Successful application of Tissue Engineered Medical Products (TEMPs) crucially depends on the generation of highly specialized cell types. Diseases that can potentially be treated or cured using TEMPs include diabetes, heart disease and neuronal degenerative diseases. A common strategy relies on instructing stem cells to differentiate towards the desired cell type through the process of directed differentiation. Conceptually, such attempts seek to provide cellular signals designed to imitate naturally occurring developmental cues and the differentiation process occur through discrete stages. Currently, such methods are developed through tedious testing, where the combinatorial space of plausible signaling inputs are inadequately explored, and the processes rarely consider cost of consumables/reagents. For instance, protein-type agonists are commonly used to imitate natural developmental cues but cost 100-1000-fold more than small molecule agonists or antagonists. Proteins also degrade over time, decreasing their overall efficacy and requiring greater QC requirements for a production process. The high cost is further amplified with each intermediate stage generated and the overall duration of the differentiation protocol. Many of the directed differentiation protocols that have been developed also suffer from poor robustness with only a percentage of the culture attaining the desired phenotype.
Current pancreatic beta cell (PBC) protocols use 6 discrete stages and can take as long as 34 days to generate beta cells that have highly variable functionality (Pagliuca et al., 2014; Rezania et al., 2014; and Velazco et al., 2020). In spite of the foregoing, PBC development has enormous potential in future health care. Specifically, TEMPs derived from PBC protocols (i.e., stem-cell derived insulin producing cells) are currently entering clinical trials. For example, Vertex, which acquired SEMMA, the origin of TEMPs, received accelerated Phase 1/2 approval (NCT04786262) for commencement of a stem cell-based therapy for metabolically unstable Type I diabetes (enrolling 17 patients).
Accordingly, while some progress has been achieved, there remains a need for efficient and robust methods and compositions for generating human pancreatic beta cells and their progenitor cells from human pluripotent stem cells.
This disclosure provides methods of generating human pancreatic beta cells (PBCs), including human dorsal foregut endodermal cells (DFECs) and human pancreatic progenitor cells (PPCs), in a three-stage protocol that can be completed in as little as 16 days. The methods use chemically defined culture media that allows for generation of DEFCs within three days of culture, PPCs within six days of culture, and PBCs within 16 days of culture. The defined culture media used to obtain the different types of progenitor cells comprises small molecule agents that either agonize or antagonize particular signaling pathways in the pluripotent stem cells such that differentiation along the endoderm lineage is promoted, leading to cellular maturation and expression of PBC-associated biomarkers. The methods of the disclosure use culture media for differentiation utilizing different components than used in earlier protocols and avoiding the need for certain components required in other protocols. The methods of the disclosure also have the advantage that use of small molecule agents in the culture media allows for precise control of the culture components, involving less differentiation stages, and less time for differentiation to PBCs compared to prior art protocols.
Accordingly, in one aspect, the disclosure pertains to a method of generating human dorsal foregut endodermal cells (DFECs) comprising: culturing human pluripotent stem cells (PSCs) in a culture media comprising a BMP pathway antagonist, an RA pathway agonist, a TGF-β pathway antagonist, and an MEK pathway antagonist on days 0-3 to obtain human DFECs.
The method can comprise further culturing the human DFECs on days 4-6 in a culture media comprising a BMP pathway antagonist, a RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist to obtain human pancreatic progenitor cells (PPCs).
The method can then further comprise culturing the human PPCs on days 7-16 in a culture media comprising a Notch pathway inhibitor (e.g., γ-secretase inhibitor), a TGF-β pathway antagonist, and a flavonoid to obtain human pancreatic beta cells (PBCs).
In one embodiment, the human pluripotent stem cells are induced pluripotent stem cells (iPSCs). In another embodiment, the human pluripotent stem cells are embryonic stem cells. In another embodiment, the human pluripotent stem cells are attached to vitronectin-coated plates during culturing.
In another embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, Dorsomorphin, K02288, LDN214117, LDN212854, folistatin, ML347, Noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the culture media at a concentration within a range of 100-400 nM. In another embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture media at a concentration of 250 nM.
In another embodiment, the RA pathway antagonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM 580, CD 1530, CD 2314, Ch 55, BMS 753, Tazarotene, Isotretinoin, AC 261066, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, All-trans Retinoic Acid (ATRA). In one embodiment, the RA pathway antagonist is present in the culture media at a concentration within a range of 0.5-4 nM. In another embodiment, the RA pathway antagonist is RA, which is present in the culture media at a concentration of 2 μM.
In another embodiment, the TGF-β pathway antagonist is selected from the group consisting of A8301, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof. In one embodiment, the TGFβ pathway antagonist is present in the culture media at a concentration within a range of 200-1000 nM. In another embodiment, the TGFβ pathway antagonist is A8301, which is present in the culture media at a concentration of 500 nM.
In another embodiment, the MEK pathway antagonist is selected from the group consisting of PD0325901, Binimetinib (MEK162), Cobimetinib (XL518), Selumetinib, Trametinib (GSK1120212), CI-1040 (PD-184352), Refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, and combinations thereof. In one embodiment, the MEK pathway antagonist is present in the culture media at a concentration within a range of 200-1000 nM. In another embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture media at a concentration of 250 nM.
In another embodiment, the TAK1 pathway antagonist is selected from the group consisting of Taki ((5Z)-7-Oxozeaenol), Takinib, Dehydoabietic acid, NG25, Sarsasapogenin, and combinations thereof. In one embodiment, the TAK1 pathway antagonist is present in the culture media at a concentration within a range of 200-1000 nM. In another embodiment, the TAK1 pathway antagonist is Taki ((5Z)-7-Oxozeaenol), which is present in the culture media at a concentration of 500 nM.
In another embodiment, the bFGF mimetic is present in the culture media at a concentration within a range of 150-600 nM. In one embodiment, the bFGF mimetic is SUN11602, which is present in the culture media at a concentration of 300 nM.
In another embodiment, the AKT pathway antagonist is selected from the group consisting of AT7867, MK2206, GSK690693, Perifosine (KRX-0401), Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, Triciribine (NSC154020), ARQ751, Miransertib (ab235550), Borussertib, Cerisertib, and combinations thereof. In one embodiment, the AKT pathway antagonist is present in the culture media at a concentration within a range of 100-400 nM. In another embodiment, the AKT pathway antagonist is AT7867, which is present in the culture media at a concentration of 250 nM.
In another embodiment, the Notch pathway antagonist is selected from the group consisting of GSI-XX, R04929097, Semagacestat, Dibenzazepine, LY411575, Crenigacestat, IMR-1, IMR-1A, FLI-06, DAPT, Valproic acid, YO-01027, CB-103, Tangeretin, BMS-906024, Avagacestat, Bruceine D, and combinations thereof. In one embodiment, the Notch pathway antagonist is present in the culture media at a concentration within a range of 50-200 nM. In another embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media at a concentration of 100 nM.
