Patent Application
20200138870
Embodiments of the disclosure concern at least the fields of cell biology, molecular biology, endocrinology, biochemistry, and medicine.
P cells are the predominant endocrine cell type of the pancreas and the only cell in the body that can generate and secrete insulin (INS) to maintain blood glucose homeostasis. Loss of functional INS-producing 3 cells causes diabetes, and the inability of available therapies to adequately stabilize blood glucose levels (Cryer, 2014; Home et al., 2014; Zaykov et al., 2016) means that even treated diabetics often develop retinopathy, neuropathy, and stroke, among other complications (Forbes and Cooper, 2013; Olokoba et al., 2012). The ideal therapy would be to replace the dysfunctional cells, and in fact, cadaveric islets can reconstitute 3 cells and restore normoglycemia. Unfortunately, each transplant requires billions of cells, and there are not enough cadaveric islets to treat the millions of people around the world with insulin-dependent diabetes. There has thus been enormous interest in regenerative medicine approaches for the treatment of diabetes.
In vitro-derived human β cells can alleviate hyperglycemia in mice (Kroon et al., 2008; Pagliuca et al., 2014; Rezania et al., 2014; Vegas et al., 2016), but human pluripotent stem cells (hPSCs) cannot yet be reliably coaxed into functional 3 cells in sufficient numbers to serve as therapy (Kroon et al., 2008; Pagliuca et al., 2014; Rezania et al., 2014; Vegas et al., 2016). The efficiency of hPSC differentiation to β cells remains low, varying from 10-30%, and the duration (4 to 5 weeks) and cost of production so far are impractical (D'Amour et al., 2006; Nostro et al., 2015; Pagliuca et al., 2014; Rezania et al., 2014; Russ and Hebrok, 2014; Russ et al., 2016). The biggest problem, however, is that in vitro-generated β cells do not behave like their mature human counterparts. Whereas mature human 3 cells secrete only INS and respond with great precision to different amounts of glucose in the blood, hESC-derived 3 cells often co-express other endocrine hormones such as glucagon and somatostatin and do not respond adequately to glucose levels. We do not know enough about how the human pancreas develops in vivo endocrine progenitors into cells that can be constantly challenged by, and responsive to glucose yet to reliably generate functional 3 cells in vitro.
Differentiation protocols commonly aim to mimic in vivo development in an in vitro system, but in vitro systems lack the neighboring cell types present in vivo. When progenitors differentiate into endocrine cells, they delaminate from an epithelial layer into the surrounding mesenchyme and thus associate closely with the pancreatic niche. Initial work in Xenopus and mouse demonstrated that endothelial cells are essential in pancreatic development (Lammert et al., 2001) and specifically for the induction of the transcription factors Pdx1 and Ptf1a, which are responsible for the formation of the organ (Jacquemin et al., 2006; Lammert et al., 2001; Yoshitomi and Zaret, 2004). Over a decade ago, Bhushan and colleagues demonstrated that fibroblast growth factor 10 (FGF10), expressed by the mesenchyme, stimulates proliferation of Pdx1+ progenitors (Bhushan et al., 2001). Since then, zebrafish and mouse studies have identified a number of signaling pathways such as retinoic acid, FGF, BMP and TGF that are important for pancreatic development (Dichmann et al., 2003; Kobberup et al., 2010; Martin et al., 2005). These interactions are temporally regulated, as blood vessels at later developmental stages restrict the outgrowth and morphogenesis of the pancreatic epithelium in mice (Magenheim et al., 2011). Irrespective of species, mature islets are highly vascularized. hPSC-derived pancreatic progenitors (PPs) are supported by ingrowing host blood vessels after transplantation. It is worth noting that transplanted islets in human patients or transplanted 3 cells in mice perdure, but survive only a short period of time in in vitro cultures. These studies suggest that something present in vivo is missing in vitro, and that identifying the signals from the surrounding niche that support the differentiation and maturation of human 3 cells could provide the missing link for the development of cell-based therapies (Negi et al., 2012).
Previously, the inventors and others demonstrated that pancreatic stage-specific mesenchyme is a source of signals that allow massive in vitro expansion of hPSC-derived definitive endoderm (DE) (Cheng et al., 2012; Sneddon et al., 2012). The role of the niche, defined here as mesenchymal and endothelial cells, at later stages, such as when human endocrine cells are developing, is not understood. It was considered that: i) interactions between human endocrine progenitors (EPs) and the pancreatic niche promote the differentiation and maturation of these progenitors into β cells, and ii) components of the human pancreatic niche change over time and differ in terms of their capability to promote β cell specification. The inventors therefore used a two-pronged strategy to identify the effect of niche on endocrine differentiation, as well as the molecular signals that collectively promote β cell specification.
First, the inventors established various human fetal pancreatic niche primary cells, comprised of mesenchymal and endothelial (M-E) cells at different stages of development, to delineate their contribution to differentiating β cells. Once the stages were identified that most strongly stimulated β cell development, the mesenchymal and endothelial signals that promote INS expression and β cell specification were then identified. Finally, it was determined that the interplay between the WNT5A/JNK and BMP signaling pathways is crucial to β cell specification and the in vitro development of functional INS-producing β cells.
The present disclosure provides a solution to long-felt need in the art to provide effective insulin-producing cells to individuals with diabetes.
The present disclosure is directed to methods and compositions related at least to the treatment of diabetes (including type 1, type 2, and gestational diabetes), diabetes-related conditions, and pre-diabetes. The disclosure concerns cell therapy for treating diabetes of any kind and its related conditions. In particular embodiments, cells are exposed to one or more factors that may or may not be endogenous to the cells such that the exposure causes the cells to produce insulin. In one embodiment the cells are exposed to certain types and amounts of one or more factors such that the exposure mimics development of β cell differentiation in vivo. In certain embodiments, effective amounts of the insulin-producing cells are provided to an individual with diabetes, diabetes-related conditions, or pre-diabetes, for example.
Particular embodiments of the disclosure concern one or more pancreatic niche-derived factors for human endocrine development. In specific embodiments, a human pancreatic niche promotes β cell differentiation via WNT5A/JNK/AP1 and BMP signaling and at least some of the agents in the pathways therein are provided to cells to cause them to become insulin-producing.
In one embodiment, there are methods of treating an individual (infant, child, adolescent, or adult) for diabetes (type I or type II), one or more diabetes-related conditions, or pre-diabetes, comprising the step of administering to the individual an effective amount of insulin-producing cells produced upon exposure of insulin-lacking cells to one or more agents, wherein the one or more agents are selected from the group consisting of Wnt5a, FGF7, WNT3a, HGF, THBS2, IGF1, PDPN, LIF, endocan, SERPINF1, EGF, and a combination thereof. The insulin-lacking cells may be stem cells, pluripotent cells, induced pluripotent stem cells, or a mixture thereof. In particular cases, the insulin-lacking cells are embryonic stem cells. The cells may be autologous or allogeneic to the individual. In some cases, the methods include the step of obtaining the insulin-lacking cells from the individual to be treated or another individual.
