pluripotency following differentiation and/or acquired abnormal epigenomic imprints or karyotypes. Although the emergence of abnormal karyotypes within conventional primed hPSC cultures is already well documented, prolonged, enzymatic single-cell passaging methods that are routinely employed in most naïve reversion methods, have also been shown to potentiate the generation of abnormal chromosomal configurations; more sensitive techniques (e.g., copy number variations, single nucleotide polymorphism) may reveal additional alterations.
In contrast, UTIRN-hPSC lines not only possessed normal karyotypes at low-medium passages (e.g., p5-p15), as well as at high passage numbers (e.g., >p30) following LIF-4i FF TIRN culture, but also improved genomic stability (as assessed by DSB DNA repair studies). Using the sensitive allele-specific Infinium methylation array platform, it was also previously demonstrated that CpG methylation marks at imprinted loci of a wide repertoire of LIF-3i/MEF-reverted hPSC lines (following 4-7 passages in LIF-3i) were found to be grossly normal in structure. Since abnormal genomic imprints and karyotypes may ultimately impair functional capacity of hPSC, prerequisite guidelines were outlined in this protocol that encourage researchers to validate hPSC cultures before and after naïve reversion, using this method as well as others.
In certain embodiments, the LIF-4i FF TIRN methods described herein can improve functional pluripotency across germ layers in a large repertoire of hESC and non-transgenic hiPSC lines. Unlike other naïve reversion protocols, the methods described herein do not require a re-priming step for subsequent differentiation of TIRN-hPSC (i.e., converting TIRN-hPSC back to conventional primed conditions prior to their use in directed differentiation assays). In certain embodiments, the LIF-4i FF-reverted TIRN-hPSC displayed significantly more efficient differentiation capacities than their isogenic conventional hPSC counterparts in both teratoma assays and directed differentiation protocols of lineages of all three germ layers. Due to assay-dependent and interline variations in functional testing of conventional hPSC, lineage-specific differentiation should be evaluated using independent directed differentiation protocols and hPSC derived from multiple genetic backgrounds. Using careful experimental design, a broad array of hPSC lines can be expected to significantly improve their multi-lineage differentiation efficiencies compared to their isogenic conventional counterparts following 4-10 passages in LIF-4i TIRN conditions.
For example, but not by way of limitation, the methods and compositions described herein have been used to generate and functionally validate pre-clinical naïve vascular progenitors (N-VP) from a test bank of representative UTIRN-hiPSC lines derived in cGMP-compliant conditions from HLA-defined CD34+ CB as a pilot for future Phase I/II clinical trials.
In summary, the TIRN reversion method rapidly and clonally expands the numbers of hPSC, improves their downstream differentiation efficiency, increases lineage-committed progenitor cell numbers following differentiation, and decreases interline variability of conventional, lineage-primed hPSC lines (
In certain embodiments, the presently disclosed subject matter provides methods for generating a population of therapeutic progenitor cells under clinical grade, current good manufacturing practice (cGMP)-compliant conditions, comprising contacting a population of human induced pluripotent stem cells (hiPSCs) with a tankyrase/PARP-inhibitor-containing composition. In certain embodiments, the tankyrasc/PARP-inhibitor-containing composition comprises (i) leukemia inhibitory factor (LIF), (ii) a Glycogen Synthase Kinase 3-β (GSK3β) signaling pathway inhibitor, (iii) a mitogen-activated protein kinase (MEK) signaling pathway inhibitor, (iv) a tankyrasc/PARP inhibitor, and (v) a protein kinase C (PKC) inhibitor. In certain embodiments, the method further comprises performing feeder-free (FF) and xeno-free (XF) culture of the hiPSCs to generate a population of these therapeutic progenitor cells under clinical grade, cGMP-compliant conditions.
In certain embodiments, the tankyrase/PARP inhibitor is selected from: XAV939, IWR-1, G007-LK, JW55, AZ1366, JW 74, NVP-TNKS656 and combinations thereof. In certain embodiments, the GSK3β signaling pathway inhibitor is selected from: 6-[[2-[[4-(2,4-Dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR 99021), 5-Ethyl-7,8-dimethoxy-1H-pyrrolo[3,4-c]isoquinoline-1,3(2H)-dione (3F8), 1-(7-Methoxyquinolin-4-yl)-3-[6-(trifluoromethyl)pyridin-2-yl]urea (A 1070722), N6-[2-[[4-(2,4-Dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine (CHIR 98014), lithium chloride (LiCl), 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), 5-iodo-indirubin-3′-monoxime (I3′M), N-(4-methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) and combinations thereof. In certain embodiments, the MEK signaling pathway inhibitor is selected from: PD032590, CI-1040 (PD184352), cobimetinib (GDC-0973, XL518), Selumetinib (AZD6244), MEK162, AZD8330, TAK-733, GDC-0623, Refametinib (RDEA119; BAY 869766), Pimasertib (AS703026), RO4987655 (CH4987655), RO5126766, WX-554, HL-085 and combinations thereof. In certain embodiments, the PKC inhibitor is Go6983.
In certain embodiments, the hiPSCs are derived from primed hiPSCs. In certain embodiments, the priming of hiPSCs comprises contacting the hiPSCs with a serum replacer-containing media. In certain embodiments, priming the hiPSCs comprises contacting the hiPSCs with a ROCK inhibitor. In certain embodiments, the ROCK inhibitor is Y-27632.