In another embodiment, the flavonoid is selected from the group consisting of quercetin, quercetin analogues, (e.g., dihydroquercetin, 6,2′,4′,5′-pentahydroxyflavone, quercetin-3-O-propionate (Q-pr), quercetin-3-O-butyrate (Q-bu), quercetin-3-O-valerate, or 3,4′-Di-O-methylquercetin), genistein, anthocyanins, catechins, gallocatechins (e.g., epigallocatechin-3-gallate (EGCG)), anthocyanidins, apigenin, luteolin, kaemferol, curcumin, myricetin, daidzein, naringin, rutin, and hesperitin. present in the culture media at a concentration within a range of 3-50 μM. In another embodiment, the flavonoid is quercetin, which is present in the culture media at a concentration of 15 μM.
In another aspect, the disclosure provides a method of generating human DFE cells comprising culturing human pluripotent stem cells in a culture media comprising a BMP pathway antagonist, a RA pathway agonist, a TGF-β pathway antagonist, and an MEK pathway antagonist on days 0-3 to obtain human DFE cells.
In another embodiment, the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, and the MEK pathway antagonist is PD0325901.
In another embodiment, LDN193189 is present in the culture media at a concentration within a range of 100-400 nM, retinoic acid is present in the culture media at a concentration within a range of 0.4-4 μM, A8301 is present in the culture media at a concentration within a range of 200-1000 nM, and PD0325901 is present in the culture media at a concentration within a range of 100-400 nM.
In another embodiment, LDN193189 is present in the culture media at a concentration of 250 nM, retinoic acid is present in the culture media at a concentration of 2 μM, A8301 is present in the culture media at a concentration of 500 nM, and PD0325901 is present in the culture media at a concentration of 250 nM.
In another aspect, the disclosure provides a method of generating human pancreatic progenitor cells (PPCs) comprising:
In one embodiment, the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, the MEK pathway antagonist is PD0325901, the TAK1 pathway antagonist is Taki ((5Z)-7-Oxozeaenol), the bFGF mimetic is SUN11602, and the Akt pathway antagonist is AT7867.
In another embodiment, LDN193189 is present in the culture media at a concentration within a range of 100-400 nM, retinoic acid is present in the culture media at a concentration within a range of 0.5-4 μM, A8301 is present in the culture media at a concentration within a range of 200-1000 nM, PD0325901 is present in the culture media in step (a) at a concentration within a range of 100-400 nM, Taki ((5Z)-7-Oxozeaenol) is present in the culture media at a concentration within a range of 200-1000 nM, SUN11602 is present in the culture media at a concentration within a range of 150-600 nM, and AT7867 is present in the culture media in step (a) at a concentration within a range of 100-400 nM.
In another embodiment, LDN193189 is present in the culture media at a concentration of 250 nM, retinoic acid is present in the culture media at a concentration of 2 μM, A8301 is present in the culture media at a concentration of 500 nM, PD0325901 is present in the culture media at a concentration of 250 nM, Taki ((5Z)-7-Oxozeaenol) is present in the culture media at a concentration of 500 nM, SUN11602 is present in the culture media at a concentration of 300 nM, and AT7867 is present in the culture media in step (a) at a concentration of 250 nM.
In yet another aspect, the disclosure provides a method of generating human pancreatic beta cells (PBCs) comprising:
In one embodiment, the BMP pathway antagonist is LDN193189, the RA pathway agonist is retinoic acid, the TGF-β pathway antagonist is A8301, the MEK pathway antagonist is PD0325901, the TAK1 pathway antagonist is Taki ((5Z)-7-Oxozeaenol), the bFGF mimetic is SUN11602, the Akt pathway antagonist is AT7867, the Notch pathway antagonist is 7-secretase inhibitor XX (GSI-XX).
In another embodiment, LDN193189 is present in the culture media in steps (a) and (b) at a concentration within a range of 100-400 nM, retinoic acid is present in the culture media in steps (a) and (b) at a concentration within a range of 0.5-2 μM, A8301 is present in the culture media in steps (a)-(c) at a concentration within a range of 200-1000 nM, PD0325901 is present in the culture media in step (a) at a concentration within a range of 100-400 nM, Taki ((5Z)-7-Oxozeaenol) is present in the culture media in step (b) at a concentration within a range of 200-1000 nM, SUN11602 is present in the culture media in step (b) at a concentration within a range of 150-600 nM, AT7867 is present in the culture media in step (b) at a concentration within a range of 100-400 nM, γ-secretase inhibitor XX (GSI-XX) is present in the culture media in step (c) at a concentration within a range of 50-200 nM, and quercetin is present in the culture media in step (c) at a concentration within a range of 5-30 μM.
In another embodiment, LDN193189 is present in the culture media in steps (a) and (b) at a concentration of 250 nM, retinoic acid is present in the culture media in steps (a) and (b) at a concentration of 2 μM, A8301 is present in the culture media in steps (a)-(c) at a concentration of 500 nM, PD0325901 is present in the culture media in step (a) at a concentration of 250 nM, Taki ((5Z)-7-Oxozeaenol) is present in the culture media in step (b) at a concentration of 500 nM, SUN11602 is present in the culture media in step (b) at a concentration of 300 nM, AT7867 is present in the culture media in step (b) at a concentration of 250 nM, GSI-XX is present in the culture media in step (c) at a concentration of 100 nM, and quercetin is present in the culture media in step (c) at a concentration of 15 μM.
The methods and compositions of the disclosure are useful in the generation of pancreatic beta cells or progenitor cells thereof for use in clinical therapy, research, development, and commercial purposes. For therapeutic applications, the in vitro generated PBCs of the present invention can be administered directly or systemically to a subject for treating or preventing type 1 diabetes, type 2 diabetes, pre-diabetes, conditions due to significant trauma (i.e., damage to the pancreas or loss or damage to islet beta cells), or other metabolic diseases or disorders associated with a deficiency in beta cell number (e.g., a reduction in the number of pancreatic cells), an insufficient level of beta cell biological activity (e.g., a deficiency in glucose-stimulated insulin secretion, or a deficiency in insulin production).
Other features and advantages of the invention will be apparent from the following detailed description and claims.
Described herein are methodologies and compositions that allow for the generation of pancreatic beta cells (PBCs) and their progenitor cells from human pluripotent stem cells under chemically defined culture conditions using a small molecule-based approach. The methods of the disclosure generate PBCs and their progenitors in a three-stage protocol in which FOXA2+HNF1b+ dorsal foregut endodermal cells (DFEs) are generated in three days, followed by generation of PDX1+PTFIA+NKX6.1+ pancreatic progenitor cells (PPCs) by day six of culture, followed by generation of pancreatic beta cells (PBCs) by day 11 of culture. Thus, the disclosure allows for obtention of PBCs in a significantly shorter time than prior art protocols using chemically defined culture conditions.
Various aspects of the invention are described in further detail in the following subsections.