In specific embodiments, the cells are administered to the individual by injection. The cells that are administered to the individual may be encapsulated. The cells that are injected may be injected into a portal vein, such as one connecting the liver and the pancreas. The cells may be administered to an individual in an encapsulation device. The cells may be administered to the individual in arginate bubbles. In certain embodiments, the cells are administered to the individual more than once. In some cases, the insulin-producing cells or insulin-lacking cells are engineered to produce one or more non-endogenous gene products. In specific embodiments, one or more cell surface receptors in the cells are modified to avoid immune system recognition of the cells.
In particular embodiments, the one or more agents comprise, consist of, or consist essentially of Endocan, SERPINF1, WNT5A, HGF, and a combination thereof. The one or more agents may comprise, consist of, or consist essentially of Endocan and SERPINF1. The one or more agents may comprise, consist of, or consist essentially of Endocan and WNT5A. The one or more agents may comprise, consist of, or consist essentially of Endocan and HGF. The one or more agents may comprise, consist of, or consist essentially of SERPINF1 and WNT5A. The one or more agents may comprise, consist of, or consist essentially of SERPINF1 and HGF. The one or more agents may comprise, consist of, or consist essentially of WNT5A and HGF. The one or more agents may comprise, consist of, or consist essentially of Endocan and SERPINF1 and WNT5A. The one or more agents may comprise, consist of, or consist essentially of Endocan and SERPINF1 and HGF.
The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organization and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.
For a more complete understanding of the present invention, reference is now made to the following descriptions taken in conjunction with the accompanying drawings.
In keeping with long-standing patent law convention, the words “a” and “an” when used in the present specification in concert with the word comprising, including the claims, denote “one or more.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.
The term “therapeutically effective amount” as used herein refers to that amount which, when administered to a subject or patient for treating a disease, is sufficient to effect such treatment for the disease, including to ameliorate at least one symptom of the disease.
Current efforts to differentiate pancreatic progenitors into mature human β cells as a treatment for diabetes are hindered by a lack of understanding of the conditions that promote differentiation and, especially, maturation of these cells. Here, the human pancreatic niche was analyzed at a number of timepoints (weeks 9-20) and it was found that the human niche changes from week to week and is unique in the factors that guide in vitro development of endocrine progenitors into physiologically competent β cells. Identified herein is a panel of secreted factors necessary for endocrine differentiation and it was found that WNT5A, in particular, markedly improved β cell differentiation and maturation in vitro. WNT5A initially acts through the non-canonical (JNK/c-Jun/AP1) WNT signaling pathway and later cooperates with Gremlin1 to inhibit BMP pathway, in particular embodiments. These factors can be used to mimic in vivo conditions in an in vitro system to generate bona fide β cells for translational applications.
I. Cells and Modifying Agents
In particular embodiments of the disclosure, cells that lack the ability to produce insulin are or were manipulated to produce insulin, and at least in some cases are provided to an individual in need thereof. The manipulation includes at least the following: exposure of the cells that lack the ability to produce insulin to one or more agents such as those selected from the group consisting of Wnt5a, FGF7, WNT3a, HGF, THBS2, IGF1, PDPN, LIF, endocan, SERPINF1, EGF, and a combination thereof, and following this exposure, the cells are capable of producing insulin. The exposure of insulin-lacking cells to the one or more agents occurs in vitro or ex vivo. As such, the cells are not products of nature and the methods do not occur in nature, such as either by accident or by standard biological processes.
In particular embodiments, the cells to which the one or more agents are exposed are stem cells, pluripotent cells, pluripotent stem cells, induced pluripotent stem cells, totipotent stem cells, and so forth. Any stem cells may or may not be embryonic.
The cells produced by methods herein are not naturally occurring and only exist because of manipulation by the hand of man.
In some embodiments, cells are manipulated to express insulin that prior to the manipulation would not express insulin at a detectable level. Following the manipulation (such as by exposure to one or more agents), the cells may express insulin but may not express one or more other endocrine hormones (such as glucagon and somatostatin). The insulin-producing cells following exposure to one or more agents may or may not express one or more certain beta cell transcription factors, such as Pdx1, Nkx6.1, MafA, and/or Nkx2.2). The insulin-producing cells following exposure to one or more agents may or may not express factors for glucose sensing and insulin processing or secretion, such as gluts, PC1/3, and Kir channels.
The agent(s) to which the cells are exposed so that the cells become insulin-producing are particular, and one or more of the agents may be sufficient to allow the developed ability of insulin production. In some cases, one or more agents may allow the onset of production of insulin and one or more agents used in addition to this may increase the level of insulin production in the cells.
In particular embodiments, the one or more agents include at least Wnt5a, FGF7, Wnt3a, HGF, THBS2, IGF1, PDPN, LIF, endocan, SERPINF1, EGF, or a combination thereof. In some cases Wnt5a is included in methods of producing insulin from cells that previously lacked the ability to produce insulin. In particular aspects, in addition to Wnt5a one or more other agents are utilized in the methods. The methods may utilize 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more agents to produce insulin-producing cells. Some methods may include at least Wnt5a and FGF7, at least Wnt5a and Wnt3a, Wnt5a and HGF, Wnt5a and THBS2, Wnt5a and IGF1, Wnt5a and PDPN, Wnt5a and LIF, Wnt5a and endocan, Wnt5a and SERPINF1 or Wnt5a and EGF, for example. In some embodiments, the methods include 2 or more of Wnt5a, FGF7, Wnt3a, HGF, THBS2, IGF1, PDPN, LIF, endocan, SERPINF1, or EGF.
In particular embodiments, a functionally active fragment of Wnt5a, FGF7, Wnt3a, HGF, THBS2, IGF1, PDPN, LIF, endocan, SERPINF1, and/or EGF are utilized in the methods instead of the entirety of the agent. Such a functionally active fragment may include an active site and/or particular functional domain of the agent, for example.
II. Methods of Producing the Cells
In particular embodiments, a group of cells that are not capable of producing insulin are exposed to one or more agents, and the exposure allows the cells then to produce insulin. In some cases, were it not for exposure of the one or more agents to the cells, the cells would not have been capable of producing insulin, such as in an in vitro setting.
Producing cells to make insulin includes exposure of certain cells to one or more agents. In specific embodiments, stem cells or pluripotent cells (or a mixture thereof) are exposed to one or more of Wnt5a, FGF7, Wnt3a, HGF, THBS2, IGF1, PDPN, LIF, endocan, SERPINF1, and EGF. The exposure may occur in a culture, for example.