In certain embodiments, the population of therapeutic progenitor cells are generated from a population of HLA-defined hiPSCs. In certain embodiments, the population of HLA-defined hiPSCs comprise human CD34+ cord blood cells.
In certain embodiments, the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-continuing composition comprises culturing the hiPSCs on vitronectin-coated substrates. In certain embodiments, the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-containing composition is performed for 1to 10 passages. In certain embodiments, passages 1-3 of the FF and XF culture of the hiPSCs in contact with the tankyrase/PARP inhibitor-containing composition comprise selection of dome-shaped colonies for each subsequent passage.
The following examples are merely illustrative of the presently disclosed subject matter and should not be considered as limitations in any way.
The instant disclosure describes strategies to efficiently revert feeder-free (FF), lineage-primed, conventional human pluripotent stem cells (hPSC) into a naïve epiblast-like pluripotent state with improved functionality, and to maintain this novel stem cell state in FF, xeno-free (XF), clinical-grade culture conditions for downstream cell therapeutic applications.
Previously disclosed feeder-dependent LIF-3i/MEF culture systems are not stable in the absence of feeders and TIRN-hiPSC undergo spontaneous differentiation. Thus, a two-step LIF-4i XF/FF TIRN method (TABLE 1) was developed following extensive media analysis of the LIF-3i/MEF protocol.
The LIF-4i FF chemical TIRN reversion method employs the classical leukemia inhibitory factor (LIF), GSK3β, and MEK/ERK inhibition cocktail (LIF-2i), supplemented with only the non-specific tankyrase/PARP inhibitor XAV939 in a novel completely FF protocol. To stabilize TIRN-hPSC without feeders, the substrate and media composition were modified from the original LIF-3i/MEF method, and a 4th inhibitor (i.e., Go6983) targeting protein kinase C (LIF-4i FF medium) was introduced, which had been shown to reinforce pluripotency in human primed and rodent ESC. This mechanism is independent from STAT3 activation and MEK/GSK3β inhibition and antagonizes lineage commitment. This inhibitor has previously been introduced into other human naïve culture systems to minimize spontaneous differentiation.
The modified LIF-4i TIRN method provides a completely defined XF/FF culture system for the derivation and long-term cryopreservation and storage (TABLE 2) of clinical-grade UTIRN-hiPSC from HLA-typed CB or PBSC donors.
Like the previously described feeder-dependent LIF-3i/MEF method, LIF-4i FF conditions revert conventional hPSC to a stable pluripotent state adopting biochemical, transcriptional, and epigenetic features of the human pre-implantation epiblast. The LIF-4i FF method, however, requires minimal cell culture manipulation and is highly reproducible in a broad repertoire of human embryonic stem cell (hESC) and transgene-free human induced pluripotent stem cell (hiPSC) lines. Moreover, this LIF-4i FF TIRN reversion method does not require a ‘re-priming’ step prior to differentiation; TIRN-hPSC can be differentiated directly with extremely high efficiencies and maintain karyotypic and epigenomic stabilities (including at imprinted loci).
To broaden the utility of the method, the LIF-4i FF TIRN protocol described herein was evaluated to promote universal naïve reversion with almost any conventional hESC or transgene-free hiPSC line cultured in standard feeder-free hPSC conditions (e.g. E8). This universalized naïve reversion method employs continuous culture on a single substrate (i.e., vitronectin), as well as selection of cell densities and bulk passaging of conventional hPSC without any requirement for tedious colony picking. Dissociation methods were selected to promote bulk enrichment of emerging naïve hPSC during the initial passages, so that adapted naïve hPSC lines could subsequently tolerate bulk clonal passaging of naïve-reverted cells in LIF-4i FF TIRN conditions. The methods described herein obviate the need for picking and subcloning of rare ‘stable’ colonies and promote rapid expansion of naïve epiblast-like hPSC.
The LIF-4i FF TIRN reversion methods described herein markedly improve the functional pluripotency of a broad repertoire of conventional hPSC by decreasing their lineage-primed gene expression and erases the interline variability of directed differentiation commonly observed amongst independent primed, conventional hPSC lines
The LIF-4i FF TIRN methods described herein have been successfully used to stably expand and maintain a broad repertoire of >10 independent, genetically diverse conventional hPSC lines for >10-30 passages using either non-enzymatic or enzymatic dissociation methods, and without evidence of induction of chromosomal or epigenomic abnormalities, including at imprinted loci.
The LIF-4i FF TIRN methods described herein have great utility in regenerative medicine and cellular therapies, and can be utilized to expand UTIRN-hiPSC derived from HLA-defined CD34+ CB or PBSC donors. Representative characterizations of LIF-4i FF-reverted TIRN-hPSC are provided, and experimental strategies for functional comparisons of isogenic hPSC in lineage-primed vs. naïve-like states are outlined herein.
The schematic, images, and reagents of the XF/FF episomal reprogramming of human CD34+ cord blood (CB) cells of pre-defined HLA type are summarized in
Episomal FF/XF reprogramming of CD34+ CB, PBSC, or BM methods can be performed in a cGMP-compliant environment with cGMP-compliant reagents from start to finish. For example, to derive HLA-defined UTIRN-hiPSC, GTP-grade HLA-defined CB, PBSC, or BM can first be obtained from a cryopreserved bank or commercial source, e.g., following prospective donor consent of HLA screened donors, from an approved IRB protocol and according to institutional requirements. In certain instances, selection of O-negative HLA-homozygous donors is associated with additional advantages.