The starting cells in the cultures are human pluripotent stem cells. As used herein, the term “human pluripotent stem cell” (“hPSC”) refers to a human stem cell that has the capacity to differentiate into a variety of different cell types. The term “pluripotent” as used herein refers to a cell with the capacity, under different conditions, to differentiate to cell types characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm). Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers, for example, using a nude mouse and teratomas formation assay. Pluripotency can be evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers.
hPSCs include, for example, induced pluripotent stem cells (iPSC) and human embryonic stem cells, such as ES cell lines. Non-limiting examples of induced pluripotent stem cells (iPSC) include 19-11-1, 19-9-7 or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, J. A. et al. (1998) Science 282:1145-1147). Human pluripotent stem cells (PSCs) express cellular markers that can be used to identify cells as being PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG and/or SOX2. Since the methods for generating the progenitor populations of the disclosure are under differentiation from the starting pluripotent stem cell population, in various embodiments the progenitor cell populations generated by the methods of the disclosure lack expression of one or more stem cell markers, including those described in the Examples.
Common practices and methods for coating TC plates or passaging of iPSCs may be used. In exemplary embodiments, PSC cultures, such as CR01 iPSC line, can be maintained and grown on vitronectin coated 6-well tissue culture (TC) plates. E8 media may be used for general maintenance of these cells. PSC cultures are generally passaged every 3-4 days using EDTA to disrupt cell-to-cell adhesion. This can be accomplished by removing the E8 media and washing each well of the TC plate with 2 ml of PBS. A 3-minute incubation in the presence of 5 mM EDTA can then be performed at 37 degrees C. Wells are then aspirated and the cells are washed off of the plates and seeded in fresh E8 media supplemented with 1X RevitaCell. Each well passaged is generally seeded onto 6 wells of a newly vitronectin-coated TC plate resulting in a 1 to 6 expansion of the iPSC line.
Passaging of PSCs may be performed using collagenase, accutase, trypsin, TyrPLE or other digestion enzymes instead of EDTA. PSCs can also be maintained and grown on substrates other than vitronectin. Commonly used substrates include gelatin, Matrigel, geltrex or other ECM or charged surface coatings. In addition, other ROCK inhibitors may be used instead of those in the RevitaCell supplement. A commonly used inhibitor is Y27632.
The pluripotent stem cells are subjected to culture conditions, as described herein, that induce cellular differentiation. As used herein, the term “differentiation” refers to the development of a cell from a more primitive stage toward a more mature (i.e., less primitive) cell, typically exhibiting phenotypic features of commitment to a particular cellular lineage.
In some embodiments, the cells generated by the methods of the disclosure are dorsal foregut endodermal cells (DFECs). As used herein, a “dorsal foregut endodermal cell” or “DFEC” refers to a cell that is more differentiated than a pluripotent stem cell in that it is committed to the endoderm lineage but still has the capacity to differentiate into different types of cells along that same lineage. The DFEC expresses the biomarkers FOXA2 and HNF1b.
The DFEC may also express additional biomarkers, including but not limited to the endodermal region markers of PROM1, CPB1, ONECUT1, and CXCL4; the endodermal dorsal markers of SFRP5, MNX1, PTCH1 and PAX6; and the midgut markers FOXA2, HNF1b, HOXA3 and ONECUT2. In contrast, the DFECs exhibit very low or undetectable expression of the ventral markers HHEX and NR5A2 and the classically defined definitive endodermal markers SOX17, GSC, MIXL1 and CER.
In some embodiments, the cells generated by the methods of the disclosure are pancreatic progenitor cells (PPCs), which are more differentiated (more mature) cells than DFECs and are committed to particular cell types within the endoderm lineage. A PPC of the present invention expresses the biomarkers PTF1A and PDX1.
The committed PPCs generated by the methods of the disclosure can be further cultured in vitro to generate mature human pancreatic beta cells (PBCs). As used herein, a “pancreatic beta cell” or “PBC” refers to a stem cell-derived pancreatic beta cell that expresses the biomarkers INS and PDX.
In some embodiments, cells can be identified and characterized based on expression of one or more biomarkers specific to or characteristic of DFECs, PPCs, or PBCs. A “positive” biomarker is one that is expressed on a cell of interest, whereas a “negative” biomarker is one that is not expressed on a cell of interest. In the embodiments described herein, the DFEC is FOXA2+HNF1b+, the PPC is PTF1A+PDX1+ and the PBC is INS+PDX+.
As used herein, expression by a cell of only “low” levels of a biomarker of interest is intended to refer to a level that is at most 20%, and more preferably, less than 20%, less than 15%, less than 10% or less than 5% above background levels (wherein background levels correspond to, for example, the level of expression of a negative control marker that is considered to not be expressed by the cell).
The methods of the disclosure for generating DFECs, PPCs, and PBCs comprise culturing human pluripotent stem cells in culture media comprising specific agonists and/or antagonists of cellular signaling pathways. In some embodiments, the culture media lacks serum, lacks exogenously added growth factors, lacks animal products, is serum-free, is xeno-free and/or is feeder layer free.
As shown in the exemplary embodiment depicted in
Further differentiation of the DFECs to pancreatic progenitor cells (PPC) can be achieved after three more days (referred to herein as “stage 2”) by culturing the DEFCs in a culture media comprising a BMP pathway antagonist, such as LDN193189; an RA pathway agonist, such as retinoic acid (RA); a TGF-β pathway antagonist, such as A8301; a TAK1 pathway antagonist, such as Taki; a bFGF mimetic, such as SUN11602; and an Akt pathway antagonist, such as AT7867. In some embodiments, the DFECs may be cultured for up to 5 days in stage 2 of the differentiation protocol.
Still further differentiation of the PPCs to pancreatic beta cells (PBCs) can be achieved in in as little as 5 days (referred to as “stage 3”) by culturing the PPCs in a culture media comprising a Notch pathway antagonist, such as γ-secretase inhibitor XX (GSI-XX); a TGF-β pathway antagonist, such as A8301; and a flavonoid, such as quercetin or a quercetin analogue thereof. In some embodiments, the DFECs can be cultured for up to 20 days in stage 3 culture media of the differentiation protocol.
As used herein, an “agonist” of a cellular signaling pathway is used with reference to an agent that stimulates (upregulates) the cellular signaling pathway. In some embodiments, stimulation of the cellular signaling pathway can be initiated extracellularly, for example by use of an agonist that activates a cell surface receptor involved in the signaling pathway (e.g., the agonist can be a receptor ligand). Additionally, or alternatively, stimulation of cellular signaling can be initiated intracellularly, for example, by use of a small molecule agonist that interacts intracellularly with one or more component(s) of the signaling pathway.
As used herein, an “antagonist” of a cellular signaling pathway is used with reference to an agent that inhibits (downregulates) the cellular signaling pathway. In some embodiments, inhibition of the cellular signaling pathway can be initiated extracellularly, for example by use of an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally, or alternatively, inhibition of cellular signaling can be initiated intracellularly, for example, by use of a small molecule antagonist that interacts intracellularly with one or more component(s) of the signaling pathway.
