Patent Application
20210115366
Organoids are stem cell-derived structures generated in vitro that mimic the three-dimensional architecture and physiology of an intact organ. Organoid development may allow for new approaches to modeling and studying normal development and disease processes, and introduce new approaches to medical research, drug discovery, and toxicology testing. In a typical organoid protocol, precursor cells, such as stem cells, are driven toward particular lineages through combinations of growth factors in culture media. Cells differentiate in a controlled manner to reiterate organ development starting from pluripotent stem cells in vitro. While organoid research holds considerable potential for investigating human development and disease and for advancing precision and regenerative medicine, in addition to usefulness for transplant into patients, the development of organoids is still in its infancy, and none of the currently available organoid models to date recapitulate the complete physiology of a human organ. In particular, while development of organoids has been described, advanced differentiation, increased size, maturity and function, particularly for use in transplantation, is an unmet need in the art. The instant disclosure seeks to address one or more of the aforementioned needs in the art.
Disclosed herein are methods of enhancing development of a three-dimensional in vitro model of an intestinal tissue (HIO), which may be derived from a precursor cell. The precursor cell may be, for example, an embryonic stem cell, an induced pluripotent stem cell (iPSC), or the like. The in vitro HIO model may be characterized in that the HIO has a lumen, in which a lengthening device may be inserted to promote development of the HIO. Compositions derived from the disclosed methods are also described.
This application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.
Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, for example within 5-fold, or within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.
The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and/or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.
As used herein, the term “pluripotent stem cells (PSCs)” encompasses any cells that can differentiate into nearly all cell types of the body, i.e., cells derived from any of the three germ layers (germinal epithelium), including endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), and ectoderm (epidermal tissues and nervous system). PSCs can be the descendants of inner cell mass cells of the preimplantation blastocyst or obtained through induction of a non-pluripotent cell, such as an adult somatic cell, by forcing the expression of certain genes. Pluripotent stem cells can be derived from any suitable source. Examples of sources of pluripotent stem cells include mammalian sources, including human, rodent, porcine, and bovine.
As used herein, the term “induced pluripotent stem cells (iPSCs),” also commonly abbreviated as iPS cells, refers to a type of pluripotent stem cells artificially derived from a normally non-pluripotent cell, such as an adult somatic cell, by inducing a “forced” expression of certain genes. hiPSC refers to human iPSCs. In some embodiments, iPSCs may be derived by transfection of certain stem cell-associated genes into non-pluripotent cells, such as adult fibroblasts. Transfection may be achieved through viral vectors, such as retroviruses. Transfected genes may include the master transcriptional regulators Oct-3/4 (Pouf51) and Sox2, although other genes may enhance the efficiency of induction. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. As used herein, iPSCs include first generation iPSCs, second generation iPSCs in mice, and human induced pluripotent stem cells. In some embodiments, a retroviral system is used to transform human fibroblasts intopluripotent stem cells using four pivotal genes: Oct3/4, Sox2, Klf4, and c-Myc. In alternative embodiments, a lentiviral system is used to transform somatic cells with OCT4, SOX2, NANOG, and LIN28. Genes whose expression are induced in iPSCs include but are not limited to Oct-3/4 (e.g., Pou5fl); certain members of the Sox gene family (e.g., Sox1, Sox2, Sox3, and Sox15); certain members of the Klf family (e.g., Klf1, Klf2, Klf4, and Klf5), certain members of the Myc family (e.g., C-myc, L-myc, and N-myc), Nanog, and LIN28.
As used herein, the term “embryonic stem cells (ESCs),” also commonly abbreviated as ES cells, refers to cells that are pluripotent and derived from the inner cell mass of the blastocyst, an early-stage embryo. For purpose of the present invention, the term “ESCs” is used broadly sometimes to encompass the embryonic germ cells as well.
As used herein, the term “precursor cell” encompasses any cells that can be used in methods described herein, through which one or more precursor cells acquire the ability to renew itself or differentiate into one or more specialized cell types. In some embodiments, a precursor cell is pluripotent or has the capacity to becoming pluripotent. In some embodiments, the precursor cells are subjected to the treatment of external factors (e.g., growth factors) to acquire pluripotency. In some embodiments, a precursor cell can be a totipotent (or omnipotent) stem cell; a pluripotent stem cell (induced or non-induced); a multipotent stem cell; an oligopotent stem cells and a unipotent stem cell. In some embodiments, a precursor cell can be from an embryo, an infant, a child, or an adult. In some embodiments, a precursor cell can be a somatic cell subject to treatment such that pluripotency is conferred via genetic manipulation or protein/peptide treatment. Precursor cells include embryonic stem cells (ESC), embryonic carcinoma cells (ECs), and epiblast stem cells (EpiSC).