CD34+-enriched cord blood (CB) cells from mixed donors were commercially obtained (AllCells), and thawed in cGMP-grade XF StemSpan Serum-Free Expansion Medium (SFEM)-XF medium (StemCell Technologies) supplemented with 50 ng/ml human recombinant FLT3 ligand, 10 ng/mL human recombinant thrombopoietin (TPO), and 50 ng/mL human recombinant Kit-ligand (FTK), and cultured for 3 days onto ultra-low attachment surfaces (Corning). Expanded CD34+ progenitors were nucleofected (AMAXA Nucleofector II, Lonza) with 6 ug per million of cells of a single EBNA-based episomal vector that expressing sequences for SOX2, OCT4, KLF4, and c-MYC.
Nucleofected cells were plated (17,500 cells per cm2) over irradiated human mesenchymal stem cells (MSC) onto a XF synthetic peptide substrate (Corning Synthemax) in SFEM-XF medium supplemented with human recombinant FTK and 10μM ROCK inhibitor Y-27632. An equal volume of XF E8 medium was added 2 days later and half of the medium was replaced with fresh E8 medium on the third day (
Stable, conventional, primed XF/FF UTIRN-hiPSC colonies arising after 10-20 days were passaged non-enzymatically and cryopreserved with 50% XF SR/40% E8/10% DMSO prior to LIF-4i naïve reversion onto vitronectin-XF-coated plates. XF/FF cGMP-compliant UTIRN-iPSC lines need to be quality control characterized for TRA+ markers, karyotypes, SNP genomic arrays, mycoplasma/endotoxin assay, tri-lineage teratoma assay, and Southern blotting/PCR studies for verification of lack of vector integration. These primed XF/FF CB-hiPSC were validated for multi-lineage functional pluripotency by teratoma assays (
The schematic, images, and reagents of the LIF-4i XF/FF TIRN reversion method of primed, conventional XF/FF hiPSC cells of a pre-defined HLA type to XF/FF UTIRN-hiPSC are summarized in
Before beginning, all conventional, primed hPSC lines growing in FF conditions can be verified to possess a normal human karyotype by G-banding, prior to beginning TIRN reversion. LIF-4i FF TIRN reversion of high-passage conventional hiPSC lines (e.g., P>40-50) should be avoided, as such cultures may already harbor genomic aberrations that can negatively impact stable, efficient, and bulk LIF-4i FF reversion of primed hiPSC. In general, an effort should be made to revert conventional hPSC lines at the lowest possible passage that they are available.
LIF-4i XF/FF TIRN reversion is compatible with conventional hPSC starting from either feeder-dependent or feeder-free primed culture conditions, but feeder-free primed culture systems (e.g., E8, mTSER) are often desirable for commercial production purposes. Non-enzymatic methods (i.e., EDTA-based dissociation buffers) are also useful for passaging of conventional hPSC prior to preparing them for reversion. LIF-4i formulation (TABLE 1) does not contain antibiotic or antifungal agents. Thus, standard operation rules for biosafety cabinet sterility and maintenance can be observed to avoid any bacterial or fungal contamination.
Before switching to LIF-4i XF/FF TIRN conditions, primed hiPSC can be passaged in a biosafety cabinet (standard passaging in E8 on vitronectin XF is recommended, but not required) and allowed to reach ˜30% confluency (i.e., 3-4 days after initial plating) in a CO2 incubator (5% CO2, humid atmosphere).
Primed hPSC cultures that are over-expanded prior to switching to LIF-4i (i.e., >50% confluency) can display pronounced differentiation (i.e., primarily extraembryonic lineages such as cytokeratin 8+ primitive endoderm) for up to 3 passages after transition in LIF-4i (
Primed hPSC cultures that were expanded using defined, serum-free medium that did not include any Knock-out Serum Replacer (e.g., E8 medium), can be supplemented with 10% Knock-out Serum Replacer in a biosafety cabinet and incubated for at least one hour (up to overnight incubation) in a CO2 incubator (5% CO2, humid atmosphere) to adapt them prior to their subsequent passage and stable reversion in LIF-4i (
Prior to passaging, conventional hPSC culture plates are placed in a biosafety cabinet, supernatant is discarded and cells are washed once with 2 mL PBS. PBS wash can be gently aspirated and 1 mL of non-enzymatic EDTA-based cell dissociation buffer (e.g., enzyme-free Gibco cell dissociation buffer) can be added to each well. The cell can then incubate for 5 min at 37° C. in a CO2 incubator.
The cells can then be gently triturated with a 1 mL pipette to obtain a single cell suspension in a biosafety cabinet. The cell suspension can then be collected in LIF-4i medium (Table 3, at least 2-fold dilution) in sterile 15 ml conical tubes.
The cell suspension collected in LIF-4i medium can then be centrifuged at 200 g for 5 min, the supernatant aspirated/discarded, and the cell pellet resuspended in 1 mL of LIF-4i medium in a biosafety cabinet for cell counting using a hemocytometer or an automatic cell counter.
3×105 cells can be distributed into 2 mL of LIF-4i medium supplemented with 5 μM Y-27632 onto 1 well of Vitronectin-XF-coated plate. Initial plating efficiency will vary between hPSC cultures and may need to be individually assessed. The plate can then be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
Stable SSEA4+TRA-1-81 LIF-4i FF UTIRN-hiPSC cultures (
The next day, the plate can be gently swirled to lift all non-attached cells, the medium can be aspirated and replaced with 2 mL of LIF-4i medium in a biosafety cabinet daily for 2-3 days or until cells are 60-70% confluent. The plate can be placed in a CO2incubator (5% O2, 5% CO2, humid atmosphere).