Agonists and antagonists used in the methods of the disclosure are known and/or are commercially available. They are used in the culture media at concentrations effective to achieve the desired outcome, e.g., generation of DFEs, PPCs, or PBCs, each characterized by specific corresponding markers. Non-limiting examples of suitable agonist and antagonist agents and effective concentration ranges are described further below.
Antagonists of the BMP (bone morphogenetic protein) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the BMP signaling pathway, which is biologically activated by binding of BMP to a BMP receptor, which is an activin receptor-like kinase (ALK) (e.g., type I BMP receptor, including but not limited to ALK2 and ALK3). In one embodiment, the BMP pathway antagonist is selected from the group consisting of LDN193189, DMH1, DMH2, Dorsomorphin, K02288, LDN214117, LDN212854, follistatin, ML347, Noggin, and combinations thereof. In one embodiment, the BMP pathway antagonist is present in the culture media at a concentration within a range of 100-500 nM, 100-400 nM, 150-350 nM or 200-300 nM. In one embodiment, the BMP pathway antagonist is LDN193189. In another embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture media at a concentration within a range of 100-500 nM, 100-400 nM, 150-350 nM or 200-300 nM. In another embodiment, the BMP pathway antagonist is LDN193189, which is present in the culture media in steps (a) and (b) of the method (i.e., stages 1 and 2) at a concentration of 250 nM.
Agonists of the retinoic acid (RA) pathway include agents, molecules, compounds, or substances capable of activating (upregulating) the RA signaling pathway. In one embodiment, the RA pathway agonist is selected from the group consisting of retinoic acid (RA), TTNPB, AM 580, CD 1530, CD 2314, CD 437, Ch 55, BMS 753, BMS 961, Tazarotene, Tamibarotene, Isotretinoin, Tretinoin, AC 261066, AC 55649, Sr11237, adapalene, EC23, 9-cis retinoic acid, 13-cis retinoic acid, 4-oxo retinoic acid, and All-trans Retinoic Acid (ATRA), AY 9944 dihydrochloride, Ciliobrevin A, Cyclopamine, or combinations thereof. In one embodiment, the RA pathway agonist is present in the culture media at a concentration within a range of 0.05-5 μM, 0.5-5 μM, or 1-3 μM. In another embodiment, the RA pathway agonist is RA, which is present in the culture at a concentration in a range of 0.2-5 μM, 0.5-4 μM, 1-3 μM. In another embodiment, the RA pathway agonist is RA, which is present in the culture media in steps (a)-(b) of the method (i.e., stages 1 and 2) at a concentration of 2 μM.
Antagonists of the TGFβ (transforming growth factor beta) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through a TGFβ receptor family member, a family of serine/threonine kinase receptors. In one embodiment, the TGFβ pathway antagonist is selected from the group consisting of A8301, SB-431542, GW788388, SB525334, TP0427736, RepSox, SD-208, and combinations thereof. In one embodiment, the TGFβ pathway antagonist is present in the culture media at a concentration within a range of 200-1000 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In one embodiment, the TGFβ pathway antagonist is A8301. In another embodiment, the TGFβ pathway antagonist is A8301, which is present in the culture media at a concentration of 200-1000 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In another embodiment, the TGFβ pathway antagonist is A8301, which is present in the culture media in steps (a)-(c) of the method (i.e., stages 1-3) at a concentration of 500 nM.
Antagonists of the MEK pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the signaling pathway of one or more of the components of the MAPK/ERK pathway (also known as the Ras-Raf-MEK-ERK pathway). In one embodiment, the MEK pathway antagonist is selected from the group consisting of PD0325901, Binimetinib (MEK162), Cobimetinib (XL518), Selumetinib, Trametinib (GSK1120212), CI-1040 (PD-184352), Refametinib, ARRY-142886 (AZD-6244), PD98059, U0126, BI-847325, RO 5126766, and combinations thereof. In one embodiment, the MEK pathway antagonist is present in the culture media at a concentration within a range of 25-1000 nM, 50-750 nM, 75-500 nM, 100-400 nM, or 150-300 nM. In one embodiment, the MEK pathway antagonist is PD0325901. In another embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture media at a concentration within a range of 25-300 nM, 50-150 nM, 50-250 nM, or 150-300 nM. In another embodiment, the MEK pathway antagonist is PD0325901, which is present in the culture media in step (a) of the method (i.e., stage 1) at a concentration of 250 nM.
Antagonists of the TAK1 (also known as MAP3K7) pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through TAK1 (MAP3K7). In one embodiment, the TAK1 pathway antagonist is selected from the group consisting of Taki ((5Z)-7-Oxozeaenol), Takinib, Dehydoabietic acid, NG25, Sarsasapogenin, and combinations thereof. In one embodiment, the TAK1 pathway antagonist is present in the culture media at a concentration within a range of 200-1000 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In another embodiment, the TAK1 pathway antagonist is Taki, which is present in the culture media at a concentration of 300-800 nM, 250-750 nM, 300-650 nM, or 400-600 nM. In another embodiment, the TAK1 pathway antagonist is Taki, which is present in the culture media in step (b) of the method (i.e., stage 2) at a concentration of 500 nM.
bFGF mimetics include agents, molecules, compounds, or substances capable of activating (upregulating) signaling through the fibroblast growth factor 2 (FGF2) signaling pathway. In an embodiment, the bFGF mimetic is SUN11602.
Antagonists of the AKT pathway include agents, molecules, compounds, or substances capable of inhibiting (downregulating) the signaling pathway of one or more of the serine/threonine kinase AKT family members, which include AKT1 (also designated PKB or RacPK), AKT2 (also designated PKBP or RacPK-β) and AKT 3 (also designated PKB7 or thyoma viral proto-oncogene 3). In an embodiment, the AKT pathway antagonist is selected from the group consisting of AT7867, MK2206, GSK690693, Perifosine (KRX-0401), Ipatasertib (GDC-0068), Capivasertib (AZD5363), PF-04691502, Triciribine (NSC154020), ARQ751, Miransertib (ab235550), Borussertib, Cerisertib, and combinations thereof. In an embodiment, the AKT pathway antagonist is present in the culture media at a concentration within a range of 25-1000 nM, 50-750 nM, 75-500 nM, 100-400 nM, or 150-300 nM. In one embodiment, the AKT pathway antagonist is AT7867. In another embodiment, the AKT pathway antagonist is AT7867, which is present in the culture media at a concentration within a range of 25-300 nM, 50-150 nM, 50-250 nM, or 150-300 nM. In another embodiment, the AKT pathway antagonist is AT7867, which is present in the culture media in step (b) of the method (i.e., stage 2) at a concentration of 250 nM.