In both directing and fostering the natural ability of stem cells to self-organize, significant advances have been made in the generation of functional human intestinal organoids. However, conventional methods for their generation are solely biological, when indeed intestinal development and morphogenesis are impacted by dynamic mechanical forces. As understanding of these mechanical forces during development deepens, the opportunity to include them in tissue engineering strategies arises. Here, Applicant has generated intestinal tissue and incorporated uniaxial strain serving to induce growth and maturation of the tissue. Using a variety of outcome measures including morphometric quantification, transcriptome profiling, and functional assays, Applicant found the newly generated tissue to be more similar to native human intestine after strain exposure. The size and complexity of the tissue was significantly improved, as was muscle tone. The novel methods incorporate a developmentally relevant mechanical cue in the development of human intestinal tissue and results in enhanced maturation and enterogenesis.
The establishment of a three-dimensional in vitro model of the human intestine has required a deep understanding of endoderm and intestinal development.1 These complex structures are created from human embryonic stem cells and/or induced pluripotent stem cells (PSCs) by the perturbation of signaling pathways through a temporal series of growth factor manipulations2, 3. This exclusively biological and mechanically static methodology to intestinal tissue generation has been successful in creating both functional intestinal lineages (e.g., Paneth, Goblet, enteroendocrine, and enterocyte) and architecture similar to that of native intestine (e.g. crypts, villi, and smooth muscle layers)2, 4. These tissues, termed human intestinal organoids (HIOs) have been shown to be functional and possess the ability to engraft in vivo4. However, the maturation status of transplanted HIOs (tHIOs) best approximates that of human fetal intestinal tissue, which ultimately limits their utility6. Current methods have yielded a powerful tool for use in both basic science and clinical applications. HIOs are an avenue to study intricate physiological interactions and personalize medicine for patients across the globe7,8. However, in order to build upon these uses it is imperative that tissues with increased size, maturity and function are generated, because creating tissue for transplantation remains an unmet clinical need.
Recently, there has been a renewed interest in not only the biological cues impacting development and morphogenesis, but the mechanics of development as well9. In particular, Shyer and Savin have elegantly demonstrated that strain plays a role in intestinal development. Gut looping, villification, and the localization of intestinal stem cells to the crypt have all been associated with mechanical strain10-13. As strain is a common contributing factor between several architectural features of the intestine, it was discovered by Applicant that incorporating this pulling or tension force within the generation of HIOs could advance differentiation, and that both mechanical and biological cues could prove advantageous in the generation of larger scale tissues with a maturation status beyond that of human fetal intestine.
In order to incorporate strain into the current protocol of HIO generation, Applicant combined in vivo transplantation with the repurposing of a lengthening device designed for the treatment of short bowel syndrome: the spring. Much like other endoluminal lengthening devices, the spring has been shown to stimulate an adaptive morphometric response in the setting of mature tissues, though additional effects have not been thoroughly characterized14-18. In one aspect, the use of a spring may be advantageous because its geometry and applied force may be scaled and may allow for the accumulation of mucous as the tHIO is a closed system14-17. In combining these transplantation strategies, Applicant found that the application of strain in a fetal setting is capable of eliciting tissue maturation and enhancing overall growth of the tHIO. Further, Applicant found that, by including a lengthening device, for example, a spring, into the lumen of an organoid, improved development of the organoid can be achieved. In particular, Applicant found that linear deployment of a spring having a suitable spring constant k was an important consideration in achieving this effect. In particular, Applicant has found that a spring constant that is too high will deploy in a nonlinear fashion and will cause obstructions resulting in the death of the host. If the spring constant k is too low, the spring/lengthening device will be too soft and the tissue will not experience sufficient strain.
Applicant has combined a common mechanic of development, uniaxial strain, with the generation of HIOs. Grafts that had undergone strain (tHIO+S) were found to have increased intestinal and maturation features compared to those that did not experience applied strain, including transcriptional, morphological, and functional shifts toward postnatal human intestine. Disclosed herein is the first description of mechanically manipulating tHIOs in vivo to result in the successful induction of maturation and enterogenesis.
In one aspect, a method of enhancing development of a three-dimensional in vitro model of an intestinal tissue (HIO) derived from a precursor cell is disclosed. The precursor cell may be, for example, an embryonic stem cell (H1 line), an induced pluripotent stem cell (iPSC), or the like. The in vitro HIO model may be characterized in that the HIO has a lumen, which method further comprises the step of inserting a lengthening device into the lumen, which applies strain to the lumen.
