WO2016138289A1 - Procédés de génération de cellules de la lignée des muscles lisses et leurs utilisations - Google Patents

Procédés de génération de cellules de la lignée des muscles lisses et leurs utilisations Download PDF

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WO2016138289A1
WO2016138289A1 PCT/US2016/019605 US2016019605W WO2016138289A1 WO 2016138289 A1 WO2016138289 A1 WO 2016138289A1 US 2016019605 W US2016019605 W US 2016019605W WO 2016138289 A1 WO2016138289 A1 WO 2016138289A1
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cells
cell
smooth muscle
subject
differentiated
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Renee A. Reijo Pera
Bertha Chen
Thomas M. Baer
Smruti PHADNIS
Yan WEN
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0661—Smooth muscle cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • Smooth muscle also called involuntary muscle and unstriated muscle, is the simplest of the three types of muscle (smooth, striated, and cardiac) . It is the muscle of the lining of the digestive tract, ducts of glands, and viscera associated with the gut. It also supplies the muscles for the genitourinary tract (including the urinary bladder) , structures of the blood vessels, connective tissues of the mucous membranes, and skin with its appendages. A typical fiber is a slender, spindle-shaped body averaging a few tenths of a millimeter in length. There is a single, centrally striated nucleus. The cytoplasm appears homogeneous.
  • the cells are arranged in bands, or bundles, with interspersed connective tissue fibers uniting them into an effective common mass. They are innervated in part by nerve fibers and in part by the contraction of adjacent muscle tissues.
  • the digestive tract particularly, demonstrates waves of contraction that pass along a band of smooth muscle.
  • the structure and function of smooth muscle cells in different organs is basically the same, but the inducing stimuli differ substantially, in order to perform individual effects in the body at individual times.
  • the glomeruli of the kidneys contain smooth muscle-like cells called mesangial cells.
  • Smooth muscle diseases, disorders, and dysfunction of smooth muscle are manifold and can be wide-spread, general diseases of the smooth muscle or may be localized, specific diseases of particular smooth muscle containing tissues. Smooth muscle diseases, disorders, and dysfunction may result in any number of different adverse symptoms including general malfunction of a smooth muscle containing organ .
  • incontinence represents the dysfunction of smooth muscle of the excretory system including urinary incontinence and fecal incontinence. Smooth muscle diseases, disorders, and dysfunction represent a significant unmet clinical challenge.
  • urinary incontinence is caused by muscle dysfunction due to a deficiency in both the skeletal and smooth muscle of the urethral sphincter.
  • U l is a major health epidemic affecting 30-50% of middle-aged and elderly women (see Lutz & Winters, 2013).
  • Surgery is the most common treatment for Ul .
  • Ul surgeries fail and the patients have no other options.
  • hMPCs human multipotent progenitor cells
  • Methods are provided for generating a homogenous population of smooth muscle progenitor cells from pluripotent progenitor cells. Methods of treating a subject having a smooth muscle defect, deficiency or disease through the use of such cells are also provided.
  • aspects of the instant disclosure relate to culturing pluripotent progenitor cells under conditions sufficient to lineage restrict the progenitor cells to smooth muscle fate and non- invasively monitoring the cells during culture, so as to allow identification of cell types based on cellular behavior parameters of cultured pluripotent progenitor cells. Some aspects of the instant disclosure further include removing and/or isolating identified cell types from a culture to allow production of a homogeneous population of cells.
  • aspects of the instant disclosure include treating an individual by administering to the individual an effective dose of homogeneous populations of lineage restricted smooth muscle precursor cells or differentiated smooth muscle cells derived from pluripotent progenitor cells.
  • aspects of the method also include administering to the individual an effective dose of a homogeneous population of lineage restricted smooth muscle precursor cells, a homogeneous population of differentiated smooth muscle cells, or a combination of homogeneous populations of smooth muscle precursor cells and differentiated smooth muscle cells.
  • Methods are also provided for treating an individual by administering to the individual an effective dose of autologously derived homogeneous populations of lineage restricted smooth muscle precursor cells or differentiated smooth muscle cells.
  • Methods are provided for generating a homogeneous population of smooth muscle precursor cells.
  • Aspects of the method include culturing pluripotent progenitor cells under conditions sufficient to expand and lineage restrict the pluripotent progenitor cells into smooth muscle precursor cells, monitoring the pluripotent stem cells during expansion and differentiation by non-invasive live imaging sufficient to identify aberrant cells based on one or more observed cellular behavior parameters, removing the aberrant cells from the culture of pluripotent progenitor cells in order to generate a homogeneous population of smooth muscle precursor cells.