Cells may be obtained from an individual to be treated with the cells, obtained from a different individual, or they may be obtained commercially, for example. The cells may come from a cell line. The cells may come from storage or a cell repository. The cells may or may not be obtained from a fetus, infant, child, adolescent, or adult. The cells may be obtained from the pancreas, duodenum, spleen, skin, blood or other organ. The cells may or may not be passaged prior to exposure to the one or more agents. In cases wherein the cells are exposed to one or more agents in culture, the culture media may or may not be changed during the culturing. The cells may or may not be reprogrammed to the pluripotency prior to exposure to the one or more agents. An example of reprogramming includes by transient overexpression of Oct4, Klf4, Sox2 and c-myc genes using modified mRNA and transfection; a few weeks after transfection of these genes, morphological distinct colonies are picked, expanded, and characterized regarding endogenous pluripotency markers like SSE4, Oct4, Nanog.
In some cases, when more than one agent is utilized to produce insulin-producing cells, the multiple agents may or may not be exposed to the cells at the same time. Exposure of insulin-lacking cells to the one or more agents may occur over a specific time, such as over the course of days, weeks, or months, for example. Such exposure may or may not be continual until the cells are to be utilized. In some cases, the cells are exposed to the agent(s) for 1, 2, 3, 4, 5, 6, or 7 or more days. In some cases, the cells are exposed to the agent(s) for 1, 2, 3, 4, or more weeks. In some cases, the cells are exposed to the agent(s) for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more months.
The concentration of the agent(s) to which the cells are exposed may be of any suitable amount and may be determined empirically for each agent using routine methods in the art. In some cases the concentration is at least or no more than 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, or 1000 or more ng/ml.
III. Methods of Treatment
In embodiments of the disclosure, there are methods of treating an individual for diabetes and/or complications from diabetes or for treating pre-diabetes. Methods include administering particular cells to an individual in need thereof. The individual may have any type of diabetes, including type I, type II, Maturity-Onset Diabetes of the Young (MODY), or gestational diabetes.
In particular embodiments, there are methods of treating an individual for diabetes, diabetes-related condition, or pre-diabetes, comprising the step of providing to the individual an effective amount of cells that were exposed to one or more agents under suitable conditions for the cells to produce insulin.
In some embodiments, there are methods of treating an individual for diabetes, diabetes-related condition, or pre-diabetes, comprising the step of providing to the individual an effective amount of cells that were exposed to one or more agents under suitable conditions for the cells to produce insulin when prior to the exposure the cells did not produce insulin.
In certain embodiments, there are methods of treating an individual for diabetes, diabetes-related condition, or pre-diabetes, comprising the step of providing to the individual an effective amount of insulin-producing cells produced upon exposure to one or more agents under suitable conditions.
In some embodiments, there are methods of treating an individual for diabetes, diabetes-related condition, or pre-diabetes, comprising the step of providing to the individual an effective amount of cells previously exposed to sufficient amounts of one or more agents, wherein the cells produce insulin.
In some embodiments, there are methods of treating an individual for diabetes, diabetes-related condition, or pre-diabetes, comprising the steps of exposing cells to a sufficient amount of one or more agents such that the cells produce insulin; and providing to the individual a sufficient amount of the cells.
In certain embodiments, there are methods of treating an individual for diabetes, diabetes-related condition, or pre-diabetes, comprising the steps of exposing cells that do not produce insulin to a sufficient amount of one or more agents such that the cells produce insulin; and providing to the individual a sufficient amount of the cells.
Methods of treating the individual may or may not include the step of producing the cells that produce insulin.
In some cases, the cells to which the one or more agents are provided lack production of insulin, whereas in alternative cases the cells prior to exposure to the agent(s) may produce insulin but at an insufficient level, and then exposure of the cells to the one or more agents increases the level of endogenous insulin.
In particular embodiments, a therapeutically effective amount of the insulin-producing cells are provided to the individual in need, and the amount may be at least 1×105 cells and may be up to or more than 1×109 cells.
The administering of the cells to the individual may occur by any suitable method. Steps may be taken to protect the administered cells from the host immune system. In some cases the cells are injected into the individual, for example through the portal vein between the liver and pancreas. In some cases the cells are encapsulated and delivered in such a form. The cells may or may not be encapsulated in a device (as an example, the Encaptra® cell delivery system) and delivered to the individual in the device. The device may be implanted under the skin of the individual. The device may be comprised of polycaprolactone, for example.
The cells may be encompassed within microbubbles (for example, alginate microbubbles) or they may be encompassed individually.
In some cases, one or more complications from diabetes are treated with cells produced by methods of the disclosure, such as neuropathy, ketoacidosis, kidney disease, Vision loss, hypoglycemia, hyperglycemia, and so forth.
In some cases, an additional therapy to the therapy encompassed herein is given to the individual, has been given to the individual and/or will be given to the individual. Such a treatment may be of any kind, such as insulin and other injectables; healthy eating and exercise; sulfonylureas; biguanides; meglitinides; thiazolidinediones; DPP-4 inhibitors; SGLT2 inhibitors; Alpha-glucosidase inhibitors; and/or bile acid sequestrants, for example.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way, however, be construed as limiting the broad scope of the invention.