In one aspect, the lengthening device may comprise a spring, for example, in one aspect, a nickel titanium (nitinol, NiTi) spring. In one aspect, the lengthening device may have a relaxed length of about 10 to about 15 mm, and may have a compressed length of about 4 to about 8 mm. In certain aspects, the lengthening device may have a diameter of about 1 to about 3 mm, or about 2 mm. The lengthening device, in some aspects, may a spring constant of from about 0.5 N/m to about 2 N/m, or from about 0.7 to about 1.7, or from about 0.9 to about 1.2 or about 1 N/m. The lengthening device may be contained in a degradable coating, for example, for a degradable coating that maintains the compressed length until deployment. An exemplary coating includes a gelatin capsule, which may further comprise a polymer in an amount and of a type sufficient to delay deployment. In one aspect, the lengthening device may comprise an enteric coating, for example, an enteric coating of cellulose acetate phthalate (C-A-P). The lengthening device may be one which provides a cavity sufficient to allow flow of lumen secretions. Other suitable devices will be readily appreciated by one of skill in the art.
In one aspect, the method may comprise a first and a second engraftment period. The first engraftment period may comprise the step of transplanting the HIO into an immune compromised animal model prior to implantation of the lengthening device, for example, in the mesentery of the animal model. The first engraftment period may be carried out for a period of time sufficient for said develop crypt regions, villi, and smooth muscle layers. In one aspect, the method may comprise the step of a second engraftment period occurs, which occurs after the first engraftment period. The second engraftment period may comprise the steps of inserting the lengthening device after the first engraftment period for a period of time sufficient to allow increased villus height and crypt depth as compared to a control HIO not subjected to said lengthening device. In one aspect, the first and second engraftment period may be for a period of time sufficient to allow the HIO to have increased villus height and crypt depth and crypt fission, and increased longitudinal and circular muscle thickness, as compared to a control HIO which does not contain a lengthening device.
In one aspect, the first engraftment period may be of a period of time of from about 6 to about 14 weeks, or from about 10 to about 12 weeks, or for a period of time sufficient to allow said HIO to obtain a blood supply and/or grow to a size sufficient for implantation of the lengthening device, for example, from about 5 mm to about 2 cm in length, or from about 10 mm to about 1.5 cm in length.
In one aspect, the precursor cell is a pluripotent stem cell or an induced pluripotent stem cell, for example, an embryonic stem cell. In one aspect, the HIO may be derived from a pluripotent stem cell, wherein said pluripotent stem cell is derived from a fetal tissue stem cell.
In one aspect, three-dimensional human intestinal organoid (HIO) composition derived from a pre-cursor cell in vitro are disclosed. The HIOs may comprise a lengthening device as described above.
In one aspect, three-dimensional human intestinal organoid (HIO) composition derived from a pre-cursor cell in vitro is disclosed, wherein said HIO is lacking one or more features native to a fully developed organ, for example, wherein said HIO is devoid of a vascular system.
The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus can be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Nitinol Spring as an Endoluminal Lengthening Device for tHIOs.
Applicant previously developed a method of stepwise growth factor manipulations to differentiate human pluripotent stem cells into intestinal organoids2-5. Upon transplantation into the mesentery of NOD-SCID IL-2Rγnull (NSG) mice these organoids indeed engraft and go on to closely resemble native intestine with well-defined crypt regions, villi, and smooth muscle layers4. After ten weeks, the tHIO has drastically grown in size. At this time, a secondary procedure was performed wherein a compressed spring was implanted inside the tHIO (
Use of the spring was based upon previous studies describing endoluminal lengthening devices14-17,20. Applicant found the nickel-titanium (nitinol, NiTi) spring as an ideal device, because it had been successfully used in rats and did not require manipulation post implantation as hydraulic devices do14,17. Another advantage in utilizing the NiTi spring is that a large amount of negative space was retained within the tHIO after insertion, which permitted mucous accumulation. The spring's geometry was based upon the constraint of a commercially available gelatin capsule used to maintain the spring in a compressed state during transplantation. An enteric coating of cellulose acetate phthalate (C-A-P) was used in order to delay the degradation of the gelatin capsule during implantation. The springs had a relaxed length of 12-13 mm, outer diameter of 2 mm and when encapsulated were 5-6 mm in length (
Morphological Maturation as a Result of Strain in the tHIO
At the time of harvest, tHIO+S had a more tubular appearance verses the saccular shape of sham or tHIO and grew to about 10-14 mm (
Strain's Impact on Transcriptional Changes in tHIOs.