  • aspects of the method also include monitoring the culture to identify the smooth muscle precursor cells and isolating the identified smooth muscle precursor cells.
  • Methods are also provided for generating a homogeneous population of differentiated smooth muscle cells.
  • Aspects of the method include culturing pluripotent progenitor cells under conditions sufficient to expand and differentiate the pluripotent progenitor cells into differentiated smooth muscle cells, monitoring the pluripotent stem cells during expansion and differentiation by non-invasive live imaging sufficient to identify aberrant cells based on one or more observed cellular behavior parameters, removing the aberrant cells from the culture of pluripotent progenitor cells in order to generate a homogeneous population of differentiated smooth muscle cells.
  • Aspects of the method also include monitoring the culture to identify the differentiated smooth muscle cells and isolating the identified differentiated smooth muscle cells.
  • Methods are provided for monitoring pluripotent stem cells during expansion, lineage restriction, and differentiation sufficient to identify particular cell types (e.g., desired cell types and undesired cell types).
  • aspects of the method include identifying particular cell types based on observed cellular behavior parameters including cluster formation, cell migration, cell velocity and direction, mitosis rate, apoptotic rate, nuclear/cytoplasm ratio, cell density, cell size, cell shape, number of neighboring cells, sequential progression in differentiation timeline and combinations thereof.
  • aspects of the method include monitoring pluripotent stem cells during expansion, lineage restriction, and differentiation sufficient to identify undesired cell types (e.g., aberrant cells) based on the observed cellular behavior parameters.
  • aspects of the method also include monitoring pluripotent stem cells during expansion, lineage restriction, and differentiation sufficient to identify desired cell types (e.g., smooth muscle precursors, differentiated smooth muscle cells, etc.) based on the observed cellular behavior parameters.
  • Methods are provided for removing and/or isolating identified particular cell types from a culture of pluripotent progenitor cells to generate a homogeneous population of a desired cell type.
  • Aspects of the method include removing aberrant cells from a culture of pluripotent progenitor cells to generate a homogeneous population of a desired cell type.
  • Aspects of the method also include isolating a desired cell type from a culture of pluripotent progenitor cells to generate a homogeneous population of a desired cell type.
  • a smooth muscle dysfunction that may be treated according to the methods described herein include but are not limited to incontinence which may include, e.g., urinary incontinence, fecal incontinence, and related dysfunctions.
  • PSCs pluripotent stem cells
  • CDM chemically defined medium
  • aspects of the methods may include wherein the culturing comprises contacting the PSCs with the first CDM for a period of one to three days and/or contacting the PSCs with the second CDM for a period of four to eight days, e.g., a period of five to seven days.
  • aspects of the methods may also include wherein the method further comprises monitoring the PSCs during the culturing by non-invasive live imaging sufficient to identify aberrant cells based on one or more observed cellular behavior parameters selected from the group consisting of cluster formation, cell migration, cell velocity and direction, mitosis rate, apoptotic rate, nuclear/cytoplasm ratio, cell density, cell size, cell shape, number of neighboring cells, sequential progression in differentiation timeline and combinations thereof.
  • aspects of the methods may also include wherein the method further comprises removing the aberrant cells from the culture in order to generate a homogeneous population of CD31 +/CD34+ smooth- muscle-cell-competent vascular progenitors.
  • aspects of the method may also include wherein the method further comprises differentiating the CD31 +/CD34+ smooth-muscle-cell- competent vascular progenitors to smooth muscle cell progenitors by culturing the CD31 +/CD34+ smooth-muscle-cell-competent vascular progenitors in a smooth muscle growth media to generate a homogeneous population of smooth muscle cell progenitors.
  • a method for treating smooth muscle dysfunction in a subject by generating homogeneous population of smooth muscle cell progenitors according to the above described methods and introducing a composition comprising smooth muscle precursor cells of the homogenous population of smooth muscle cell progenitors into the subject in an amount effective to treat the subject for the smooth muscle dysfunction.
  • the pluripotent progenitor cells are derived autologously from the subject.
  • the subject is a mammal.
  • the smooth muscle dysfunction is incontinence, including e.g., urinary incontinence or fecal incontinence.
  • the method further comprises introducing into the subject differentiated smooth muscle cells.