Human EPs are first detected during development between weeks 7 and 8 (Wk7-8) by expression of the pro-endocrine gene NGN3. EPs expand around Wk12-13 when islets become vascularized, and mature around Wk26-29 (Piper, 2004). The inventors therefore obtained human pancreas and other endodermal organs at Wk9.1, 10.6, 13, 14.6, 16.3 (separated as body and head of pancreas, 16.3b and 16.3h), 17.5 (17.5b and 17.5h), and 20.1, and isolated M-E cells to derive 9 stage- and organ-specific human primary cell lines (
To characterize the de novo established M-E cell primary lines, immunofluorescence was used to assess expression of mesenchymal (Vimentin) and endothelial markers (PECAM1, CFIII) (FIG. 1B). qPCR was also used to quantify mesenchymal markers (Vimentin, FSP1) (Franke et al., 1982; Franke et al., 1978; Strutz et al., 1995) and endothelial markers (PECAM1, FLK1, VE Cadherin, ICAM, and VWF) (Albelda et al., 1991; Breier et al., 1996; Durieu-Trautmann et al., 1994; Jones et al., 1981; Lawson and Wolf, 2009; Yamaguchi et al., 1993) in the derived primary cells. The inventors then compared the observed expression levels to those of mesenchymal character (human dermal fibroblasts, HDFs) and endothelial character (human umbilical vein endothelial cells, HUVECs (Jaffe et al., 1973) and murine pancreatic endothelial cells, Mile Sven1, MS1 line (Arbiser et al., 2000)(
To determine whether the niche influences endocrine cell formation and differentiation, particularly in promoting progenitors to differentiate into β cells, and at which developmental stage, each of the M-E cells were co-cultured with hESC-derived PPs. The hESCs were guided in a stepwise manner towards pancreatic fate as outlined in
Two criteria were used to determine the maturity of hESC-derived β cells after 7-day co-culture with Wk17.5 and Wk20.1: flow cytometry to determine the amount of polyhormonal or non-INS+ cells, and GSIS to determine their functionality. The inventors obtained 10.1% INS+ cells, 2.3% glucagon (GCG)+ cells, and importantly no cells co-expressing both hormones (
In vitro-derived β cells do not yet show comparable to native cells glucose-induced INS secretion (GSIS), which is the key function of the cells lost in diabetes. To assess the physiological capacity of these cells, GSIS was performed after coculturing progenitors with the Wk17.5 and Wk20.1 niche for 3, 7, and 14 days and the amount was determined of secreted human C-peptide, a byproduct of insulin production. The GSIS is designed to mimic basal blood glucose levels (2.8 mM), a level at which naïve β cells are not stimulated, and elevated blood glucose levels (16.7 mM) to stimulate β cells to secrete C-peptide. After 7 and 14 days of co-culture, but not as early as 3 days, cells gradually responded to stimulation with a high concentration of glucose (
To better evaluate the maturity of P cells differentiated in coculture with niche cells, β cells were stimulated with 30 mM KCl, which hyperpolarizes the cell membrane, allowing measurement of stored c-peptide. Co-culturing PPs with selected M-E cells for 3 days is insufficient to form mature β cells, though the cells did produce a small amount of C-peptide after KCl stimulation (1.2 μIU/103 C-peptide/cell for Wk17.5 and 1.8 μIU/103 C-peptide/cell for Wk20.1) (
Cells co-cultured with stage-specific M-E gained the ability to sense glucose and respond by secreting the appropriate amount of C-peptide by day 14. At that day, only β cells derived in co-culture with Wk17.5h and 20.1 niche responded adequately to fluctuating glucose levels. Cells cultured with Wk17.5h niche had the greatest sensitivity to low and high glucose levels, with a 20-fold difference compared to no-coculture or 10-fold to mefs-coculture controls (
Crosstalk between the pancreatic niche and progenitors can be achieved through secreted factors, cell-cell interactions, and ECM. Because the co-culture experiment permitted direct cell-cell contact, the inventors performed separate co-culture of PPs with ECM and secreted factors of M-E primary cells to avoid cell-cell contact and to dissect how M-E cells promote β cell differentiation. To evaluate the effect of ECM, M-E cells were cultured for one week and then removed from the plate, leaving only the secreted ECM on the dish (ECM matrix). PPs were later seeded on the same dish and co-cultured for 10 days (
In the weeklong co-culture of PPs with M-E primary cells, the PPs must traverse through the EP stage to become β cells. Therefore, it was next tested when the Wk17.5 and 20.1 M-E cells had greatest impact on β cell differentiation by co-culturing PPs or EPs with M-E cells for 7 days or 3 days, respectively. Co-culturing EPs with Wk17.5 or 20.1 M-E primary cells for 3-days leads to a 7-8-fold increase in β cell induction, as measured by increase in C-peptide+ cells and compared to EPs differentiated without co-culture (
In summary, the molecular environment within the human pancreatic niche varies at each stage, with niche cells from specific developmental stages promoting endocrine maturation through the secretion of soluble factors and ECM.
A gene expression array was conducted to identify candidate factors enriched in these pancreatic niche cells compared to HDFs. Pearson's correlation (R2=0.7878) was used to illustrate the degree of linear dependence between Wk17.5h and 20.1 cell gene expression profiles, and the similarity was confirmed between these two times points, yet some distinctions in gene expression profiles did exist (
Based on the microarray analysis, the inventors selected factors upregulated in both Wk17.5h and 20.1 M-E cells, including FGF7, HGF, PDPN, SERPINF1, WNT5A, THBS2, IGF, Endocan, LIF, and WNT3A, to test their role further in human β cell differentiation in vitro. As the co-culture with M-E had a positive effect on EP differentiation and the signals responsible for endocrine cell maturation are not well understood, hESC-derived EPs were used to study the role of niche derived-secreted factors in differentiating these cells into mature β cells (
hESC-derived EPs were treated separately with selected factors at two different concentrations for 3 days before assessing CHGA, ISL1, INS and C-peptide expression by immunofluorescence (
Cells treated with Endocan, SERPINF1, WNT5A, and PDPN had increased numbers of ISL1-eGFP, CHGA+, INS+ and C-peptide-+ cells over untreated controls (
As the pancreatic niche exhibits complex signal signatures in a temporally and spatially specific manner in vivo, it was investigated how these factors cooperate to promote differentiation of human endocrine cells. To assess the cooperation between factors in an efficient manner, four factors were selected that promoted endocrine differentiation (Endocan, SERPINF1, WNT5A and HGF) and their effect was tested on hESC-derived EPs in combinations of two or three and at various concentrations (Table 4). After 3 days of treatment, the number was evaluated of INS+ cells compared to untreated cells or treated with single growth factor (
WNT5A is expressed in the mouse pancreatic mesenchyme at e11.5 and is thought to play a role in islet formation (Heller et al., 2003; Kim et al., 2005). Recent studies showed that WNT5A induces proliferation of some β cells and β cell maturation (Bader et al., 2016), but the role of WNT5A in EP differentiation is not well understood. Immunostaining was performed for WNT5A and other pancreatic markers on human fetal pancreatic tissue from Wk16.3 to 20.1, as well as qPCR with different M-E primary cells (
To characterize WNT5A expression throughout different stages of human endocrine development in vitro, its expression was examined in hESCs and hESC-derived DE, PPs, EPs and β cells using qPCR (