Notch pathway antagonists include agents, molecules, compounds, or substances capable of inhibiting (downregulating) signaling through or activity of the Notch transcription factor, including gamma secretase inhibitors (GSIs). In an embodiment, the Notch pathway antagonist is selected from the group consisting of GSI-XX, GSI-X, R04929097, Semagacestat, Avagacestat, Dibenzazepine, LY411575, LY450149, DAPT, Crenigacestat, MK0752, BMS-708163, BMS-906024, CB-103, AL101, Compound E, Compound X (CX), IMR-1, IMR-1A, FLI-06, Valproic acid, YO-01027, Tangeretin, Bruceine D, and combinations thereof. In an embodiment, the Notch pathway antagonist is present in the culture media at a concentration within a range of 25-200 nM, 50-150 nM, or 75-125 nM. In another embodiment, the Notch pathway antagonist is GSI-XX. In another embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media at a concentration of 25-200 nM, 50-150 nM, or 75-125 nM. In another embodiment, the Notch pathway antagonist is GSI-XX, which is present in the culture media in step (c) of the method (i.e., stage 3) at a concentration of 100 nM.
Flavonoids include agents, molecules, compounds, or substances selected from the group consisting of quercetin, quercetin analogues, (e.g., dihydroquercetin, 6,2′,4′,5′-pentahydroxyflavone, quercetin-3-O-propionate (Q-pr), quercetin-3-O-butyrate (Q-bu), quercetin-3-O-valerate, or 3,4′-Di-O-methylquercetin), genistein, anthocyanins, catechins, gallocatechins (e.g., epigallocatechin-3-gallate (EGCG)), anthocyanidins, apigenin, luteolin, kaemferol, curcumin, myricetin, daidzein, naringin, rutin, and hesperitin. In an embodiment, the flavonoid is quercetin. In one embodiment, the flavonoid is present in the culture media at a concentration within a range of 3-50 μM, 5-30 μM or 10-20 μM. In another embodiment, the flavonoid is quercetin, which is present in the culture media within a range of 3-50 μM, 5-30 μM or 10-20 μM. flavonoid is quercetin, which is present in the culture media at a concentration of 15 μM.
When an agonist or antagonist is used in more than one step of the method, the agonist or antagonist may be the same or different for one or more of the steps in which the agent is present in the culture media. Further, when an agonist or antagonist is used in more than one step of the method, the concentration of the same agonist or antagonist may be the same or different for each step in which the agent is present in the culture media.
In combination with the chemically defined and optimized culture media described in subsection II above, the methods of generating DFEs, PPs, and PBCs of the disclosure utilize standard culture conditions established in the art for cell culture. For example, cells can be cultured at 37° C. and under 5% 02 and 5% CO2 conditions. In one embodiment, cells can be cultured in standard culture vessels or plates, such as 96-well plates. In certain embodiments, the starting pluripotent stem cells are adhered to plates, preferably coated with an extracellular matrix material, such as vitronectin. In one embodiment, the stem cells are cultured on a vitronectin coated culture surface (e.g., vitronectin coated 96-well plates).
Pluripotent stem cells can be cultured in commercially available media prior to differentiation. For example, stem cells can be cultured for at least one day in Essential 8 Flex media (Thermo Fisher #A2858501) prior to the start of the differentiation protocol. In a non-limiting exemplary embodiment, stem cells are passaged onto vitronectin (Thermo Fisher #A14700) coated 96-well plates at a cell density of 150,000 cells/cm2 and cultured for one day in Essential 8 Flex media prior to differentiation.
To initiate the differentiation protocol, the media in which the stem cells are cultured is changed to a basal differentiation media that has been supplemented with signaling pathway agonists and/or antagonists as described above in subsection II. A basal differentiation media can include, for example, a commercially available base supplemented with additional standard culture media components needed to maintain cell viability and growth, but lacking serum (the basal differentiation media is a serum-free media) or any other exogenously added growth factors, such as bFGF (FGF2), PDGF or HGF. In a non-limiting exemplary embodiment, a basal differentiation media contains 1× IMDM (Thermo Fisher #12440046), 1× F12 (Thermo Fisher #11765047), poly(vinyl alcohol) (Sigma #p8136) at 1 mg/ml, chemically defined lipid concentrate (Thermo Fisher #11905031) at 1%, 1-thioglycerol (Sigma #M6145) at 450 μM, Insulin (Sigma #11376497001) at 0.7 ug/ml and transferrin (Sigma #10652202001) at 15 ug/ml (also referred to herein as “CDM2” media, as used in the exemplary differentiation protocols shown herein and as depicted in
To generate DFECs, PPCs, and PBCs, the starting stem cells are cultured in the optimized culture media for sufficient time for cellular differentiation and expression of committed DFE, PPC, or PBC markers. As described in the Examples, it has been discovered that culture of pluripotent stem cells in a three-stage method, one optimized for generation of DFECs, a second optimized for generation of PPCs, and a third optimized for the generation of PBCs so as to produce PBCs in as little as 16 days of culture. The culture period for the first stage (leading to DFECs) corresponds to days 1-3, the culture period for the second stage (leading to PPCs) corresponds to days 4-6, and the culture period for the third stage (leading to PBCs) corresponds to days 7-16.
Accordingly, in the first stage of the method for generating DFECs, also referred to herein as “step (a)” or “stage 1”, pluripotent stem cells are cultured in the stage 1-optimized culture media on days 0-3, or starting on day 0 and continuing through day 3, or for 72 hours (3 days), or for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or for 60 hours, or for 64 hours, or for 68 hours, or for 70 hours, or for 72 hours. In some embodiments, the pluripotent cells may be cultured for up to 5 days in stage 1 of the differentiation protocol. Therefore, in certain embodiments, the pluripotent stem cells may be cultured in the stage 1-optimized culture media for 72 hours followed by: at least 8 additional hours, at least 8 additional hours, at least 16 additional hours, at least 24 additional hours, at least 32 additional hours, at least 40 additional hours; for cultured for 72 hours followed by: 8 additional hours, 16 additional hours, 24 additional hours, 32 additional hours, 40 additional hours, or 48 additional hours (i.e., up to 5 days total).
In the second stage of the method, which generates the PPCs, also referred to herein as “step (b)” or “stage 2”, the DFECs generated in step (a) are further cultured in the stage 2-optimized culture media on days 4-6, or starting on day 4 and continuing through day 6, or starting on day 4 and continuing for 72 hours (3 days), or starting on day 4 and continuing for at least 60 hours, or at least 64 hours, or at least 68 hours, or at least 70 hours, or at least 72 hours, or starting on day 4 and continuing for 60 hours, or for 64 hours, or for 68 hours, or for 70 hours, or for 72 hours. In some embodiments, the DFECs may be cultured for up to 5 days in stage 2 of the differentiation protocol. Therefore, in certain embodiments, the DFECs may be cultured in the stage 2-optimized culture media for 72 hours followed by: at least an additional 8 hours, at least an additional 16 hours, at least an additional 24 hours, at least an additional 32 hours, at least an additional 40 hours; or cultured for 72 hours followed by: 8 additional hours, 16 additional hours, 24 additional hours, 32 additional hours, 40 additional hours, or an additional 48 hours (i.e., up to 5 days total).