Applicant examined the differential gene expression in tHIO+S compared to tHIO and human jejunum (all full thickness samples) using RNA sequencing. Out of 23,366 genes annotated in the genome, 4,537 genes were significantly differentially regulated amongst the samples. Samples were analyzed using a scaled centered principle component analysis (PCA) to visualize the multi-dimensional variation between samples. Principal component 1 (36.14%), discriminated the samples among their types and suggested a higher degree of similarity between tHIO+S and human infant tissue (
In order to examine the biological processes induced by strain in tHIO, Applicant compared the differentially expressed genes (DEGs) between the tHIO+S and tHIO groups. In tHIO+S compared to tHIOs, 454 and 243 genes were respectively up- and down-regulated. Next, a functional enrichment analysis was performed to highlight biological processes within these gene sets. 14 d after the initial application of strain implantation, cell cycle and overall catabolic and protein assembly processes were captured, suggesting an impact of strain upon cell proliferation and tissue formation (
To broadly investigate some of the pathways enriched with exposure to strain beyond the level of transcription, protein analysis was performed. A protein phosphorylation array for MAPK signaling was executed and quantified (
Pan ERBB receptor expression was also measured and quantified (
Strain's Impact on Proliferation and the Stem Compartment in tHIOs
To examine whether strain could impact the intestinal stem cell/transit-amplifying compartment, Applicant analyzed proliferation in the crypts of tHIOs and tHIO+S and human adult jejunum. Dual chromogenic staining for Marker of Proliferation K167 (MK167) and Cadherin-1 (CDH1) was performed for the purpose of quantification (
Epithelial Response of tHIOs to Strain
To evaluate if strain impacted cell fate, secretory epithelial cell types were quantified. Alcian blue staining was performed for the identification of Goblet cells, human specific Lysozyme (LYZ) for Paneth cells and Chromogranin-a (CHGA) for enteroendocrine cells (EECs) which were all subsequently quantified (
Epithelial topography of tHIO, tHIO+S, and adult jejunum samples was observed to see if strain impacted features of the surface ultrastructure (
In addition to larger scale structural features, Applicant also wanted to examine aspects related to epithelial function to see if strain had modulated them. On top of overall villus length, the brush border's size impacts the available surface area for nutrient exchange within the gut22. TEM micrographs depict the brush border in tHIO, tHIO+S and adult jejunum (
An Ussing chamber was used to measure the epithelial characteristics of short circuit current, fluorescein isothiocyanate (FITC)-dextran flux and transepithelial resistance to observe any functional changes related to strain. All values were corrected using a correction factor calculated from morphometric observations (
Ex Vivo Muscle Function in Response to Strain in tHIO
Applicant next tested ex vivo muscle properties to see if the increased muscle thickness further correlated to improved muscle function. In a previous study, the presence of intestinal cells of Cajal (ICCs) was confirmed in tHIOs5. Here, with Anoctamin1 (ANO1), a marker more specific for ICCs than CD117 which also stains mast cells, Applicant observed their presence in all sample types24. In both tHIO and tHIO+S, ICCs were localized primarily within the smooth muscle layers, though not always at the interface of the longitudinal and circular muscle layers as was typically observed in adult jejunum (
After an equilibration period, muscle force contraction was assayed using a logarithmic series of bethanechol doses ranging in concentration from 1 nM to 10 mM. Representative tensile response recordings are plotted for tHIO, tHIO+S and adult jejunum (
Discussion
Applicant has combined the principles of embryonic intestinal development with the mechanics of development to successfully engineer human pluripotent stem cell-derived intestinal tissue with maturity and function, which exceeds that of those produced with a strictly biological approach. Applicant has shown the application of strain induced gross, microscopic, and ultrastructural changes in the morphology of transplanted organoids making them more similar to native human samples. These structural changes were reflected transcriptionally, where a closer correlation to human tissue was observed with the application of strain. When testing function, Applicant also saw a positive effect of strain in the tHIO. Increasing trends in TER coupled with increases in tight junction protein transcripts suggest that barrier function increased as a result of strain. Applicant also observed an increase in overall muscle activity and tone indicating that strain promoted not only muscle growth, but strength as well. This dual approach to HIO generation seemed an improvement over the singular approach that is common in the field.
While the present study has not clearly elucidated the specific mechanism of mechanotransduction in the tHIO+S system that results in maturation, there is evidence that the TGFβ and MAPK pathways contribute as they were both enriched for transcriptionally. Protein analysis revealed an increase in TGFβ signaling activity as marked by an overall elevation in protein phosphorylation across the board, while the data was less convincing for MAPK signaling. However, with further investigation of pJUN, which is a transcription factor downstream in both MAPK and TGFβ signaling, expression was observed to be visually increased with strain by immunohistochemistry. Furthermore, pJUN has been described as one of the transcription factors controlling responses to mechanical strain in mechanotransduction pathways25, making these pathways worthy candidates for investigation.
Vertebrate gut development and organogenesis involves not only specification and growth, which have been well described, but also mechanical processes26-30. Briefly, the gastrointestinal tract and its adjacent organs initially develop from an endodermal sheet, which folds to form a midline tube along the anterior-posterior axis of the embryo which can be divided into the foregut, midgut and hindgut regions28. Following tube formation in humans is a significant elongation process which results in a hairpin fold, subsequent rotations and looping of the gut in the human embryo31. Evidence in animal models suggests that strain between the gut tube and mesentery, caused by differential growth rates, influences loop formation13. When this interaction is disrupted by detaching a portion of the mesentery from the gut tube looping fails to occur in that area of the gut13. Furthermore, the development and growth of the gut's smooth muscle layers have been shown to influence the epithelial architecture within the gut tube. Prior to the presence of smooth muscle, the luminal surface of the gut is smooth. It is with the development of the underlying circular muscle layer that longitudinal ridges and buckling of the epithelium occurs9,11. These ridges go on to form zigzags, in chick, which ultimately resolve into villi with further growth of the longitudinal muscle layer9,11. The growth of these muscle layers provides a compressive force limiting any outward development driving buckling of the luminal surface11. These changes in geometry have been successfully modeled mathematically and found to hold true across several vertebrate species with slight variations in progression11.