  • Systems for generating homogeneous populations of smooth muscle precursor cells or differentiated smooth muscle cells.
  • Systems are provided for the generation of a homogeneous population of smooth muscle precursor cells, a homogeneous population of differentiated smooth muscle cells, and combinations of homogeneous populations of smooth muscle precursor cells and differentiated smooth muscle cells.
  • aspects of the systems include a cell culture chamber configured for the expansion, lineage restriction, and/or differentiation of pluripotent progenitor cells under conditions sufficient to expand, lineage restrict, and/or differentiate pluripotent progenitor cells into smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • aspects of the systems also include an imaging component configured for non-invasive live imaging sufficient to identify particular cell types (e.g., desired cell types and undesired cell types) based on observed cellular behavior parameters.
  • aspects of the systems also include a cell removal component configured to remove the identified particular cell types.
  • Systems include an imaging component configured to monitor (e.g., detect, measure, etc.) cellular behavior parameters of cultured pluripotent stem cells during expansion, lineage restriction, and differentiation sufficient to identify particular cell types (e.g., desired cell types and undesired cell types).
  • aspects of the system include configurations sufficient to identify particular cell types based on observed cellular behavior parameters including cluster formation, cell migration, cell velocity and direction, mitosis rate, apoptotic rate, nuclear/cytoplasm ratio, cell density, cell size, cell shape, number of neighboring cells, sequential progression in differentiation timeline and combinations thereof.
  • aspects of the system include an imaging component configured to monitor (e.g., detect, measure, etc.) pluripotent stem cells during expansion, lineage restriction, and differentiation sufficient to identify undesired cell types (e.g., aberrant cells) based on the observed cellular behavior parameters.
  • aspects of the system include an imaging component configured to monitor (e.g., detect, measure, etc.) pluripotent stem cells during expansion, lineage restriction, and differentiation sufficient to identify desired cell types (e.g., smooth muscle precursors, differentiated smooth muscle cells, etc.) based on the observed cellular behavior parameters.
  • compositions and kits for practicing the methods and/or for use with the systems of the disclosure are also provided.
  • a subject composition includes agents and/or solutions for culturing pluripotent progenitor cells.
  • a subject composition includes agents and/or solutions for lineage restricting cultured pluripotent progenitor cells to smooth muscle precursor cells.
  • a subject composition includes agents and/or solutions for differentiating cultured pluripotent progenitor cells to differentiated smooth muscle cells.
  • a subject kit includes one or more compositions as described herein and a system for generating homogeneous populations of smooth muscle precursor cells or differentiated smooth muscle cells.
  • a subject kit includes cells (e.g., pluripotent precursor cells) or a device and/or instructions for obtaining a sample of cells from a subject and one or more compositions and/or systems as described herein.
  • a subject kit includes one or more cell culture devices (e.g., cell culture arrays) or components and instructions for producing one or more cell culture devices useful in practicing the methods and/or for use with the systems of the disclosure.
  • Figures 1A-1 H depict the characterization of cells based on dynamic properties,
  • hESCs human embryonic stem cells
  • FIG. 1A-1 H depict the characterization of cells based on dynamic properties
  • Percent cell survival for first 24hrs for cells seeded at high and low densities (n 6 fields of view with around 800 cells each in high density and 25-30 cells at low density)
  • Figures 2A-2I depict the influence of neighbors on isolated hESCs.
  • Figures 3A-3C depict the dynamics during early differentiation of hESCs.
  • (b) Rather than aggregating to form a tight colony, the cells accumulated cytoplasm, thereby increasing cell surface area (n 15). Starting from a single cell, two daughter cells were traced for about 96 hours in medium supporting pluripotency.
  • (iii) Representative lineage plot of hESCs exposed to differentiation medium indicates lengthier cell cycle as compared to the pluripotent cells.
  • FIG. 4 depicts a Flow Diagram for Cell Moment Tracker (CMT).
  • CMT Cell Moment Tracker
  • the overall purpose of CMT is to act as a user interface to analyze large time-lapse data sets of cells.
  • CMT can save prior work or export for post processing, accurately track cells, and compute information about cell behaviors, including, e.g. , cell shape and migration patterns, cluster formation, morphology of cells, and the like.
  • Figures 5A-5F depicts microfluidic probe cell extraction.