In the third stage of the method, which generates the PBCs, also referred to herein as “step (c)” or “stage 3”, the PPCs generated in step (b) are further cultured in the stage 3-optimized culture media on days 7-16, or starting on day 7 and continuing through day 16, or starting on day 7 and continuing for 240 hours (10 days), or starting on day 7 and continuing for at least 216 hours, or starting on day 7 and continuing for at least 220 hours, or starting on day 7 and continuing for at least 224 hours, or starting on day 7 and continuing for at least 228 hours, or starting on day 7 and continuing for 216 hours, or for 220 hours, or for 224 hours or for 228, or for 232 hours. In some embodiments, the PPCs may be cultured for up to 20 days in stage 3 of the differentiation protocol. Therefore, in certain embodiments, the PPCs may be cultured in the stage 3-optimized culture media for 240 hours (10 days) followed by: at least 1 additional day, at least 2 additional days, at least 3 additional days, at least 4 additional days, at least 5 additional days, at least 6 additional days, at least 7 additional days, at least 8 additional days, at least 9 additional days, or for 240 hours (10 days) followed by: 1 additional day, 2 additional days, 3 additional days, 4 additional days, 5 additional days, 6 additional days, 7 additional days, 8 additional days, 9 additional days, or 10 additional days (i.e., up to 20 days total).
In an exemplary embodiment, TC plates are seeded in Essential 8 (E8) media at an initial cell density of 62,500 cells/cm2 and grown overnight. The following day, growth media is replaced with a stage 1 media composed of a basal media (CDM2) supplemented with 250 nM LDN193189, 2 μM retinoic acid, 500 nM A8301 and 250 nM PD0325901. The CDM2 media can be used in all stages of the differential protocol and has been described by Loh et al 2014. The culture is maintained in a stage 1 media for 3 consecutive days. On the third day the differentiation media is changed to a stage 2 media composed of the basal media CDM2 supplemented with 250 nM LDN193189, 500 nM (5Z)-7-Oxozeanol, 2 μM retinoic acid, 300 nM SUN 11602, 250 nM AT7867 and 500 nM A8301. Cultures are incubated in the stage 2 media for three consecutive days until the media is changed to a stage 3 media. The stage 3 media is composed of CDM2 supplemented with 100 nM gamma secretase, 500 nM A8301 and 15 μM quercetin. Cultures are incubated in the presence of the stage 3 media for 5-10 consecutive days.
The foregoing differentiation protocol has been shown to be highly reproducible and results in consistent differentiation regardless of the adherent system used and has been successfully employed in most TC format including 96-well, 24-well, 6-well and T75 flasks. This protocol has also been shown to function in 3D suspension cultures as described below. Throughout all stages, the differentiated PSC culture is provided fresh media daily through a media change.
In some cell suspension embodiments, a shaker flask expansion method may be employed, which consists of filling a flask approximately ⅓ of the way with growth media, adding PSCs, and placing the entire flask on a shaker so that PSCs do not settle and stay in suspension. The media solution is shaken so that the liquid is constantly circulating without splashing. This method, originally developed for bacterial or yeast growth, has been adapted for mammalian cell growth and subsequently for PSC aggregate growth. In other cell suspension embodiments, spinner flasks may be employed. Spinner flasks have a propeller attached to a rod that reaches down into the flask for stirring. The propellers are horizontal and are designed to be driven by magnetic stir plates that the flasks sit on.
In some embodiments, a bioreactor system is used for culturing the cells. An exemplary bioreactor system is the PBS Vertical Wheel Bioreactor system (https://www.pbsbiotech.com/uploads/1/7/9/9/17996975/2021_borys_et_al._-_overcoming_bioprocess_bottlenecks.pdf). The PBS VW Bioreactors include an enclosed vesicle that can accommodate suspension cultures of various sizes, including 100 ml, 500 ml, 3,000 ml, and 15,000 ml. The culture vessels have a corresponding motorized unit that they sit on and spin the magnetized vertical wheel allowing for constant stirring of the suspension culture. In one embodiment, StemScale media is used for the general expansion of PSCs within the bioreactor.
In an exemplary embodiment, the initial seeding into the VW Bioreactors comes from an adherent culture, though subsequent passaging events can be achieved from bioreactor to bioreactor. Multiple wells of a 3-4 day 6-well plate are washed with PBS followed by a 3-minute incubation in the presence of TryPLE to remove cells from the plate. Three to four of these wells are then washed with a basal media to remove any cells that are not completely de-attached from the plate. Cells are counted and are seeded at a concentration of 150,000 cells per ml into a PBS VW Bioreactor in StemScale media supplemented with 10 μM Y27632. The following day, and every 2nd day after that, a demi-depletion is performed on the bioreactor. The demi-depletion is achieved by removing the bioreactor from its base and setting it in a hood for 5 minutes allowing aggregates to settle to the bottom of the bioreactor at which time half of the volume of the bioreactor is removed without disturbing the bottom of the reactor. This volume is then replaced with fresh Stem Scale media. Bioreactors are passaged every 4-5 days. All media containing aggregates are removed from the bioreactor and placed into tubes for centrifugation. Samples are spun at 400×g for 4 minutes followed by aspiration of the supernatant media leaving pellets of the cellular aggregates. This pellet is then incubated in the presence of accutase for 10 minutes to break the aggregates up into smaller pieces and individual cells. The accutase is then diluted centrifuged so that it can be aspirated out of the sample. Passaging within the bioreactor content can be done in a scale up model or in a continuous culture model. Scale up occurs when all biomaterial is placed into a larger bioreactor for subculturing, such as the next size bioreactor for passaging a 100 ml sample into a 500 ml bioreactor, etc. The continuous model may be employed when only a portion of the sample is sub-cultured in a subsequent bioreactor, such as when the sample is processed from a 100 ml run and only a portion of it is then seeded back into a 100 ml bioreactor.
The methods and compositions of the disclosure for generating DFECs, PPCs, and PBCs allow for efficient and robust availability of these cell populations for a variety of uses in clinical therapy, research, development, and commercial purposes. For therapeutic purposes, the in vitro generated PBCs of the present invention can be administered systemically or directly to a subject for treating or preventing type 1 diabetes, type 2 diabetes, pre-diabetes, conditions due to significant trauma (i.e., damage to the pancreas or loss or damage to islet beta cells), or treating other metabolic diseases or disorders associated with a deficiency in beta cell number (e.g., a reduction in the number of pancreatic cells), an insufficient level of beta cell biological activity (e.g., a deficiency in glucose-stimulated insulin secretion, a deficiency in insulin production).