Not only does the gut experience an array of mechanical cues and forces during embryonic and fetal development, but is also undergoing mechanical manipulations postnatally. On a daily basis during the digestive process, the gut is experiencing internal and external forces. Both segmentation and peristalsis result in physical forces. Segmentation, attributed to focused coordinated contractions of the surrounding circular muscle, results in compressive force32. Peristaltic contractions proximal to the food bolus coupled with distal relaxation are then responsible for its forward movement, of which generates an internal sheer force and radial pressure on the epithelium32, 33. Furthermore, it has been shown that an absence of mechano-luminal forces impact the gut homeostasis in humans34. It is logical to believe the mechanical forces of digestion generated by the smooth muscle in coordination with the enteric nervous system play a role in the homeostasis of gut architecture, though this interplay has not been well described34-36.
With the advent of microfluidic devices, like the various “on-a-chip” bioreactors, Applicant is able to both model and modulate specific mechanical forces present in native tissues37,38. This new category of devices may serve as a basis for a more developmental approach in constructing what Applicant might term as organogenesis chips, as they grow to include the biological, mechanical, and electrical facets required. Perhaps the well-described patterning protocols for deriving various organoids can be translated within a context of dynamic forces3,39-41. Applicant would speculate that as the understanding of mimicry of natural developmental processes improves, the tissues yielded will better approximate native ones.
These experiments support the importance of creating interdisciplinary marriages between the biological and mechanical developmental processes in tissue engineering. The present study provides a platform which may be translated to bigger animal models to engineer more mature, larger scale human intestinal tissue segments, with the hopeful application of transplantation.
Methods
Human Tissue
Human tissue collection was performed with the prior approval of Cincinnati Children's Hospital Medical Center's (CCHMC) Institutional Review Board (Tissue Characterization, Study No. 2014-0427). Surgical samples of pathologically normal adult human jejunum were obtained from patients undergoing bariatric procedures between the ages of 16 and 25 years old. Informed consent or assent was obtained from all patients and/or parent/legal guardians as appropriate. Pathologically normal infant jejunum surgical samples and/or paraffin sections of ages three months and younger were obtained as de-identified samples from the Cincinnati Biobank, part of Cincinnati Children's Research Foundation as well as the Better Outcomes for Children Biorepository.
Animals
Adult immune-deficient NOD-SCID IL-2Rγnull (NSG) mice with ages between day of life 56 and 84 were used in all experiments (Comprehensive Mouse and Cancer Core Facility, Cincinnati, Ohio). Mice were housed in CCHMC's pathogen-free animal vivarium and handled humanely in accordance with the NIH Guide for the Care and Use of Laboratory Animals. NSG mice were fed antibiotic chow (275 p.p.m. Sulfamethoxazole and 1,365 p.p.m. Trimethoprim; Test Diet). Both food and water were provided ad libitum before and after surgeries. All experiments were performed with the prior approval of CCHMC's Institutional Animal Care and Use Committee (Signaling Pathways associated with Intestinal Stem Cell Expansion, Protocol No. 2016-0014).
Generation of Human Intestinal Organoids
Human Intestinal Organoids (HIOs) were generated and maintained as previously described2-4. Briefly, line H1 embryonic stem cells (WiCell Research Institute, Inc.) were grown in feeder-free conditions in Matrigel (BD Biosciences) coated six-well Nunclon surface plates (Nunc) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), cells were passaged with Accutase (Stem Cell Technologies) and plated at a density of 65,000 cells per well in 24-well Nunc plates. Cells were allowed to grow in mTESR1 media for two days before treatment with 100 ng/ml of Activin A for three days as previously described. DE was then treated with hindgut induction medium (RPMI 1640, 100×NEAA, 2% dFCS), for four days with 100 ng/ml FGF4 (R&D) and 3 μM Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Growth Factor Reduced (GFR) Matrigel and maintained in intestinal growth medium (Advanced DMEM/F-12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng/ml EGF (R&D) to generate human intestinal organoids (HIOs). Media was changed twice weekly thereafter. HIOs were replated in fresh Matrigel every 14 days. HIOs were utilized for surgical transplantation between days 28 and 36.