  • the probe uses two capillaries with an inner diameter of 100 urn. One capillary injects fluid (enzyme) and the other withdraws it. When the injection to withdrawal ratio is 1 :7 or more and the probe is less than 100 urn above the surface, hydrodynamic forces form a laminar flow, thus preventing the injected fluid from mixing with the media. Once the fluid injection is stopped it can be completely removed from the dish with minimal contamination. This allows the formation of a zone of influence next to the probe where the injected fluid is present (indicated by the green area).
  • (F) Preliminary RT-qPCR analysis showing average expression of certain genes in extracted cells. Type 1 cells seem to cluster well cells in a colony (C) and also with Type 2 cells. Type 3 cells show a distinct separation. Gene expression normalized to GAPDH, CENTB3, EEF1a and CNNTB1. Data represent mean ⁇ SEM (n 2 technical replicates) .
  • FIGS 6A-6D depict the dynamic properties of iPSCs with karyotypic abnormality (HUF43 c5, Trisomy 12).
  • A Single cells derived from HUF43 iPSC clone 5.
  • B HUF43 clone 5 shows trisomy of chromosome 12.
  • D Overall HUF43 c5 cells are (mean difference in % and p- value) smaller in area than H9 single cells even after forming colonies.
  • Figure 7 depicts the uniform differentiation of stenciled controlled cell culture.
  • Figure 8 depicts results obtained using the described common-path quantitative phase imaging (QPI) system.
  • Raw image is a hologram (Left) in which the fringes bend with 3D shape and density (Left inset) .
  • the data is extracted using Fourier analysis to yield a phase map (right).
  • Figure 9 depicts cell segmentation using QPI and the described cell segmentation and tracking software.
  • the software finds cell boundaries (red) and an algorithm creates "cutting lines" (blue) for more accuracy.
  • Figure 10 demonstrates the use of QPI biomarkers.
  • Figure 11 depicts the use of pattern recognition via automated texture analysis for identification and laser microdissection.
  • Laser microdissection using fluorescent markers yields 90% purity (Left).
  • Texture analysis identifies areas of different cell types, PSCs in red and differentiating cells in green (Right).
  • Figures 12A-12B depict the improvement of urethral sphincter function following treatment of urinary incontinence rats with pluripotent stem cell derived smooth muscle precursors (P-SMC).
  • P-SMC pluripotent stem cell derived smooth muscle precursors
  • A Urethral sphincter function was measured 5 weeks after treatment. P-SMC treated animals were compared to other treatment groups including treatment with adult bladder smooth muscle cells (B-SMC) and collagen (Sham-U). The collagen treatment group represents what is currently used in clinical practice for humans. This study shows that P-SMC treatment restores urethral function to the control (no treatment and no surgery) and is comparable to current treatment with collagen.
  • B A confirmatory study of the study presented in (A) without the use of collagen.
  • Figures 13A-13I depict modulation of elastin metabolism by PSMC therapy.
  • A-C Untreated controls display long, thin and well organized elastin fibers in (A) 20X and (B) 40X magnification images of H&E stained sections and (C) 2.5X magnified cross sections of rat urethra.
  • D-F Sham treated controls (collagen I) show scant, short, thin and broken elastin fibers in corresponding images, whereas (G-l) PMSC treated rats show abundant and this elastin fibers in corresponding images.
  • Figure 14 depicts the restoration of elastin expression to the level of normal controls (Pure control) in PSMC treated animals. Saline and BSMC treatment did not restore elastin expression to normal levels. PCR analysis of urethral tissues was used to measure elastin gene expression.
  • Figure 15 depicts the in vivo survival of tagged-pSMC derived from H9 or iPSCs.
  • Figure 16 depicts the in vivo survival of tagged-pSMC derived from H9 showing a decrease in signal from the tagged cells between Days 7 and 18 and a reemergence of signal at Day 35.
  • Figure 17 depicts the in vivo survival of tagged-pSMC derived from H9 showing signal at Days 49-58 (exposure time 25 min.).
  • Figure 18 depicts the in vivo survival of tagged-pSMC derived from H9 showing signal at time of sacrifice (Day 66). Injection site was dissected, frozen, and sectioned for histological and immunohistochemical analysis.
  • Figure 19 depicts the survival of nucleofection tagged-pSMC derived from H9 showing signal decay over 15 days post transplantation.
  • Figure 20 depicts H&E histological staining of the dissected hindleg skeletal muscle of a mouse injected with tagged-pSMC derived from H9.