In one embodiment, the PBC cells of the invention are directly injected into an organ of interest (e.g., pancreas). Alternatively, compositions comprising beta-like cells of the invention are provided indirectly to the organ of interest, for example, by administration into the circulatory system (e.g., the pancreatic vasculature). Expansion and differentiation agents can be provided prior to, during or after administration of the cells to increase production of cells having insulin-producing potential in vitro or in vivo. In some embodiments, the PBCs of the present application may be genetically modified to increase their therapeutic and/or safety profiles prior to administration. The cells can be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into another convenient site where the cells may find an appropriate site for regeneration and differentiation.
In one embodiment, at least 100,000, 250,000, or 500,000 cells are injected. In other embodiments, 750,000, or 1,000,000 cells are injected. In other embodiments, at least 1×105 cells, at least about 1×106, at least about 1×107, or as many as 1×108 to 1×1010, or more are administered. Dosages can be readily adjusted by those skilled in the art (e.g., a decrease in purity may require an increase in dosage). The PBCs cells can be introduced by localized injection, including catheter administration, systemic injection, intravenous injection, or parenteral administration. When administering a therapeutic composition of the present invention (e.g., a pharmaceutical composition containing a selected cell), it will generally be formulated in a unit dosage injectable form (solution, suspension, emulsion).
In other embodiments, the PBCs cells of the disclosure can be used for screening potential drugs or for the development of novel cell therapies for treatment of diseases or disorders involving dysfunction of PBCs.
In other embodiments, the methods and compositions can be used in the study of pancreatic beta cell progenitor development and biology, including differentiation into PBCs, to assist in the understanding and potential treatment of diseases and disorders associated with abnormal pancreatic beta cell function, such as diabetes.
In certain embodiments, DFECs, PPCs, and PBCs generated using the methods of the disclosure can be further purified according to methods established in the art using agents that bind to surface markers expressed on the cells. Accordingly, in one embodiment, the disclosure provides a method of isolating DFECs, PPCs, and PBCs, the method comprising: contacting DFECs, PPCs, or PBCs generated by a method of the disclosure with one or more binding agent(s) (e.g., monoclonal antibodies (mAbs)) binding to one or more cell surface marker(s) expressed in DFECs, PPCs, or PBCs; and isolating cells that bind to the binding (s) to facilitate isolation of the DFECs, PPCs, or PBCs. Cells that bind the antibody can be isolated by methods known in the art, including but not limited to fluorescent activated cell-sorting (FACS) and magnetic activated cell sorting (MACS).
In other aspects, the disclosure provides compositions related to the methods of generating DFECs, PPCs, and PBCs, including culture media and cell cultures, as well as isolated progenitor cells and cell populations thereof.
In one aspect, the disclosure provides a culture media for obtaining human DFEs comprising culture media comprising a BMP pathway antagonist, a RA pathway agonist, a TGF-β pathway antagonist, and an MEK pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In another aspect, the disclosure provides a culture media for obtaining human PPCs comprising a BMP pathway antagonist, a RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In another aspect, the disclosure provides culture media for obtaining human pancreatic beta cells (PBCs) comprising a Notch pathway antagonist, such as a γ-secretase inhibitor, a TGF-β pathway antagonist, and a flavonoid, such as quercetin or a quercetin analogue. Suitable agents, and concentrations therefor, include those described in subsection II.
In another aspect, the disclosure provides an isolated cell culture of human DFECs, the culture comprising human DFECs cultured in a culture media comprising a BMP pathway antagonist, a RA pathway agonist, a TGF-β pathway antagonist, and an MEK pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In another aspect, the disclosure provides an isolated cell culture of human PPCs, the culture comprising human PPCs cultured in a culture media comprising a BMP pathway antagonist, a RA pathway agonist, a TGF-β pathway antagonist, a TAK1 pathway antagonist, a bFGF mimetic, and an Akt pathway antagonist. Suitable agents, and concentrations therefor, include those described in subsection II.
In another aspect, the disclosure provides an isolated cell culture of human pancreatic beta cells (PBCs) the culture comprising human PBCs cultured in a culture media comprising a Notch pathway antagonist, such as a γ-secretase inhibitor, a TGF-β pathway antagonist, and flavonoid, such as quercetin or a quercetin analogue. Suitable agents, and concentrations therefor, include those described in subsection II.
In another aspect, the disclosure provides human DFECs generated by a method of the disclosure (i.e., step (a) or stage 1 of the culture protocol).
In another aspect, the disclosure provides human PPCs generated by a method of the disclosure (i.e., steps (a) and (b), or stages 1 and 2 of the culture protocol).
In another aspect, the disclosure provides human PBCs generated by a method of the disclosure (i.e., steps (a), (b) and (c), or stages 1, 2 and 3, of the culture protocol).
The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.
The Dorsal Foregut Endoderm (DFE) is defined as a pluripotent derived endodermal population patterned to a region of the early developing embryo occurring on the dorsal side of the midgut of the primitive gut-tube. Expression patterns within this population are marked by robust expression of the endodermal dorsal markers of SFRP5, MNX1, PTCH1 and PAX6 and the midgut markers FOXA2, HNF1b, HOXA3 and ONECUT2. The high expression of these markers contrasts with the absence of these markers of the ventral and more posterior endodermal regions. DFE was found to exhibit very low or no expression of the ventral markers HHEX and NR5A2, as well as the classically defined definitive endodermal markers SOX17, GSC, MIXL1 and CER. It was previously demonstrated that the DFE state could be achieved through the dual optimization of HNF1b and FOXA2 and that this induction could be achieved through the retinoid signaling occurring in the absence of BMP signaling. Previous work performed on the DFE population used a single iPSC line and embryonic stem cells.
To assay the efficacy of a potentially clinical grade iPSC line the NCRM1 cell line was chosen. An HD-DoE based optimization was used to validate the HNF1b and FOXA2 optimizers for this cell line. The HD-DOE method was applied with the intent to find conditions for induction of endoderm-expressed genes, directly from the pluripotent stem cell state. This example utilizes a method previously described by Bukys et al. (2020) Iscience 23:101346. The method employs computerized design geometries to simultaneously test multiple process inputs and offers mathematical modeling of a deep effector/response space. The method allows for finding combinatorial signaling inputs that control a complex process, such as dauring cell differentiation. It allows testing of multiple plausible critical process parameters, as such parameters impact output responses, such as gene expression. Because gene expression provides hallmark features of the phenotype of, for example, a human cell, the method can be applied to identify, and understand, which signaling pathways control cell fate.
To develop a cell culture recipe for each stage, the impact of agonists and antagonists of multiple signaling pathways (herein called effectors) on the expression of pre-selected genes was tested and modeled. The impact of each effector on gene expression level is defined by a parameter called factor contribution that is calculated for each effector during the modeling. These effectors are small molecules or proteins that are commonly used during stepwise differentiation of stem cells to specific fates. Choice of the effectors were based on current literature on differentiation of stem cells to endoderm progenitors.