To test the necessity and sufficiency of WNT5A in promoting human β cell differentiation, the inventors disrupted WNT5A expression in Wk17.5h and 20.1 cells by targeting the first constitutive exon (exon 3) using CRISPR-Cas9 nickase (WNT5A-KO). A neomycin cassette was inserted at exon 3 to introduce the frame-shift and to select for positive clones before confirmation of the knockout by external and internal PCRs (
To determine the sufficiency of WNT5A to influence the development of β cells, the inventors overexpressed and repressed WNT5A signaling in hESC-derived EPs. Overexpression of WNT5A in a dose-dependent manner was transiently introduced by 1 μg and 2 μg of pCDNA-WNT5A plasmid in EPs. Increased WNT5A+ cells were observed after 3 days of WNT5A overexpression, with verified dosage efficiency (
To determine whether WNT5A treatment increased INS+ cell numbers through proliferation or differentiation, the mitotic marker phospho-histone 3 (pH3) was utilized (
WNT5A activates the non-canonical and canonical WNT pathway (Mikels and Nusse, 2006; Torres, 1996). Here, it was first determined of the activity of the canonical β-catenin-dependent pathway in EPs after WNT5A treatment using the TOPFLASH reporter system (Veeman et al., 2003). EPs were transfected with either TOPFLASH or FOPFLASH and treated with WNT5A or GSK inhibitor CHIR99021 as a positive control. Untreated EPs had low TOPFLASH activity, and WNT5A treatment did not significantly activate or antagonize the 3-catenin-dependent pathway (
For islet formation, EPs must lose adherence and migrate from the epithelial layer (Kesavan et al., 2014; Kesavan et al., 2009). Because the non-canonical WNT pathway often affects cell motility and polarity downstream, it is possible that WNT5A affects EP migration and subsequently islet formation in specific embodiments, but there was observed no increase in cell mobility in scratch assays after WNT5A treatment (
To investigate the downstream targets of WNT5A in EPs, RNA-sequencing was performed from cells treated with WNT5A over the short term (12h) and long term (5 days) (
The inventors next looked at the JUN and PCP pathway as a putative downstream effector of WNT5A during β cell differentiation. Activity of JUN transcription factors is regulated by JNK-mediated phosphorylation. Short-term (12h) EPs treatment with WNT5A caused increased JNK expression and phosphorylation as determined by Western blot (
RNA-sequencing data analysis also revealed potential link between WNT5A signaling and BMP suppression during β cell differentiation (
To determine the relationship between the BMP pathway and WNT5A, the inventors first performed a novel triple luciferase reporter assay to investigate whether WNT5A treatment caused simultaneous activation of AP-1 and down-regulation of Smad, a downstream effector of BMPs (
The inventors then tested for Smad1/5 activation in hESC-derived β cells using imunofluorescence and single cell flow cytometry imaging. There was correlation between the cellular localization of phosphorylated Smad1/5 and INS expression, with nuclear vs. cytosolic Smad1/5 indicating ability vs. inability to activate the BMP pathway (
To mimic an in vivo source of BMP inhibition, the inventors next treated EPs with Gremlin1 (
Lastly, to test whether WNT5A-treated hEPs differentiate into β cells in vivo, the inventors implanted cells into the kidney capsule of SCID-Beige mice. Immunofluorescent analysis 12 weeks after transplantation demonstrated significantly more PDX1, INS, and C-peptide positive cells in grafts of WNT5A-treated EPs, compared to untreated EPs (
These results indicate that M-E cells are a source of WNT5A and BMP inhibitors. The WNT-BMP signaling crosstalk between the pancreatic niche and EPs profoundly and specifically influences EP differentiation into β cells in stage specific manner (
Growth factors with the fold increase in number of ins+ cell generate from hESCs:
Endocan+SERPINF1(4.5 fold)
Endocan+WNT5A(5.5 fold)
Endocan+HGF(4 fold)
SERPINF1+WNT5A (5.5 fold)
SERPINF1+HGF(3 fold)
WNT5A+HGF (3 fold)
Endocan+SERPINF1+WNT5A(6 fold)
Endocan+SERPINF1+HGF (5 fold)
WNT5A (6 fold)
Material and Methods
Derivation of Human M-E Cell Lines
To derive M-E cells, human fetal pancreas, duodenum, and spleen, were obtained from 9.1 to 20.1 weeks after fertilization, in accordance with Institutional Review Board guidelines. Each tissue was chopped into approximately 4 mm3 cubes. Samples were transferred to 6-well plates and kept at 37° C. for 10 min to allow attachment of the tissue to the plate surface. Then, DMEM:F12 media (+10% FBS, 1 xpenicillin-streptomycin, 1 xGlutamax (all Invitrogen)) was added. Over the subsequent 2 weeks, media was changed every other day, and wells were monitored for outgrowth of M-E cells. Once 50% confluent, the cubes were removed and M-E cells were trypsinized using 0.25% trypsin-EDTA and expanded until passage 3. For each stage, there were at least two independent cell line derivations completed. To derive primary murine M-E cells, ICR embryos were collected at e13.5, 14.5 and 18.5 and pancreas was processed as described above. In addition, previously established cell lines were used: human dermal fibroblasts (HDFs, ATCC), human umbilical vein endothelial cells (HUVECs, ATCC), mouse embryonic fibroblasts (mefs, E12.5 ICRs, Taconic) and mouse islet endothelial cells (MS1, ATCC).
hESC Maintenance and Pancreatic Differentiation
hESC, ISL1-EGFP Hues8 and H1, were maintained under a feeder-free system on Geltrex (Invitrogen) in TeSR-E8 media (Stemcell Technologies). Cells were passaged at 70-80% confluence using TrypLE (Invitrogen). After dissociation, cells were seeded in TeSR-E8 media+10 μM Y-27632 for 24h. Differentiation was started when cells were 90% confluent. The following media and growth factors/small molecules (see also Table 3) were used: Day1: MCDB-131 media with 0.1% BSA+10 mM glucose+ActivinA and CHIR99021. Day2-3: MCDB-131 media+0.1% BSA+10 mM glucose+ActivinA. Day4-5: MCDB-131+0.1% BSA, 10 mM glucose, VitC+KGF. Day6-9: MCDB-131 2% BSA+5.5 mM glucose+Vit.C+ITS (Invitrogen)+ActivinA+KGF+RA+SANT-1+Noggin. Day10-12: MCDB-131+2% BSA+5.5 mM glucose+VitC+ITS+SANT-1+Noggin+PdBU. Day13-15: MCDB-131+2% BSA+5.5 mM glucose+VitC+ITS+Noggin+AlK5i. EPs were dissociated and seeded as 25,000 cells/well on Geltrex-coated 96-well plates in DMEM media (Invitrogen) with B27 Supplement (Thermo Fisher Scientific), later called B27 media, supplemented with 10 M Y-27632 for 24h. Growth factors (Table 3) were added to B27 media, at two concentrations, and tested on EPs for 3 days. After 3 days, cells were PBS washed, fixed with 4% PFA/PBS for 20 min at room temperature (RT), washed twice with PBS and stained.
Co-Culture of hESC-Pancreatic Progenitors and Mesenchymal-Endothelial Cell Lines
Co-culture of M-E cells and hESC-derived progenitors was performed in three settings. The first was cell-cell interaction, the second was culture of PPs or EPs in conditional media collected from M-E cells, and the third was culture of EPs on the M-E ECM matrix. For the first assay, M-E cells were plated on 6 well-plates 24h in advance. Mitotic inactivation was performed for 2h with 10 g/ml mitomycin C (Sigma-Aldrich) followed by three washes with PBS. In the meantime, hESCs were differentiated to either the PP or EP stage and plated on M-E cells at a density of 60,000 cells per cm2. As controls, mefs, laminin or gelatin-coated plates were used. Conditional M-E media was prepared from 40-80% confluent M-E cells cultured in the same media as for hESC differentiation, and media were collected every day for 6 days. The collected medium, “conditional medium” was later used as a base medium to differentiate PPs or EPs. For the third assay, M-E ECM matrix plates were prepared as follows: confluent M-E cells were cultured on 6-well plates for 6 days, after which cells were removed by short non-enzymatic, EDTA treatment, leaving the ECM matrix behind. PPs or EPs were plated on these ECM-plates and differentiated into β cells.