Following initial adaptation to LIF-4i, subsequent LIF-4i TIRN cultures are passaged every 2-3 days, or when cultures become 60-70% confluent in a biosafety cabinet (
Typically, LIF-4i XF/FF TIRN cultures require rigorous maintenance and allowing UTIRN-hPSC cultures to reach high confluency/cell density from prolonged culture (e.g., >4 days) decreases subsequent clonal re-plating efficiency, and promotes spontaneous differentiation (
While LIF-4i XF/FF TIRN reversion of high-passage (e.g., >p40) lineage-primed, conventional hPSC lines is possible, generally an effort should be made to revert conventional hPSC lines at the lowest possible passage that they are available. Additionally, the use of LIF-4i-reverted hPSC that have undergone greater than 15 LIF-4i passages is not recommended for functional studies, since such TIRN-hPSC cultures may harbor karyotypically-abnormal clones due to prolonged clonal cell culture selection. Fresh LIF-4i reversions of low-passage conventional hPSC lines should be conducted for functional studies, if stocks of TIRN-hPSC with <10 passages in LIF-4i are not available.
The small molecule Go6983 is auto-fluorescent. Cells may be recovered and resuspended for counting using LIF-4i medium that does not include Go6983 before passaging in complete LIF-4i FF medium when fluorescence-based assays are required. Multiple washes in medium without Go6983 and PBS are recommended to permit cellular exclusion of Go6983 and minimize carry-over of red auto-fluorescence.
UTIRN-hiPSC are typically passaged for least 4-7 continuous bulk passages in LIF-4i FF TIRN medium prior to use of UTIRN-hiPSC in functional studies or cryopreservation. In general, it is desirable to record the number of passages of TIRN-hPSC in either conventional or LIF-3i media.
The use of LIF-4i-reverted UTIRN-hiPSC that have undergone greater than 10 LIF-4i passages is not recommended for functional studies, since such TIRN-hiPSC cultures might harbor karyotypically-abnormal clones due to prolonged clonal cell culture selection. Fresh LIF-4i reversions of low-passage conventional hiPSC lines should preferably be conducted for functional studies, if stocks of UTIRN-hPSC with <10passages in LIF-3i are not available.
The initial LIF-4i FF passage will often only display minimal differentiation (
In a biosafety cabinet, the culture medium can be discarded and each well washed of initial LIF-4i passage cultures by gently adding 2 mL of PBS. PBS can be discarded and 1 mL of non-enzymatic EDTA-based dissociation buffer added. This can then be incubated for 5 min at 37° C. in a CO2 incubator (5% CO2, humid atmosphere).
Cold PBS for washing cells as TIRN-hPSC is generally disfavored as it may detach from the plate. In addition, extended exposure to PBS or leaving the plate at room temperature are also disfavored, as these conditions may also result in some colony detachment.
Using an inverted microscope or other appropriate means, it can be useful to verify that most dome-shape colonies have detached and ensure the cell suspension is collected, e.g., in a biosafety cabinet. If flat colonies are present, exposure to detachment solution should be limited, so that carry-over of differentiated cells is minimized during the next passages. LIF-4i FF TIRN medium can be added (TABLE 1, at least 2-fold dilution) and the cells gently triturated by pipetting to recover all hPSC in a single cell suspension. The cells can then be transferred, e.g., in sterile 15 ml conical tubes. If a large number of detached cells are still detected in the well under microscope, 1 mL of LIF-4i medium can be added and combined with the TIRN-hPSC in the 15 ml conical.
The 15 ml conical can be centrifuged at 200 g for 5 min and the supernatant aspirated/discarded. The cells can be re-suspended in LIF-4i medium in a biosafety cabinet for cell counting using a hemocytometer or an automatic cell counter.
˜2×105 cells can be plated into 2 mL of LIF-4i FF medium supplemented with 5 μM Y-27632 per well onto Vitronectin-XF-coated 6-well plates in a biosafety cabinet. The plate can be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
The next day, the plate can be gently swirled to lift all non-attached cells, the medium aspirated and replaced with 2 mL of LIF-4i medium in a biosafety cabinet daily for 2-3 days. The plate can then be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
The 2nd and 3rd passages in LIF-4i FF require rigorous maintenance (
In a biosafety cabinet, culture medium can be discarded and each well of 2nd or 3rd LIF-4i passage cultures washed by gently adding 2 mL of PBS. The PBS can be discarded, and 1 mL of non-enzymatic EDTA-based cell detachment solution can be added. The cells can then be incubated for 5 min at 37° C. in a CO2 incubator (5% CO2, humid atmosphere).
Using an inverted microscope or other appropriate means, it can be advantageous to verify that most dome-shape colonies have detached. The sides of the plate can be gently tapped to maximize colony detachment.
Without trituring cells in the well, the cell suspension can be gently transferred in sterile 15 mL conical tubes in a biosafety cabinet. 1 mL of LIF-4i medium can be added (TABLE 1; at least 2-fold final dilution) without disturbing the monolayer areas of differentiated cells that are still attached in the wells. Cells can be gently triturated in the conical tubes by pipetting to obtain a single cell suspension.
The conical tubes can be centrifuged at 200 g for 5 min and the supernatant aspirated/discarded. The cells can be re-suspend in LIF-4i medium in a biosafety cabinet for cell counting using a hemocytometer or an automatic cell counter.