  • Figure 21 depicts, at 10X magnification, double immunofluorescence staining of smoothelin-human nuclei in the dissected hingleg muscle of a mouse injected with tagged- pSMC derived from H9 as compared to negative control.
  • Figure 22 depicts, at 20X magnification, double immunofluorescence staining of smoothelin-human nuclei in the dissected hingleg muscle of a mouse injected with tagged- pSMC derived from H9 as compared to negative control.
  • FIGS 23A-23D depict episomal reprogramming and characterization of iPSC lines.
  • A Schematic overview of episomal reprogramming.
  • B Reprogrammed cell lines show no expression of epsiomes after 5+ passages. Top figure shows detection of transgene oriP at 544bp. Bottom figure shows detection of trangene EBNA-1 at 666bp. Lane M 1 & M2: 100kb molecular weight ladder. Lane NC: negative water control. Lanes PC: positive epsiomal DNA control. Lane ID and CAF - representative clones from ID and CAF lines respectively.
  • C Characterization of iPSCs.
  • C1 EiPSCs show presence of pluripotency related proteins and gene expression and also have the ability to differentiate in vitro and in vivo to all three germ layers.
  • D Karyotyping of iPSC clones.
  • Figures 24A-24C depict directed differentiation of hPSCs into smooth muscle cells in chemically defined medium.
  • A Stepwise differentiation strategy of hPSCs to mature SMCs and phase contrast images of cells during differentiation.
  • CDM Chemically defined medium
  • D day
  • VEGF vascular endothelial growth factor
  • FACS fluorescence-activated cell sorting
  • hPSC human pluripotent stem cell
  • PDGF-BB platelet-derived growth factor-BB
  • SMC smooth muscle cell
  • TGFbl transforming growth factor b1
  • SMGS Smooth Muscle Growth Supplement
  • SMDS Smooth Muscle Differentiation Supplement.
  • B Time course analysis to quantify highest percentage of CD34+/ CD31 + vascular progenitor cells.
  • C Efficiency of differentiation across various hPSC lines on D8.
  • Figures 25A-25D depict the characterization of pSMCs.
  • A Expression of smooth cell markers, Calponin, SM22a, desmin and aSMA in derived pSMCs in comparison with the HASMC and BDSMC line (scale bar: 25 and 50 ⁇ ).
  • B Gene expression of SMC markers in derived pSMCs versus undifferentiated hPSCs.
  • C Comparison of the highly proliferative potential of SMCs derived from H9s, tagged-H9s, and all iPSCs lines.
  • D Karyotype of SMCs derived from H9s, tagged-H9s, and all iPSCs lines.
  • FIGS 26A-26C depict functional assessment of derived SMCs.
  • Mature SMCs ubiquitously express terminal markers Elastin (green) and Myosin Heavy Chain (red) , co- stained with dapi (blue).
  • B Ca2+ influx in SMCs was detected using the AM Fluoro-4 Ca2+ indicator (C) upon exposure to carbachol, and the observed contraction (B) was quantified by % change in cell surface area. Representative fields of view are shown next to the graph, and the white arrows denote cells or groups of cells that contracted.
  • FIGS 27A-27D depict that E-cadherin expression drives the dynamic behavior of hESCs.
  • Figures 28A-28E depict that socially-networked hPSCs still retain their pluripotency in somatic cell medium, (a) Coculture of hESCs and smooth muscle cells (SMCs) in SMC growth medium. hESCs were labeled with CFSE dye (green) and mixed with SMCs at a 5: 1 ratio. White arrows indicate hESCs in clumps and red arrows indicate single hESCs.
  • Figures 29A-29C depic the Dynamic behaviors during early differentiation of hESCs to vascular progenitors,
  • Methods are provided for generating a homogenous population of smooth muscle progenitor cells from pluripotent progenitor cells and treating a subject having a smooth muscle defect, deficiency or disease through the use of such cells. Methods are also provided for treating a subject for a smooth muscle deficiency, defect, and/or disease with cellular therapy using homogenous populations of smooth muscle precursors and/or differentiated smooth muscle cells derived according to the methods described herein. Some aspects of the treatment include methods of generating homogenous populations of smooth muscle precursor cells and/or differentiated smooth muscle cells from pluripotent progenitor cells using non-invasive automated identification of cell types based on cellular behavior parameters of cultured pluripotent progenitor cells and removal and/or isolation of the identified cell types. Also provided are systems, compositions, and kits for practicing the methods of the disclosure.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s) , i.e. , arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e. , causing regression of the disease and/or symptom(s).