GSIS
Cells were washed with Krebs buffer (128 mM NaCl, 5 mM KCl, 2.7 mM CaCl2, 1.2 mM MgCl2, 1 mM Na2HPO4, 1.2 mM KH2PO4, 5 mM NaHCO3, 10 mM HEPES, 0.1% BSA) and then pre-incubated in 2.8 mM D-glucose Krebs buffer for 2h. Cells were then incubated in fresh-low glucose Krebs, followed by 16.7 mM and then in Krebs buffer with 2.8 mM glucose and 30 mM KCl for 30 min at each condition. After incubation, supernatant was collected. Between incubations cells were washed 2 times with Krebs buffer. This procedure was repeated at least 3 times for different time points and coculture combinations. At the end, cells were dispersed into single cells using TrypLE Express and quantified by Countess (Invitrogen). C-peptide was measured using the Human Ultrasensitive C-peptide ELISA (Mercodia). The C-peptide amount was normalized to the cell number.
Detailed information of human islets isolation is described elsewhere herein. Human islets donor data: 67-year-old male, 44-year-old and 54-year-old female. Upon arrival, islets were seeded in 804G-coated 96-well plates and incubated in CMRL1066 media (Mediatech Inc.) supplemented with 10% human serum overnight. After 3 days, GSIS was performed.
Whole mRNA Sequencing
Total RNA was extracted as described elsewhere herein and RNA quality was assessed using 2100 Bioanalyzer (Agilent Genomics). Samples with RIN≥9 preceded to library preparation using TruSeq stranded mRNA Library Prep Kit LT (Illumina) according to the manufacturer's protocol. Library concentration was determined by qPCR (KAPA Library Quantification Kit) to pool equal amounts of libraries with different adaptor indexes. Sequencing was performed using NextSeq500 (Illumina).
Dual-Pathway Luciferase Vector and Multicolor Luciferase Assay
The dual-pathway luciferase reporter vector was generated using the GoldenBraid2.0 Assembly Platform (Sarrion-Perdigones et al., 2011; Sarrion-Perdigones et al., 2013). Briefly, transcriptional units comprising the promoter elements, the CDS of the corresponding luciferase and the bovine growth hormone terminator were first assembled, and then these were latter combined in successive rounds of assembly to build the multigenic vector used in this assay. For the multicolor luciferase assay, H1-derived EPs were first dissociated into 96-well plates using method previously described and incubated overnight to allow cell attachment. Transient transfections were then performed using 0.75 μl Lipofectamine2000 with 150 ng of dual-pathway luciferase vector for each well of the 96-well plate and incubated for 24h. Positive controls (CMV:FLuc:bGHT, CMV:RedF:bGHT and CMV:Renilla:bGHT) were transfected in separate wells to adjust the transmission constants for each luciferase. Transfected EPs were further treated with 500 ng/ml WNT5A, 200 ng/ml BMP4, 1 ng/μl Anisomycin dissolved in basal media, as previously described. At the determined harvesting point, culture media was removed and wells were washed with PBS. 35 μL of passive lysis buffer (PLB) were added to the wells. Culture plates were incubated at room temperature for 15 min on a rocking platform and stored at −80° C. for further assay until all data points were collected. After thawing the lysates, they were transferred to a 384-well plate and the luciferase assay was performed in a CLARIOstar illuminometer. 10 μL of LARII reagent was added with the built-in injectors and after 2 seconds, the total light and the BP filtered light emitted by the FLuc and RedF mixture were measured for 1 second. Finally, 15 μL of Stop & Glo® reagent were injected and after 4 seconds the emitted light by Renilla luciferase was measured. The activity corresponding to FLuc and RedF that were simultaneously measured after the LARII reagent was added were calculated according to the method proposed by Nakayima et al (Nakajima et al., 2005) that is adjusted to this particular assay and explained in
Immunofluorescent Analysis
Cells were incubated with 5% donkey serum (Jackson ImmunoResearch Laboratories) in PBST (PBS+0.1% Triton-X) for 30 min to avoid nonspecific binding of the antibody and to permeabilize the cells. Primary antibodies, diluted in 5% donkey serum in PBST, were then added and incubated at 4° C. overnight with shaking. After primary antibody incubation, cells were washed 3 times with PBST and secondary antibodies conjugated with Alexa-Fluor Dyes (Jackson ImmunoResearch Laboratories) and diluted in 5% donkey serum in PBST were added to the cells for 30 min at RT. Then, cells were thrice washed with PBST and nuclei were stained with Dapi (Roche Diagnostics). Antibody sources, catalog numbers and dilutions are listed in Table 2. For imaging, Leica DMI6000 or confocal Leica TCS SPE was used. Images were initially processed by LAS X software and then further analyzed and quantified using ImageJ software (NIH, W Rasband, http://rsb.info.nih.gov/ij) using cell counter plug-in. Typically, at least 5 randomly selected images were counted per condition.
Flow Cytometry
Cell were dissociated, washed with PBS, filtered through 40 μm cell strainer and fixed with 4% PFA for 10 min at RT. 5% donkey serum in PBST (PBS+0.2% Triton-X) was used to block unspecific binding of antibody and to permeabilize cells by 30 min incubation on ice. Primary antibodies were then added as described in Table 2 for 30 min at 4° C. with shaking. After primary antibody incubation, secondary antibodies conjugated with fluorophore, were added and incubated for 30 min at 4° C. with shaking. Then cells were centrifuged at 1500 rpm for 5 min and washed with FACS buffer (PBS+2% FBS+10 mM EDTA) twice. Stained cells were filtered through 40 m cell strainer before flow cytometry. FACS analysis was performed using LSRII (BD Biosciences) and Diva software package. For all the samples, 10,000 events were captured and FlowJo was used for gating and analysis.