˜2×105 cells can be plated into 2 mL of LIF-4i medium supplemented with 5 μM Y-27632 per well onto Vitronectin-XF-coated 6-well plates in a biosafety cabinet. The plate can be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
The next day, the plate can be gently swirled to lift all non-attached cells, the medium aspirated and replaced with 2 mL of LIF-4i medium in a biosafety cabinet daily for 2-3 days. The plate can be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
Note: After the 2nd and 3rd passages in LIF-4i using manual picking or the protocol steps detailed above, successful reversion will result into homogenous TIRN-hPSC LIF-4i cultures with only minimal residual differentiation carry-over. Stable LIF-4i cultures will tolerate bulk passaging thereafter.
Once homogeneous dome-shape colony cultures have been established (i.e., after the third passage in LIF-4i), cells bulk can be passaged every 2-3 days (
In a biosafety cabinet, culture medium can be discarded and each well of LIF-4i cultures washed by gently adding 2 mL of PBS. PBS can be discard and 1 mL of non-enzymatic EDTA-based dissociation buffer added. The cells can be incubated for 5 min at 37° C. in a CO2 incubator (5% CO2, humid atmosphere).
LIF-4i FF medium can be added (TABLE 1, at least 2-fold dilution) and the cells gently triturated by pipetting to recover all UTIRN-hiPSC in a single cell suspension. The cells can then be transferred into sterile 15 ml conical tubes. 1 mL of LIF-4i medium can be added to recover remaining cells in the well and combined UTIRN-hiPSC.
The conical tubes can be centrifuged at 200 g for 5 min and the supernatant aspirated/discarded. The cells can be re-suspend in LIF-4i medium in a biosafety cabinet for cell counting using a hemocytometer or an automatic cell counter.
˜2×105 cells can be plated into 2 mL of LIF-4i medium supplemented with 5 μM Y-27632 per well onto Vitronectin-XF-coated 6-well plates in a biosafety cabinet. The plate can be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
The next day, the plate can be gently swirled to lift all non-attached cells, the medium aspirated and replaced with 2 mL of LIF-4i medium in a biosafety cabinet daily for 2-3 days. The plate can then be placed in a CO2 incubator (5% O2, 5% CO2, humid atmosphere).
Typically, reverted conventional, primed hiPSC are expanded for at least 5-10 passages in LIF-4i FF, as indicated above, prior to use in functional studies or long-term cryopreservation. In general, the number of passages in conventional conditions and in LIF-4i FF conditions are recorded on each cryopreserved vial.
Excess LIF-4i FF UTIRN-hPSC not used in functional assays can be cryopreserved at each passage (TABLE 2), but freezing of lower post-reversion passages (e.g., <p5) will be hindered by low cell number and contamination from differentiated cells that may result in poor, or highly variable post-thaw recovery efficiencies.
In a biosafety cabinet, culture medium can be aspirated, cells washed in PBS (2 mL per well), PBS aspirated, and hPSC colonies dissociated into single cells using enzyme-free EDTA-based cell detachment solution (1 mL per well). The plate can then be placed for 5 min at 37° C. in a CO2 incubator (5% CO2, humid atmosphere).
The cell detachment solution can be diluted with LIF-4i medium (2-fold) in a biosafety cabinet and the TIRN-hPSCs collected in a sterile 15 mL conical tube.
The cells can be centrifuged at 200 g for 5 min and the cell pellet resuspended in LIF-4i medium (1-2 mL per well-equivalent) in a biosafety cabinet. The number of cells can be counted using a hemocytometer or an automatic cell counter.
UTIRN-hPSCs can be centrifuged in LIF-4i medium (200 g for 5 min) and the cells can be resuspend in a biosafety cabinet in freezing solution (TABLE 2), at a density of at least 1×106 cells/mL.
The cells can be transferred into long-term storage cryogenic tubes and placed into a slow-freezing container. The samples can be allowed to freeze overnight in a −80° C. freezer.
The next day, the cryovials can be transferred into a liquid nitrogen freezer for long term storage.
For thawing, the frozen vial can be placed into a 37° C. water bath for ˜2 min. The vial can be sterilized (i.e., ethanol spray), hPSCs transferred in a sterile 15 ml conical and the cells slowly diluted 10-fold in LIF-4i medium (TABLE 1) supplemented with 5 μM of Rho-associated protein kinase (ROCK) inhibitor Y-27632 within a sterile biological safety hood cabinet.
The cells can be centrifuged at 200 g for 5 min. In a biosafety cabinet, cell-free supernatant can be discarded and the cell pellet resuspended in LIF-4i medium (1-2 mL) supplemented with 5 μM ROCK inhibitor Y-27632.
Exclusion of Y-27632 will generally result in poor post-thawing recovery efficiencies.
The thawed cells resuspended in LIF-4i/ROCK Inhibitor can be transferred onto Vitronectin-XF-coated wells. LIF-4i cultures are routinely cryopreserved at a density of 1×106 cells per vial. Each of these vials is thawed in one well of a Vitronectin-XF-coated 6-well plate.
The next day, regular LIF-4i FF UTIRN-hiPSC expansion can be started.
The starting primed, conventional hPSC cultures can be screened for possession of a normal karyotype (e.g., with Giemsa-band staining analysis using methods known in the art) before initiating LIF-4i reversion, to eliminate conventional hPSC populations that may harbor abnormal genomic alterations that may drive artefactual selective survival advantage in clonal LIF-4i FF conditions (
Conventional hPSC cultures can be freshly reverted to a naïve-like state with LIF-4i several weeks prior to their use in functional studies or directed differentiations.