  • Those in need of treatment can include those already inflicted (e.g. , those with smooth muscle dysfunction or deficiency, e.g.
  • those having incontinence as well as those in which prevention is desired (e.g. , those with increased susceptibility to smooth muscle dysfunction or deficiency; those with incontinence; those suspected of having smooth muscle dysfunction or deficiency or incontinence; those having one or more risk factors for smooth muscle dysfunction or deficiency or incontinence, etc.) .
  • the terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans.
  • "Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, camels, etc. In some embodiments, the mammal is human.
  • pluripotent progenitor cells refer to cells that are capable of differentiating into two of more different cell types and proliferating.
  • pluripotent precursor cells include but are not limited to embryonic stem cells, blastocyst derived stem cells, fetal stem cells, induced pluripotent stem cells, ectodermal derived stem cells, endodermal derived stem cells, mesodermal derived stem cells, neural crest cells, amniotic stem cells, cord blood stem cells, adult or somatic stem cells, neural stem cells, bone marrow stem cells, bone marrow stromal stem cells, hematopoietic stem cells, lymphoid progenitor cell, myeloid progenitor cell, mesenchymal stem cells, epithelial stem cells, adipose derived stem cells, skeletal muscle stem cells, muscle satellite cells, side population cells, intestinal stem cells, pancreatic stem cells, liver stem cells, hepatocyte stem cells, endothelial progenitor cells, hemangioblasts, gonadal stem cells, germline stem cells, and the like.
  • pluripotent progenitor cells may be acquired from public or commercial sources or may be newly derived.
  • pluripotent progenitor cells of the subject disclosure are those cells capable of giving rise to smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • a cell may be naturally capable of giving rise to smooth muscle precursor cells and/or differentiated smooth muscle cells or may be artificially made (e.g., reprogrammed, dedifferentiated, transdifferentiated, etc.) to be capable of giving rise to smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • naturally capable is meant that giving rise to smooth muscle precursor cells and/or differentiated smooth muscle cells represents part of the natural developmental lineage or the natural differentiation potential of the cell.
  • cells made capable of giving rise to smooth muscle precursor cells and/or differentiated smooth muscle cells artificially are generally cells that do not have such capability naturally.
  • smooth muscle precursors refers to precursor and/or progenitor cells capable of giving rise to differentiated smooth muscle and/or differentiating into smooth muscle cells either before or after engraftment into smooth muscle.
  • smooth muscle precursor may refer to any cell that is restricted to the smooth muscle lineage excluding terminally differentiated smooth muscle cells.
  • differentiated smooth muscle cells refers to smooth muscle precursor cells that have terminally differentiated and are readily identifiable as smooth muscle cells.
  • smooth muscle precursor cells are identified by the expression of one or more marker genes and/or expression of one or more marker proteins, e.g., smooth muscle markers, including but not limited to e.g., smooth muscle myosin heavy chain, smooth muscle actin, calponin, SM22, lipoma preferred partner (LPP), smoothelin-b, cpi-17, myocardin, cysteine rich protein 1 (CRP1), and the like.
  • smooth muscle markers including but not limited to e.g., smooth muscle myosin heavy chain, smooth muscle actin, calponin, SM22, lipoma preferred partner (LPP), smoothelin-b, cpi-17, myocardin, cysteine rich protein 1 (CRP1), and the like.
  • smooth muscle markers including but not limited to e.g., smooth muscle myosin heavy chain, smooth muscle actin, calponin, SM22, lipoma preferred partner (LPP), smoothelin-b, cp
  • progenitor cells and/or precursor cells may be identified, e.g., in comparison to differentiated cell types, by the expression of one or more progenitor/precursor marker genes and/or expression of one or more progenitor/precursor marker proteins, including but not limited to, e.g., pluripotency markers (including e.g., Oct-4, Sox2, Nanog, KLF4, etc.), sternness markers (including e.g., those described in Ramalho-Santos et al. (2002) Science, 298(5593):597-600, the disclosure of which is incorporated herein by reference), proliferation and cell cycle markers (e.g., Ki-67, proliferating cell nuclear antigen (PCNA), etc.), and the like.
  • pluripotency markers including e.g., Oct-4, Sox2, Nanog, KLF4, etc.
  • sternness markers including e.g., those described in Ramalho-Santos et al.