Imaging Flow Cytometry to Analyze pSmad1/5 Localization
To determine cellular localization of pSmad1/5 and INS, one million of EPs was dissociated and filtered to single cell suspension as previously described and then fixed with 4% PFA/PBS with 0.1% Saponin for 30 min at 40 C. After fixation, cells were centrifuged at 3,000 g for 3 min and washed with 0.1% Saponin, 1% BSA in PBS followed by incubation with primary and then secondary antibody diluted with 0.1% Saponin, 1% BSA/PBS. ImageStreamX MarkII (Millipore) was used to capture high-resolution single cell images to detect Dapi, INS and pSMAD1/5 cellular localization. 10,000 events were acquired and compensation was adjusted to minimize spectral overlap between the fluorophores, used in the experiment, which are Dapi, Alexa488 and TRITC. Data were analyzed by IDEAS software (Millipore).
qPCR-Based Gene Expression Analysis
Total RNA was isolated using TRIzol (Thermo Fisher Scientific) according to the manufacturer's protocol. DNAase (Qiagen) treatment was performed to remove genomic DNA. cDNA was synthesized using iScript (Biorad) by using one g of RNA. For qPCR, KAPA SYBR FAST (Kapa Biosystems) and Connect CFX light cycler (Biorad) for PCR reaction (<40 cycles were used). Primers were designed using qPrimerDepot in such way that the PCR product spans across exons junction. Primer sequences are listed in Table 1. Primer specificity was checked using CFX manager software v3.1 (Applied Biosystems) and PCR product electrophoresis. Threshold data were analyzed by CFX manager software v3.1 using Comparative Ct relative quantitation method with TBP as internal control.
Microarray Analyses
50 ng of total RNA combined with RNA spike mix were reverse-transcribed using a T7 Primer Mix to produce cDNA. The cDNA product was transcribed using T7 RNA Polymerase, producing cyanine-3-labeled cRNA. The labeled cRNA was purified using a Qiagen RNeasy Mini Kit. Purified products were quantified using the NanoDop spectrophotometer for yield and dye incorporation, and tested for integrity on the Agilent Bioanalyzer. 600 ng of the labeled cRNA were fragmented. 480 ng of fragmented cRNA was loaded onto each of the Human G3 v2 8×60K Agilent Expression arrays. The arrays were hybridized in an Agilent Hybridization Chamber for 17h at 65° C. with 10 rpm rotation. The arrays were washed using the Agilent Expression Wash Buffers 1 and 2, followed by acetonitrile, as per the Agilent protocol. Once dry, the slides were scanned with the Agilent Scanner (G2565BA) using Scanner Version C and Scan Control software version A.8.3.1. Data extraction and quality assessment of the microarray data was completed using Agilent Feature Extraction Software Version 11.0.1.1. Pearson's correlation was created using Prism 6. Heatmaps were generated in R (version 3.2.3) using heatmap.2 from gplots package (version 2.17.0) with viridis (version 0.4.0), and ggbiplots (Wickham, 2009). The data is submitted at NCBI under GEO number GSE102877.
RNA-Seq Data Analysis
After preliminary analyses, showing a significant Pearson Correlation Coeficient for gene expression, two biological replicates were concatenated and analysed as single samples. This step yielded four samples, two for each treatment (5 days and 12h), as well as untreated samples. All reads were considered single-end in the bioinformatic analyses.
For Venn diagram, TFactS analysis and Gene enrichment analysis (GSEA), sequencing reads were first aligned using TopHat and the gene differential expression were assembled and analyzed using Cufflinks. Significantly up- and down regulated genes were determined by comparing Fragments Per Kilobase of transcript per Million mapped reads (FPKM) between untreated and WNT5A treated samples with p less than 0.05. Genes upregulated >2 fold and downregulated <0.5 fold were used to generated Venn diagram from BioVenn and the gene function categorization was refer from Hrvatin et al., 2013. To predict which transcription factors are responsible to the gene changes in the sequencing result, TFactS analysis was performed by inputting up and down regulation gene lists. Significantly regulated transcription factors were determined with p, e, and q<0.05, as default setting of the software. For GSEA (http://software.broadinstitute.org/gsea/index.jsp), all input files were generated through GenePattern and the analysis was performed based on the instruction from Broad institute GSEA user guide with the following parameter: phenotype labels as 5dUT versus 5dWNT5A, 1000 genes set of permutations, weighted enrichment statistic, gene sets between 15 to 500, with log 2 ratio of class as metric for ranking genes. Significantly regulated pathways have p<0.01. Data GEO submission number: GSE 90785.
WNT5A Expression and Inhibitions in EPs and HDFs
pCDNA-WNT5A plasmid was obtained from Dr. Marian Waterman (Addgene #35911). Nucleofection was used for DNA delivery. One million EPs or HDFs were dissociated with TrypLE and resuspended in 20 μl P3 solution with supplement and 1 or 2 μg of DNA. Cells were nucleofected using Amaxa-4D nucleofector (Lonza) CM113 program. After nucleofection, the inventors added pre-warmed medium to the cells and incubated them at 37° C. for 5 min before plating. The transfection efficiencies were evaluated using the pmaxGFP (Lonza): the efficiency was 17.5% and was 15.7% for EPs and HDFs, respectively. To blockWNT5A autocrine signal from EPs, 1 μg of WNT5A antibody was added to every 25,000 of EPs for three days before fixation and further immunofluorescence analysis.
Generation of WNT5A KO in Wk17.5h and Wk20.1 Cell Lines
Methods and design of WNT5A KO was described in Yang et al., 2016. Three days after the nucleofection, Wk17.5h and 20.1 cells were selected with 50 μg/ml G418 (Sigma-Aldrich) and the dosage was increased to 100 μg/ml after 2 day. After 8 days of selection, cells were cultured in DMEM+10% FBS+Glutamax+3-mercaptoethanol (Invitrogen) to evaluate efficiency of KO.
Generation of SC-β Cells and WNT5A Treatment
SC-β cells were generated using the protocol as previously described (Pagliuca et al., 2014). WNT5A were introduced into the differentiation from EP stage (EN in the original paper) together with T3, ALK5i in CMRL media for the first two days and then change into T3, ALK5i in CMRL media from the third day. Samples were collected at the 4th day and 12th day counting from EP stage and were fixed with 4% PFA and thrice washed with PBS for 10 min. For whole-mount staining, samples were first blocked with 5% donkey serum in PBST overnight and incubated with antibodies overnight as described above.
TOPFLASH Reporter Assay
For TOPFLASH reporter assay, 80,000 EPs were transfected with 0.5 μg of TOPFLASH (Addgene #12456) or FOPFLASH (Addgene #12457) from Dr. Randall Moon, together with 0.25 μg pRLTK using Lipofectamine 2000 (Invitrogen) for 48h. Cells were then treated with CHIR99021 (as positive control), DMSO (mock control) or WNT5A for 3 days before collecting the samples. Luciferase assay was performed using Dual luciferase assay system (Promega) which Luciferase and Renilla signal were measured by TD20/20 Luminometer (Turner designs).