Generation of Nitinol Springs
Nickel titanium (nitinol, NiTi) springs were formed as previously described20. Briefly, NiTi wires of diameter 0.152 mm were wrapped around a mandrel and heat set in order to impart the spring's desired geometry (Nitinol Devices & Components). The resulting springs were then cut down to have a relaxed length of approximately 12 mm. Springs were compressed to half their relaxed length prior to implantation and placed within a gelatin capsule (Torpac, Inc.) which was subsequently double coated with C-A-P (Eastman Chemical Company) as previously described14.
Transplantation of Human Intestinal Organoids and Implantation of Springs
HIOs were prepared for transplantation as previously described4, 5, 42. Briefly, single matrigel embedded HIOs were transplanted into the mesentery of the mice at the most distal arcade before the ileocecal junction. Mice were anesthetized with 2% inhaled isoflurane (Butler Schein), and the abdomen shaved and prepped in sterile fashion using isopropyl alcohol and povidine-iodine. A 2 cm midline incision was made and approximately 4 cm of the intestines pulled out. A small pocket was created in the mesentery and the HIO placed within. The abdominal cavity was irrigated with normal saline with Zosyn (2 mg/ml; Pfizer Inc.) and the intestine placed back within the abdominal cavity. The abdominal wall muscles and skin were then closed in a double layer fashion and the mice were given a subcutaneous injection of Buprenex (0.05 mg/kg; Midwest Veterinary Supply) for pain management. Ten to twelve weeks following engraftment, the mice then underwent a secondary surgery with similar preparations. During this procedure, the tHIO was incised along a length of approximately 3 mm using Vannas scissors to gain access to the lumen. Directionality of the incision varied between tHIOs based on their shape, vascularization and proximity to the mouse's bowel. Any mucous plugs were manually removed. Then, using an 18 G blunt tip fill needle positioned at the incision of the lumen, the tHIO was irrigated with normal saline. Care was taken during this process to avoid damaging the epithelium. With the luminal space cleared, the gelatin capsule was placed within the lumen of the tHIO to allow for spring deployment parallel to the adjacent mouse intestine. This was done to reduce the risk of obstruction. The tHIO was then closed using a 9-0 silk suture in a simple interrupted fashion. Sham operated mice underwent the same aforementioned procedure except that the C-A-P coated, gelatin capsule was empty, without spring. Mice were sacrificed and tissue harvested 14 d postoperatively.
MicroCT Imaging System
An IVIS Spectrum (PerkinElmer) coupled to a computer with Living Image software (Caliper Life Sciences) was used in the acquisition of microCT images. Briefly, live mice were anesthetized with 2% inhaled isoflurane prior to and during imaging which was used to monitor post-operative spring deployment.
Tissue Processing, Immunohistochemistry, and Light Microscopy
Segments of human jejunum, tHIO and tHIO+S grafts were fixed overnight in 4% paraformaldehyde (PFA), processed and embedded in paraffin. Sections of 5 μm thickness were deparaffinized and either stained immediately with a histological kit (Pentachrome, Newcomer Supply) or subjected to antigen retrieval, and stained. For both primary and secondary antibodies, incubations took place at 4° C. overnight in 1% bovine serum albumin in phosphate buffered saline (PBS). Antibodies, their respective dilutions are listed in Table 1. The Vectastain ABC system (Vector Laboratories) was used for amplification. The following kits were used for signal detection: diaminobenzidine substrate kit, Immpact SG substrate kit, and Vector Red substrate kit (Vector Laboratories). Lillie-Mayer's Hematoxylin (Dako North America, Inc.) or Nuclear Fast Red (PolySciences, Inc.) was used as a counterstain. Images were acquired using a Nikon Eclipse Ti microscope and analyzed using Nikon Elements Imaging Software (Nikon).
Electron Microscopy
Scanning Electron Microscopy. Segments of human jejunum, tHIO and tHIO+S grafts were fixed overnight in 3% gluteraldehyde in 0.175 M sodium cacodylate buffer with pH 7.4. Samples were then buffer rinsed and post fixed in 1% osmium tetroxide in 0.175 M cacodylate buffer for 1 hour at 4° C. After another buffer rinse, samples were put through a graded ethanol series (25, 50, 75, 95, 3×100%) for dehydration. Specimens were then critical point dried in a Leica EM CPD300, stub-mounted and sputter-coated 10 nm thick with 60/40 gold palladium using a Leica EM ACE600. A Hitachi SU8010 transmission electron microscope was used to image samples.
Transmission Electron Microscopy. Segments of human jejunum, tHIO and tHIO+S grafts were fixed and dehydrated in the same fashion as those for scanning electron microscopy. Infiltration was performed with 2× propylene oxide followed by a graded infiltration with LX-112. Samples were polymerized into blocks overnight at 37° C. and subsequently kept at 60° C. for 3 days. Thicks were sectioned and stained with Toluidine Blue to verify tissue orientation. Blocks were then sectioned at 10 nm, skipping 10 μm between grids. Sections were stained with urinyl acetate and lead citrate before imaging with a Hitachi H7600 transmission electron microscope.