  • lineage restricted refers to a cell that is not totipotent and has limited or defined differentiation potential. By limited or defined differentiation potential it is meant that the cell is incapable of differentiating or being differentiated into one or more particular cell types without the use of methods of dedifferentiation or transdifferentiation. Linage restricted cells may or may not be proliferative and may or may not be pluripotent, as such lineage restricted cells may be progenitor or differentiated cell types.
  • a cell may be lineage restricted at any point in development or cellular differentiation and the lineage restriction of cells will vary widely. For example, a cell may be lineage restricted to the progeny of a particular germ layer and thus may be endoderm restricted, mesoderm restricted or ectoderm restricted.
  • a cell may be lineage restricted to generate the progeny of a particular tissue type or cell type, e.g. , cells may be smooth muscle progenitors restricted to smooth muscle cell types or progeny.
  • Lineage restriction of a cell may be determined in a variety of ways known to the ordinary skilled artisan including but not limited to culturing the cells under various conditions to determine lineage potential, transplantation of the cells into various environments to determine lineage potential, gene or protein expression assays including transcriptomics, and proteomic assays, and the like.
  • a cell may be determined to be lineage restricted based on the expression of one or more lineage markers, e.g. , a smooth muscle lineage marker may indicate smooth muscle lineage restriction.
  • a cell may be determined to be lineage restricted based on a cellular behavior that is characteristic of a one or more particular cell lineages, e.g. , a smooth muscle lineage cellular behavior characteristic, or the loss of cellular behavior that is characteristic that is characteristic of a one or more particular cell type, e.g. , loss of characteristic of an iPS cell or an embryonic stem cell, e.g. , loss of totipotency or a morphological feature characteristic of an iPS or of an embryonic stem cell.
  • a cellular behavior that is characteristic of a one or more particular cell lineages e.g. , a smooth muscle lineage cellular behavior characteristic
  • the loss of cellular behavior that is characteristic that is characteristic of a one or more particular cell type e.g. , loss of characteristic of an iPS cell or an embryonic stem cell, e.g. , loss of totipotency or a morphological feature characteristic of an iPS or of an embryonic stem cell.
  • population means a grouping (i.e. , a population) of two or more cells that are separated (i.e. , isolated) from other cells and/or cell groupings.
  • a 6-well culture dish can contain 6 cell populations, each population residing in an individual well.
  • the cells of a cell population can be, but need not be, clonal derivatives of one another.
  • a cell population can be derived from one individual cell. For example, if individual cells are each placed in a single well of a 6-well culture dish and each cell divides one time, then the dish will contain 6 cell populations.
  • the cells of a cell population can be, but need not be, derived from more than one cell, i.e. non- clonal.
  • the cells from which a non-clonal cell population may be derived may be related or unrelated and include but are not limited to, e.g. , cells of a particular tissue, cells of a particular sample, cells of a particular lineage, cells having a particular morphological, physical, behavioral, or other characteristic, etc.
  • a cell population can be any desired size and contain any number of cells greater than one cell.
  • a cell population can be 2 or more, 10 or more, 100 or more, 1 ,000 or more, 5,000 or more, 10 4 or more, 10 s or more, 10 6 or more, 10 7 or more, 10 8 or more, 10 9 or more, 10 10 or more, 10 11 or more, 10 12 or more, 10 13 or more, 10 14 or more, 10 15 or more, 10 16 or more, 10 17 or more, 10 18 or more, 10 19 or more, or 10 20 or more cells.
  • significant amount in this context, is meant an amount of undesired or contaminating cell types that negatively impacts the use of the isolated desired cell population.
  • the actual amount of undesired or contaminating cells that defines a significant amount will vary and depend on the particular type of undesired or contaminating cells. For example, in a population of smooth muscle precursor cells used in the treatment of a subject, a significant amount of pre-cancerous or cancer causing contaminating cell types will be small as such cells have a high capacity to negatively impact the use of the isolated desired cell population.
  • a homogenous population may refer to a highly enriched population. Levels of homogeneity will vary, as described, and may, in some instances, be greater than 60% pure, including e.g., more than 65%, more than 70% , more than 75%, more than 80% , more than 85%, more than 90%, more than 95%, more than 96%, more than 97% , . more than 98%, more than 99% , more than 99.5% , more than 99.6%, more than 99.7%, more than 99.8%, and more than 99.9%.
  • heterologous means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • a promoter removed from its native coding sequence and operatively linked to a coding sequence with which it is not naturally found linked is a heterologous promoter.