Routine prolonged ‘maintenance’ culture in LIF-4i FF conditions for more than 10 passages following naïve reversion is not recommended. Routine expansion and maintenance of hESC and hiPSC lines should be performed using conventional culture systems (e.g., in E8, or MEF/hESC medium with bFGF).
Post-reverted TIRN-hPSC lines can be assessed for retention of normal karyotypes 5-7 passages after LIF-4i FF reversion (e.g., with Giemsa-band staining analysis, or other method of choice,
All reverted TIRN-hPSC lines can be assessed for retention of normal parental genomic imprints by a DNA methylation analysis of choice (e.g., protocols for CpG DNA microarray analysis of parental imprints in LIF-3i-reverted TIRN-hPSC are known in the art) after 5-10 passages of LIF-4i FF TIRN reversion.
LIF-4i FF UTIRN-hiPSCs can be directly utilized in established directed differentiation protocols without any extended cell culture manipulations. For example, but not by way of limitation, “re-priming” (i.e., converting TIRN-hPSC back to conventional primed conditions prior to their use in directed differentiation assays) is not necessary with the LIF-4i FF methods. However, the PKC inhibitor Go6983 requires washing out prior to starting differentiation protocols (successful differentiation has also resulted with additional removal of the MEK inhibitor PD0325901). LIF-4i FF TIRN-hPSC are still capable to differentiate directly without washing Go6983, but the presence of the PKC inhibitor and its intracellular retention can interfere with directed differentiation protocols. The medium can be switched to LIF-4i without PKC inhibitor for at least one hour (intracellular retention of the small molecule Go6983 is easily detectable in a red channel by flow cytometry without any additional staining).
To control for the impacts of assay and interline variability in the functional testing of individual hPSC lines, in certain embodiments, cross-validation of lineage-specific differentiation potencies can be ensured by employing independent differentiation protocols with at least three hPSC lines derived from independent genetic backgrounds (i.e., multiple donor-derived hiPSC and hESC).
For functional comparisons of conventional vs UTIRN-hiPSC, parallel sibling cultures can, in certain embodiments, be employed, at equivalent passage number, and from the same (isogenic) hPSC line in parallel between conventional lineage-primed and LIF-3i-reverted hPSC cultures. In such embodiments, it can be useful to maintain primed/naïve sibling isogenic hPSC cultures in parallel in their respective media (e.g., E8vs. LIF-4i FF), and simultaneously differentiate in parallel using identical differentiation protocols and materials, to eliminate experimental bias.
For isogenic primed vs. TIRN hPSC comparisons, it can, in certain embodiments, be useful to adjust initial plating densities for each individual differentiation assay. Suitable detailed protocols for neural progenitor, definitive endoderm and hemato-endothelial directed differentiations of LIF-4i FF-reverted TIRN-hPSC are known in the art.
LIF-4i-reverted UTIRN-hiPSC can exhibit a more robust proliferative and differentiation capacity in directed differentiation assays than conventional hPSC. UTIRN-hiPSC typically require a lower initial plating concentration than conventional hPSC, and unlike their conventional primed hPSC counterparts, typically do not require the use of anti-apoptotic reagents to enhance their clonal survival following enzymatic digestion in differentiation assays.
The transition between primed, conventional, and LIF-4i FF TIRN culture systems is accompanied by distinct physical changes in hPSC colony morphology. Conventional hPSC cells proliferate as flat, wide monolayer colonies that expand rapidly from small cell clumps (on MEF or feeder-free conditions), but poorly as single cells. Exposure of conventional hPSC lines to LIF-4i promotes the growth and expansion and of smaller, tightly-packed, dome-shaped colonies that arise clonally from single cells. These morphological changes are completely reversible, and LIF-4i-reverted dome-shaped colonies can spontaneously transition back to a conventional monolayer morphology if LIF-4i FF is withdrawn and cells are re-cultured in standard conventional hESC medium supplemented with bFGF. Additionally, expansion of LIF-4i FF-reverted cells at high confluent densities (or prolonged culture without frequent passaging) results in spontaneous reacquisition of the flat, conventional morphology with reduced clonal efficiency; emphasizing the need for diligent maintenance and care of LIF-4i FF-reverted hPSC (e.g., <40-60% confluence).
Evaluation of pluripotency markers during the transition from conventional to a naïve-like UTIRN-hiPSC state following continuous LIF-4i FF culture can be monitored by live antibody staining without detecting negative effects on UTIRN-hiPSC expansion (e.g., live-staining fluorochrome-conjugated antibodies against TRA-1-81, TRA-1-60, and SSEA4).
Retention of naïve pluripotency during LIF-4i FF TIRN reversion can also be routinely monitored by using qRT-PCR of naïve-specific transcripts (
LIF-4i FF cultures maintain similar expressions of TRA-1-81, TRA-1-60, and SSEA4 to those observed in conventional and LIF-3i/MEF conditions. The retention of these markers can be routinely monitored by flow cytometric analysis. Results are presented for the hiPSC line 6.2 (
Because the genetic background of hPSC lines has been characterized as a strong contributor to interline variability, it is important to rigorously assess isogenic cultures at matching culture time points when comparing conventional hiPSC to TIRN-hiPSC culture systems (
Accordingly, most studies of human naïve culture systems have focused on assaying molecular pluripotency of TIRN-hPSC at 1) the epigenetic level (e.g., histone marks by ChIP sequencing or ChIP-PCR, global DNA methylation by immunoblots or whole genomic bisulfite sequencing, allele-specific CpG methylation microarrays, OCT4enhancer predominant usage by reporter systems, global activity at regulatory elements by DNAse I hypersensitivity, and repeat clement profiling by RNA-sequencing), 2) transcriptomic level (RNA-sequencing, expression microarrays, and quantitative RT-PCR), protein expression analysis (e.g., FACS, immunofluorescent microscopy, and Western blotting) and 3) via metabolic studies (e.g., glycolysis, oxidative phosphorylation and nicotinamide metabolism).