Morphometric Analysis, Cell Type Quantification and Statistical Analysis
Morphometric analysis was performed on hematoxylin and eosin stained tissue sections. Crypt depth, crypt width, villus height, villus width, and mucosal thickness were measured for a minimum of 20 well-oriented crypt-villus units per tissue sample and then averaged using Nikon NIS imaging software (Nikon). Microvilli were measured using ImageJ; at least 150 microvilli per sample across three different grids of at least 10 μm apart were quantified. Profiles of proliferation by position within the crypt were determined using dual MKI67 and CDH1 immunostaining. A minimum of 10 intact crypts were analyzed per sample and averaged. For quantification of cell types along the crypt-villus axis the following were utilized: CHGA immunostaining for quantification of endoendocrine cells, Alcian Blue pH 2.5 staining for goblet cells, LYZ for Paneth cells. For semi-quantitative analysis of the stem cell compartment OLFM4 immunostaining was used. In each case a minimum of 20 well-oriented crypt-villus units per tissue sample were quantified and averaged. All data are represented as mean±standard deviation. For analysis of proliferative profiles, Gaussian curves were fit to each data set and a comparison of fits was performed using Prism software (GraphPad). Two-way ANOVAs and post-hoc Tukey's tests were completed using Prism software as appropriate. The statistical significance cutoff was p<0.05 and is further denoted as follows: *=0.01<p<0.05, **=0.001<p<0.01, ***=p<0.001, and ****=p<0.0001.
Ex Vivo Epithelial Permeability
The epitheliums of freshly harvested tHIO, tHIO+S grafts and human surgical samples were carefully dissected through a technique similar to seromuscular stripping23, 43. tHIOs and fresh surgical samples were opened longitudinally. All further dissection was done in ice cold Kreb's buffer (NaCl, 117 mM; KCl, 4.7 mM; MgCl2, 1.2 mM; NaH2PO4, 1.2 mM; NaHCO3, 25 mM; CaCl2, 2.5 mM and glucose, 11 mM). Tissue segments were then pinned in a dish containing 0.5 cm thick cured Sylgard (Electron Microscopy Sciences). Unlike mouse tissue, the seromusculature layers (serosa, longitudinal, and circular smooth muscle) are relatively thick in both tHIOs and human jejunum. These layers are then micro-dissected as one unit from the epithelium using Dumont #5 and #7 forceps along with Vannas scissors taking care to only handle the edges of the tissue (Fine Science Tools, Inc.). The layers are gently separated and cut in small increments. After separation, gross tissue integrity was assessed using the stereoscope's bottom lighting for evenness and uniformity in appearance. The edges of the tissue, experiencing the majority of manipulation, were discarded along with any portions of the epithelium that appeared damaged. The dissection set up can be found in
Ex Vivo Muscle Strength
tHIO and tHIO+S grafts were harvested and placed in ice-cold Hank's Balanced Salt Solution (HBSS). Human surgical samples were maintained overnight at 4° C. in HBSS prior to assaying in order to minimize the effects of anesthetics. Muscle strips (4-6 mm in length and 1-2 mm in width) were dissected from the samples. Strips were then suspended vertically in an organ bath chamber (Radnoti) filled with freshly prepared Krebs-Ringer solution (Sigma; supplemented with 2.5 mM CaCl and 15 mM NaHCO3; pH 7.4), warmed to 37° C. and gassed with 95% O2+5% CO2. Segments were allowed to equilibrate for an hour at an initial tension of 0.5 g. The contractile response of the muscle was continuously recorded, using 4-chamber tissue-organ bath chambers with isometric force transducers (0-25 g; AD Instruments) coupled to a multi-channel bridge amplifiers and data recorder (AD Instruments; PowerLab 4/35) linked to a computer equipped with LabChart Pro software (AD Instruments). A logarithmic dose response to Carbamyl-p-methylcholine chloride (Bethanechol; Sigma-Aldrich) was obtained through the administration of exponential doses with concentrations of 1 nM to 10 mM at 2 minute intervals before the administration of 10 μM scopolamine (Tocris Bioscience).
RNA Isolation and Sequencing
RNA was extracted using an RNeasy Plus Micro Kit (Qiagen) according to manufacturer guidelines. Samples were then quantified and submitted to CCHMC's DNA Sequencing and Genotyping core for Next Generation Sequencing. All samples were assayed to have RNA integrity numbers greater than eight. After quality control, a cDNA library was created and sequenced using an IlluminaHiSeq2000 (Illumina) with 20 million paired-end reads per sample.