Protein Extraction and Western Blotting for Phosphorylated JNK and Total JNK
ISL1-EGFP hESCs were differentiated into EPs and first balanced with basal media (DMEM with 1% BSA and NEAA) for 6h and then treated with 500 ng/ml WNT5A in basal media. After 12h, cell lysates were collected as imilion of EPs were pelleted, PBS washed and resuspended in 250 μl of lysis buffer (10 mM HEPES pH7.5, 10 mM MgCl2, 5 mM KCl, 0.1 mM EDTA pH8, 0.1% TritonX-100, 0.2 mM PMSF, 1 mM DTT, 1 tab of Complete Protease Inhibitor Cocktail (Roche)). Cell lysates were centrifuged 12,000 g at 4° C. for 15 min and their supernatants were collected. BCA assay were performed using Pierce BCA protein assay kit (Thermo) to determine protein concentration. For Western blot, 30 g of protein were denatured with 4x Laemmeli buffer (40% glycerol, 8% SDS, 240 mM Tris-HCl pH6.8, 5% β-mercaptoethanol, 12.5 mM EDTA, 0.04% bromophenol blue) at 95° C. 3 min and resolved in 8% SDS-PAGE. PVDF membranes (BioRad) were used for transfer and membrane were blocked with 5% BSA in Tris-buffered saline with 1% Tween 20 (TBST) for 1h before applying primary antibody. Membranes were washed thrice for 10 min and anti-rabbit IgG-HRP (GE Life Science) were added for 3h at RT, followed by three washes. HyGLO™ Quick Spray Chemiluminescent HRP Antibody Detection Reagent (Denville Scientific Inc.) was used to detect antigen and the membrane was developed using CL-XPosure Film (Thermo Scientific). Membrane stripping was performed using mild stripping buffer (200 mM glycine, 0.1% SDS, 1% Tween20, pH 2.2) according to the Abcam's instructions.
Human Islet Isolation
Human pancreata were obtained with informed consent for transplant or research use from relatives of heart-beating, cadaveric, multi-organ donors through the efforts of The National Disease Research Interchange (NDRI), Tennessee Donor Services, the Mid-South Transplant Foundation, and the United Network for Organ Sharing. Donor demographics were collected at the time of acceptance and included age in years, gender, race, body mass index, history of alcohol intake, and history of hypertension. Donor-related laboratory data included donor blood glucose, serum amylase, lipase, liver function tests (ALT, AST), cytomegalovirus infection status, and procurement and preservation parameters such as pancreatic warm and cold ischemia times, ventilation time, pancreas weight and adequacy of pancreas perfusion were also recorded. All pancreata in this study were perfused using University of Wisconsin (UW) solution. Human islets were isolated from cadaver donors using an adaptation of the automated method described by Ricordi et al. (Ricordi et al., 1988). Liberase (Boehringer Mannheim, Indianapolis, Ind.) was the enzyme used in all the isolations in this study. Liberase was dissolved in cold (4° C.) Hank's balanced salt solution (HBSS) (Mediatech, Inc., Herndon, Va.) that was supplemented with 0.2 mg/ml Dnase (Sigma Chemical Co., St. Louis, Mo.), 1% penicillin-streptomycin (Sigma Chemical Co.), 20 mg/dl calcium chloride (J.T. Baker, Inc., Phillipsburg, N.J.), and HEPES (Sigma Chemical Co.). Once dissolved, the pH was adjusted to between 7.7 and 7.9. The enzyme preparation was then sterile filtered, warmed to 37° C., and used for the intraductal distension of the pancreas. The distended pancreas was cut into several pieces, placed in the Ricordi's chamber, and the Heating circuit started. Pancreatic digestion was performed at 37° C. until more than 90% free islets were observed in the sample. Digested tissue was collected into cold HBSS supplemented with 20% human serum and 1% penicillin-streptomycin solution and centrifuged at 400 g at 4° C. for 5 min. Tissue pellets were pooled into cold UW solution and held at 4° C. for 1h with periodic mixing. Islet purification was performed on a COBE 2991 Cell Processor (COBE BCT, Lakewood, Colo.) using OptiPrep (Nycomed Pharma AS, Oslo, Norway) as a step-gradient based on a modification of the procedure of London et al (Robertson et al., 1993). Islet culture: Aliquots from human islet isolations were cultured in SFM containing 1% ITS, 1% L-glutamine (Life Technologies, Gaithersburg, Md.), 1% antibiotic antimycotic solution (Sigma Chemical Co.), and 16.8 μM/L zinc sulfate. ITS (1%; Collaborative Biomedical Products, Bedford, Mass.) as described (Fraga et al., 1998). Human islets donor data: 67-year-old male, and 54-year-old female. For wholemount immunofluorescence staining, islets were first fixed 15 min in 4% PFA at room temperature shaker, followed by three 30 min washes in PBS. Islets were incubated at 4° C. overnight rotor for the blocking, primary and secondary incubation, as described before.
Cell Migration Assays:
a) Scratch Assay.
EPs were dissociated and seeded into 12 well-plate to reach 90% confluence before a scratch was made. 200 μl pipette tip was used to create a smooth scratch and followed by 3h of mitomycin C treatment. Cells were then washed thrice with PBS and B27 media with or without WNT5A was added. Pictures of the scratch wound were taken at 0, 6, 12, 18, 24 and 30h after scratch to observe cell migration. Pictures were analyzed by counting cells migrating into the gap using ImageJ.
b) Transwell Assays.
Transwell assays were performed using FluoroBlock Insert, 8 μM pore size (Corning). The bottom 24 wells and the insert of transwell were coated with Geltrex to retained cells. At the experiment day, 1.5×105 hESC-derived EPs were seeded in each well at the transwell insert in B27 media with 10 μM Y-27632 and the well bottoms were replenished with either B27 media as control, B27 media+100 or 500 ng/ml WNT5A, or conditional media from Wk9.1, 17.5h and 20.1. After one week, the cell attached to the bottom wells were stained with Dapi and were counted.
In Vivo Transplantation
WNT5A treated hESC-derived EPs were treated with 500 ng/ml WNT5A for 3 days and cultured in B27 for 11 days before transplantation. Control EPs were maintained in B27 for 2 weeks. 1.5 million of hESC-derived EPs were mixed with equal volumes of matrigel and injected into the kidney capsule into 6 week-old SCID-Beige mice (Taconic Bioscience). For control N=6 and WNT5A treated N=8 in 2 cohorts. Grafts were collected at X weeks, washed in PBS, and fixed with 4% PFA for 1h. After fixation, tissues were washed with PBS and incubated with 30% sucrose/PBS overnight before embedding into OCT for cryosectioning. All animal experiments were approved by Baylor College of Medicine Institutional Animal Care and Use Committee.
All patents and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents and publications herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in their entirety.
Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.
This application claims priority to U.S. Provisional Patent Application Ser. No. 62/423,471, filed Nov. 17, 2016, which is incorporated by reference herein in its entirety.
This invention was made with government support under P30-DK079638 awarded by National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2017/062097 | 11/16/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62423471 | Nov 2016 | US |