Representative examples of immunofluorescence stains and Western blot detection of expression for key markers of molecular pluripotency are shown for non-XF and XF hPSC lines (
LIF-4i FF colonies maintained uniform expression of the pluripotency markers NANOG, OCT4 and TRA-1-81, acquire expression of naïve markers (i.e., NR5A2) and display elevated levels of the activated isoform of beta-catenin (
The data also further illustrate that the LIF-4i FF system reproduces mechanistic regulation of active beta-catenin via tankyrase inhibition, as previously described for the LIF-3i/MEF culture system (
Additional evidence of molecular naïve pluripotency of UTIRN-hiPSC was provided by evaluating the epigenetic functionality of the OCT4 promoter in conventional, primed vs UTIRN-hiPSC (
The most rigorous assay of functional pluripotency of PSC is the blastocyst injection chimera assay, which is limited in the testing of UTIRN-hPSC lines for ethical reasons. Alternatively, several groups that reported the generation of TIRN hPSC derived with various other methods have also attempted to generate interspecies chimeras. However, these attempts have yielded extremely low or unsuccessful contribution of differentiated TIRN-hPSC lineages to murine or porcine embryos, in comparison to the chimera-generating capacity of standard mouse ESC. Additional functional studies have investigated directed in vitro differentiation of putative TIRN-hPSC derived via various methods, but have revealed biased, defective, or diminished multi-lineage differentiation capacity, with concomitant harboring of epigenetic abnormalities. Similar epigenomic aberrations, especially at imprinted loci, have been detected in mouse ESC following prolonged exposure to the LIF-2i cocktail. Interestingly, some culture systems that included the non-specific tankyrase/PARP inhibitor XAV939 have reported global improvements in specific attributes of functional pluripotency of PSC, such as improved chimera contribution and an enhanced capacity for in vivo trophectoderm contribution.
An isogenic approach can be employed to compare directed differentiation to ectodermal, mesodermal, and endodermal lineages hPSC in primed (i.e., E8) vs. feeder-free naïve LIF-4i FF TIRN conditions. However, initial plating density and conditions should be adjusted for each individual assay. Additionally, certain LIF-4i FF protocols are compatible with rapid in-bulk reversion of multiple primed hPSC lines.
Using a broad collection of independently-derived UTIRN-hPSC, multilineage differentiation assays have been employed to show that the LIF-4i FF system dramatically improved the functional pluripotency of conventional, primed hPSC lines. In addition, a systematic analysis of conventional hiPSC vs UTIRN-hiPSC lines in isogenic pairs has been performed to eliminate interline-dependent variations. UTIRN-hiPSC lines do not require a re-priming step prior to EB differentiation. However, UTIRN-hiPSC proliferate at significantly higher clonal rates than isogenic cells expanded in E8, and thus, initial lower plating densities require adjustment to allow each culture to reach confluence at a similar time point.
Investigators can utilize multiple assays to demonstrate improved functionality of LIF-4i FF-reverted UTIRN-hiPSC, including not only in vivo teratoma assays, but also in vitro directed differentiation assays to neural (ectodermal), definitive endoderm and hematovascular (mesodermal) lineages using multiple assays (e.g., 2D APEL 2 and 3D embryoid body systems). To control for assay-dependent reproducibility, at least two different differentiation methods should typically be performed in replicate for each isogenic pair of primed LIF-4i FF hPSC cultures. The experimental design should include a robust number (e.g., >3-5) primed LIF-4i FF isogenic pairs of hPSC lines from multiple, independent donor genetic backgrounds.
LIF-4i FF cultures do not promote spontaneously-arising chromosomal defects as was reported in other human naïve reversion systems. Nevertheless, the use of low passage LIF-4i FF hPSC for functional assays (i.e., directed differentiations) for prevention of genomic aberrations that may arise from prolonged clonal culture is recommended. During the performance of functional assays, primed cultures should typically be maintained in parallel conditions and differentiated using the same materials and methods as isogenic TIRN-hPSC, to eliminate potential experimental bias. Excluding the protein kinase C inhibitor from LIF-4i FF TIRN cultures for at least one hour at the initiation of directed differentiation protocols is also recommended. Initial plating densities should also be adjusted to allow each culture to reach confluence at a similar time point.