RNA Sequencing Bioinformatics
Pre-Processing
Data pre-processing was performed in Galaxy public server (https://usegalaxy.org). fastq datasets were encoded in Phred+33 to fastqsanger under the attributes for each dataset (FASTQ Groomer, Galaxy v1.0.4). Read quality was assessed using FastQC (Galaxy, v0.67). Adapter and low quality (<20) sequences were removed using Trimmomatic (SLIDINGWINDOW) (Galaxy v0.32.3). Reads were mapped against the human genome GRCh37Hg19 using Tophat2/Bowtie2 with default settings (Galaxy v2.1.0). The number of reads uniquely mapping to each gene was counted using feature Counts (Galaxy v1.4.6.p5) using the Illumina iGenomes (http://support.illumina.com/sequencing/sequencing_software/igenome.ilmn) annotation file. Alternatively, each sample was independently processed with Cufflinks44 in order to generate an initial transcriptome. Applicant used the Cuffmerge tool to merge the private transcriptomes into a single reference, and at the same time annotated known genes and extended partial transcripts45. This common transcriptome was used in a second pass with Cufflinks to quantify each transcript and gene (known or novel) in each sample46. The reference annotation used was based on the UCSC knownGenes table47.
Analysis of RNA-Seq Data
RNA-seq analysis was performed using Strand NGS 2.9 software (Strand Life Sciences). Count tables generated in Galaxy were processed and normalized using the DESeq2 package48 within the Strand NGS 2.9 R console. Applicant performed a PCA using Strand NGS 2.9 software. From the PCA, the gene with the highest loadings for each principal component were extracted and plotted using the standalone hi_loadings function in pcaExplorer package. A functional enrichment analysis using the limmaquickpca2go routine provided by the limma package was performed on the 10000 genes with the highest loadings for each principal component. ANOVA and moderated t-Test were used to perform statistical analysis amongst samples and between groups. Functional enrichment analysis was performed in the ToppGene suite (https://toppgene.cchmc.org). Plots were generated using ggplot2 v2.2.1 and GOplot v1.02 in Rstudio v1.0.14.
Statistics on FPKM Data
For statistics on normalized FPKMs, either a Student T test or Mann-Whitney test was used according to data distribution. The statistical significance cutoff was p<0.05 and is further denoted as follows: *=0.01<p<0.05, **=0.001<p<0.01, ***=p<0.001, and ****=p<0.0001.
Analysis of RNA-Seq Data with Publicly Available Datasets
Publicly available datasets of fetal and adult full thickness intestinal tissues were downloaded from EBI-AE and NCBI-GEO databases using the Galaxy public server (Table 2). Datasets were selected upon RNA extraction and library preparation methods and sequencing platform (Illumina HiSeq). Only forward reads were used from paired-end files. Datasets were encoded to fastqsanger and read quality was assessed using FastQC (Galaxy, v0.67). Adapter and low quality (<20) sequences were removed using Trimmomatic (SLIDINGWINDOW). Reads were mapped against the human genome GRChb37\Hgl9 using HISAT2 with default settings (Galaxy v2.0.5.1)49. Count tables were generated in Galaxy using feature Counts using the Illumina iGenomes annotation file. The count matrix was processed in R 3.4.1 using the functions exported by the pcaExplorer package50 for a standalone usage. Briefly, the count matrix was transformed and normalized using a variance stabilizing transformation (VST) to the count data. Applicant visualized the sample PCA using the pcaplot function. The genes with the highest variance were selected to compute the PCA i.e. from 500 to 10000 genes. From the PCA, the gene with the highest loadings for each principal component were extracted and plotted using the hi_loadings function. A functional enrichment analysis using the limmaquickpca2go routine provided by the limma package was performed on the 10000 genes with the highest loadings for each principal component. The detailed analysis and R scripts are provided in the following Github repository (www.github.com\maximemahe\Poling2017). The accession number for data generated for this paper is ArrayExpress: E-MTAB-6017 and includes the transplanted HIO, with or without lengthening device, the human adult and infant data shown in
Protein Phosphorylation Arrays
C-Series phosphorylation arrays were performed according to manufacturer recommendations (RayBiotech, Inc). Briefly, protein was extracted from flash frozen full thickness tHIO and tHIO+S tissues, quantified, and normalized between samples. Protein was incubated on antibody array nitrocellulose membranes, followed by horseradish peroxidase based amplification and detection. Chemiluminescent readings were taken using a ChemiDoc MP imaging system (Bio-Rad Laboratories, Inc.) and densitometry data extracted using ImageJ software. Readings were normalized to the positive loading controls and membrane background signal subtracted.
All percentages and ratios are calculated by weight unless otherwise indicated.
All percentages and ratios are calculated based on the total composition unless otherwise indicated.
It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”
Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.
While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
This application claims priority to and benefit of 62/488,984, filed Apr. 24, 2018, entitled “Mechanically Induced Enterogenesis of Human Intestinal Organoids,” the contents of which are incorporated in its entirety for all purposes.
This invention was made with government support under P30 DK078392 and 1K99DK110414-02 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/029083 | 4/24/2018 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62488984 | Apr 2017 | US |