In both optimizations, it was shown that BMP pathway inhibition was the most crucial component of the optimization with factor contribution factors of 31.26 and 31.11 for LDN193189 in both the optimization of HNF1b (
Performing a dual optimizer for the combined expression of FOXA2 and HNF1b further demonstrated the importance of simultaneously controlling these four pathways as the optimizer predicted that active retinoid signaling was needed in the presence of BMP, TGF-β and MEK pathway inhibition (
As shown in
As shown in
Use of the previously identified critical process parameters retinoic acid and LDN193189 in the presence or absence of A8301 and PD0325901 showed the dependence of robust FOXA2 and HNF1b on the addition of either of these compounds (
As shown in the heatmap depicted in
As shown in
To assay the capacity to generate pancreatic field from the DFE culture, genes associated with this endodermal region were evaluated for expression in pancreatic bud generation. These genes include PROM1, CPB1 and ONECUT1. As shown in the dynamic profiling depicted in
To determine the best way for inducing a pancreatic fate from a DFE progenitor, a subsequent HD-DoE analysis was performed. Exposing a DFE culture to a perturbation matrix composed of all previously identified effectors shown to have a positive effect on PDX1 activation was used to define optimal conditions for PDX1 induction. As shown in
Coefficient plots comparing the input logic of the pancreas specific genes PTF1A and PDX1 showed that the combined effects of active retinoid signaling (retinoic acid) in the presence of MEK pathway inhibition (PD0325901) are the two pathways most critical to control for the activation of the pancreatic field from a regionalized dorsal foregut endodermal field (
As shown in
Comparing the PDX1 optimizer to markers of the pancreatic endocrine fates showed that the predicted conditions were compatible with the generation of endocrine fates (
Since PDX1 positive cells can give rise to all cell types of the pancreas and gall bladder, as well as cells of the stomach and intestines, optimal conditions for beta cell induction were explored. This was accomplished through a parallel experiment exposed to the same perturbation matrix that was differentiated an additional 5 days afterwards in the presence of both a Notch pathway inhibitor (gamma secretase inhibitor (GSI)-XX) and a TGF-β pathway inhibitor (A8301). It is well established that inhibition of these pathways induces a beta cell fate from a proendocrine progenitor field. Optimization of this culture for the INS predicted a similar culture condition as the original PDX1 optimizer. Notably, as shown in
Comparing the regulatory inputs of the effectors used in the insulin optimizer to PDX1 activation showed almost identical input logic for the two genes. As shown in
As shown in
To define factors contributing to the activation of INS, a series of HD-DoE perturbations were performed using factors chosen to target different aspects of beta cell biology. As shown in
To ascertain which factors could potentially favor the beta cell over other endocrine cell types, optimizers for markers of the other endocrine cell types and beta cell specific markers were compared. The relative factor contribution from the different optimizers were compared. As shown in
To define factors contributing to the activation of INS, a series HD-DoE perturbations were performed using factors chosen to target different aspects of beta cell biology. Of the factors tested, the ones determined to have strong INS activating capacity included the Ax1 inhibitor R248, the SHH inhibitor SANT1, the retinoid agonist TTNPB, the TPH1 inhibitor LP533401, and the amino acids tryptamine and glutamine (
Optimizers for the main pancreatic endocrine hormones and beta cell-specific transcription factors were generated to compare their contribution factors for this series of HD-DoE. Compounds that could selectively favor the differentiation of the beta cell over that of the alpha and delta cells were identified in this manner. Notably, the retinoid agonist TTNPB was shown to strongly activate all beta cell specific transcription factors assayed in this manner while strongly inhibiting both the alpha and delta cell fates (
Dynamic profiling of the crucial beta cell specific markers INS and PDX1 was used to compare the regulatory profile of the beta cell to that of the alpha and delta cells (
Dynamic profiling of the crucial beta cell specific markers INS and PDX1 was used to compare the regulatory profile of the beta cell to that of the alpha and delta cells (
Dynamic profiling of the crucial beta cell specific markers INS and PDX1 was used to compare the regulatory profile of the beta cell to that of the alpha and delta cells. As shown in
Dynamic profiling of the crucial beta cell specific markers INS and PDX1 was used to compare the regulatory profile of the beta cell to that of the alpha and delta cells. As shown in
The addition of quercetin to the stage 3 media increased overall endocrine conversion with an overall higher induction of de novo beta cell generation as shown in the immunohistochemical analysis of a day 16 culture shown in
As shown in
A glucose stimulated insulin secretion assay was performed to evaluate the functional maturity of the de novo beta cells generated. Assay samples had growth media removed and were washed once with PBS to remove any trace of media that may have been previously conditioned with secreted C-PEP. All samples were then incubated in the presence of basal media at 37 degrees Celsius for 15 minutes. Basal media consisted of RPMI containing 3 mM glucose. This media was then changed to either media supplemented with 17.5 mM glucose or 30 mM KCl and incubated for an additional 15 minutes. As shown in
A bioreactor system depicted in
Cultures displayed an initial proliferative phase that tapered off within 4-5 days followed by a declining number of cells surviving the sequential stages of the protocol (
The continued growth in the bioreactors resulted in a significant increase in the biomass being produced, with an estimated 20-50 fold expansion capability for the iPSC culture. For the initial establishment of the bioreactor-based protocol, a reduction of biomass throughout the process was used to maintain an optimal cellular density within the bioreactors. To confirm a similar phenotype between cells differentiated in the bioreactors as compared to cells differentiated using the nascent protocol, transcript levels of some key endocrine genes were compared. The endocrine products INS, GCG and SST representative of the pancreatic alpha, beta and delta cells, respectively, were measured throughout the different stages of the bioreactor runs. As expected, these hormones were undetectable in the early stages, but increased significantly by stage 3. Two highly specific beta specific genes NEUROD and NKX2.2 were also measured showing a similar pattern of expression. No significant differences in transcript levels were noted when comparing the bioreactor run to the control culture differentiated on adherent TC conditions.
Stage 3 bioreactor runs consistently produced average densities of 100 aggregates/ml with aggregates consisting of approximately 500 cells each. C-peptide was detected in both the stage 3 media used to differentiate the cellular aggregates and within cellular aggregates (
In this example, additional experiments were performed to characterize the insulin producing cells.
As demonstrated in
As shown in
The average insulin content per cell was determined, the results of which are shown in
As shown in
As shown in
As shown in
As shown in
RNA sequencing results for gene expression are shown in
In addition, the iPSC-derivatives have the continued expression of HK1, HK2 and SLC16A1. The down-regulation of these three genes has been shown to be crucial for achieving a fully functional phenotype. The iPSC derivatives also show decreased expression of other crucial maturation markers FFAR1 and KIR6-2. All together this demonstrates that the iPSC-derivatives are not fully functional and have a limited response to glucose.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims
This application claims priority to U.S. Provisional Application No. 63/400,349, filed Aug. 23, 2022, the entire contents of which is hereby incorporated by reference.
Number | Date | Country | |
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63400349 | Aug 2022 | US |