Teratoma Assays. Functional pluripotency of UTIRN-hiPSC was assayed using teratoma differentiation protocols, without any requirement for re-priming LIF-4i FF hPSC before injection into animals (
In Vitro Directed Vascular Differentiation. LIF-4i FF UTIRN-hPSC cultures routinely demonstrated more robust differentiation capacities than their isogenic primed hPSC counterparts in not only teratoma assays, but also in XF directed differentiation assays (APEL system). For example, directed UTIRN-hiPSC differentiation from LIF-4i in XF vascular lineage differentiation conditions resulted in more rapid kinetics of pericytic vascular progenitor (VP; CD31+CD146+) and endothelial progenitors (CD34+CD140b+), and other mesodermal vascular populations (KDR+, CD144+) than from their isogenic conventional hPSC counterparts (
Preserving genomic stability is critical during early embryonic development. Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are the main mechanisms that eukaryotic cells use for double-strand DNA break (DSB) repair and preservation of genomic integrity. Pre-implantation embryos and mouse embryonic stem cells (mESC) favor the more accurate HDR rather than the fast, but error-prone NHEJ. As a result, mESC exhibit poor efficiency for rejoining radiation-induced DNA double-strand breaks in comparison to human primed ESC that similarly to mouse post-implantation embryos predominantly utilize NHEJ for DSB repair. Various strategies have been recently developed to overcome limited precise editing in CRISPR-Cas9-mediated genome editing, including altering HDR or NHEJ machinery. The predominant DSB repair strategy in naïve hPSC remains unclear, but superior HDR efficiencies would be expected if naïve hPSC adopt developmental expressions that are closer to preimplantation embryos. However, HDR efficiencies were actually impaired using the human naïve method 4iLA, which also exhibits elevated aneuploidy frequency and erased parental imprints.
LIF-4i stably reverts conventional, primed hPSC to a functional human naïve epiblast-like state that recapitulates molecular and epigenetic signatures of the human pre-implantation epiblast. More importantly, UTIRN-hPSC maintained normal karyotypes and epigenomic imprints. If UTIRN-hPSC possess improved HDR, this would greatly impact developmental biology and regenerative medicine. While mESC may harbor deficiencies for NHEJ and radiation-induced DSB repair, retention of NHEJ in UTIRN-hPSC would also be important to support efficient and rapid repair in DSB-inducing environment. The study of HDR/NHEJ mechanisms in UTIRN-hiPSC may functionally validate the DSB repair strategies utilized by UTIRN-hPSC. Thus, the HDR machinery of LIF-3i-reverted TIRN-hPSC vs. their isogenic conventional, primed hPSC counterparts was investigated.
Methods. Conventional (primed) XF CB-hiPSC lines and control H9 hESC were grown in E8 medium. Isogenic TIRN-hPSC were cultured in parallel using LIF-4i. For Western blotting, whole cell lysates were prepared in RIPA buffer, 1.5 mM EDTA with protease inhibitor. Protein lysates were quantified using the BCA assay. Equal amounts of total protein lysate (e.g., 20 μg) were loaded into precast polyacrylamide gradient gels, size-separated using the Mini-PROTEAN electrophoresis system and blotted onto PDVF membranes for chemiluminescent analysis. Primed hPSC and isogenic TIRN-hPSC were assayed for endogenous DNA damage during routine culture (
Results. To explore the effects of LIF-4i on DSB repair, the levels of major components of NHEJ and HDR were evaluated, particularly the catalytic subunit of DNA-PK and BRCA1. which are two critical components of the NHEJ and HDR machineries respectively, and that are both regulated by both PARP1 and tankyrase PARylating and non-PARylating activities. The tankyrase inhibitor XAV939 has been shown to not only downregulate tankyrase PARylating activity, but also PARP1-mediated PARylation in 293T cells. Inhibition of PARP1-mediated PARylation has been shown to not only promote DNAPK auto-phosphorylation and activation, but also controls and activates BRCA1-mediated HDR. Tankyrase knock-down or XAV939 inhibition increases DNAPKc activity. Alternatively, tankyrase controls the activity of BRCA1 complexes and HDR by interacting with the DNA damage sensor MDC1. MDC1 is a DNA checkpoint protein that is essential to spread the DNA response machinery and that is cleaved by caspase3 during apoptosis. This tankyrase activity on MDC1 is independent of tankyrase-mediated PARylation and is not diminished in the presence of the tankyrase/PARP inhibitor XAV939 in the 293T cell line.
Unlike mESC, UTIRN-PSC that were grown in LIF-4i continued to express high levels of DNA-PKc (
Previous studies suggested that mESC were deficient in repairing radiation-induced DSB. Consequently, whether reduced H2AX levels in LIF-4i UTIRN-hiPSC reflected a DSB repair deficiency rather than efficient repair in endogenous condition was investigated. Primed E8 and LIF-4i UTIRN-hiPSC were exposed to the radiomimetic neocarzinostatin to induce DSB (
These studies provide evidence that UTIRN-PSC can efficiently employ HDR as a DSB repair strategy, yet remain competent to repair radiation-induced DSB. The inclusion of XAV939 in LIF-3i appeared to interrupt tankyrase auto-PARylation-mediated proteolysis. Elevated tankyrase protein in TIRN-hPSC correlated with stabilization of the non-cleaved isoform of its partner MDC1 and reinforcement of RAD54 and BRCA1.Accordingly the data described herein indicates that modifying the balance between NHEJ and HDR events by manipulating naïve vs. primed pluripotent states can enhance genome editing strategies (e.g., CRISPR-Cas9, plasmid-based HDR), and allow for more facile gene targeting of hPSC.
The contents of all figures and all references, patents and published patent applications and Accession numbers cited throughout this application are expressly incorporated herein by reference.
This application is a continuation of International Patent Application No. PCT/US2023/011688, filed Jan. 27, 2023, which claims priority to U.S. Provisional Patent Application Ser. No. 63/303,611 filed Jan. 27, 2022, the content of which are incorporated by reference in their entireties, and to which priority is claimed.
This invention was made with government support under grant HD082098awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63303611 | Jan 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2023/011688 | Jan 2023 | WO |
| Child | 18785343 | US |