  • autologous means derived from the subject that is to be treated or is to receive the cells as a cellular transplant.
  • Autologously derived cells used in an autologous transplantation need not be unaltered and, in many instances, may be modified or used to derive progeny that are ultimately used in the transplant.
  • modified cells or cell progeny may be referred to as autologously derived cells if the modified cells or cell progeny are used in a treatment of a subject from which cells used to derive the modified cells or cell progeny were derived.
  • aspects of the disclosure include methods for lessening the symptoms of and/or ameliorating a smooth muscle cell dysfunction or deficiency in an individual using homogenous populations of smooth muscle precursors and/or homogenous populations of differentiated smooth muscle cells generated from pluripotent precursor cells. Because such methods can be used to treat an individual, such methods can also be referred to as methods of treating an individual for a smooth muscle cell dysfunction or deficiency.
  • Treatment methods described herein include therapeutic treatments, in which the subject is inflicted prior to administration, and prophylactic treatments, in which the subject is not inflicted prior to administration.
  • the subject has an increased likelihood of becoming inflicted or is suspected of having an increased likelihood of becoming inflicted (e.g., relative to a standard, e.g., relative to the average individual, e.g., a subject may have a genetic predisposition to a smooth muscle disease or deficiency and/or a family history indicating increased risk of a smooth muscle disease or deficiency), in which case the treatment can be a prophylactic treatment.
  • the individual to be treated is an individual with a smooth muscle disease or deficiency.
  • a smooth muscle disease or deficiency and “a smooth muscle cell dysfunction or deficiency” are used interchangeably and include any form of smooth muscle disease or deficiency whether acute or chronic and whether at the cellular , tissue, organ, or organism level.
  • diseases, deficiencies, and dysfunctions include but are not limited to, e.g., disease or deficiency of the smooth muscle of the excretory system, disease or deficiency of the smooth muscle of the vascular system, disease or deficiency of the smooth muscle of the respiratory system, disease or deficiency of the smooth muscle of the cardiovascular system, and the like) and symptoms resulting from smooth muscle disease or dysfunction including both medically relevant symptoms and cosmetic symptoms.
  • Smooth muscle dysfunction also includes a smooth muscle disease or deficiency as a result of any other primary condition, e.g., resulting a symptom of a smooth muscle system or tissue.
  • the individual has recently undergone treatment for a smooth muscle disease or deficiency (e.g., corrective surgery, etc.).
  • Any and all forms of a smooth muscle disease or deficiency, whether treated or untreated, or a smooth muscle disease or deficiency resulting from any primary condition, whether treated or untreated, are suitable conditions to be treated by the subject methods described herein.
  • treatment of a smooth muscle disease or deficiency may be described in specific terms, e.g., treatment of incontinence, however, such specific references are not necessarily limited to treatment of the specific disorder and may be applicable, as will be readily apparent to the ordinary skilled artisan, to treatment of other smooth muscle disease or deficiency.
  • a smooth muscle disease or dysfunction that may be treated according to the methods described herein includes but is not limited to incontinence.
  • incontinence as it refers to smooth muscle disease or dysfunction may refer to urinary incontinence (Ul), fecal incontinence (Fl) (i.e., bowel incontinence, anal incontinence, etc.), and combinations thereof.
  • individuals to be treated according to the methods describe herein include individuals with incontinence.
  • aspects of the disclosure may be described in relationship to a particular type of incontinence, the ordinary skilled artisan will readily understand wherein such aspects apply generally to incontinence, e.g., urinary incontinence and fecal incontinence.
  • the individual to be treated is an individual with Ul .
  • Ul Ul
  • "Urinary Incontinence” includes any form of U l (including, e.g. , stress Ul , urge Ul , overflow Ul , total Ul , functional Ul , anatomical Ul , trauma induced Ul , mixed Ul , and the like) and Ul as a result of any other primary condition.
  • the individual has recently undergone treatment for Ul (e.g. , Ul corrective surgery, etc.). Any and all forms of Ul , whether treated or untreated, or Ul resulting from any primary condition, whether treated or untreated, are suitable Ul conditions to be treated by the subject methods described herein.
  • Conditions that are associated with or cause Ul include but are not limited to, e.g. , cystitis, urinary tract infection (UTI) (including, e.g. , recurrent UTI , relapse UTI , reinfection, UTI , chronic UTI , uncomplicated UTI , complicated UTI , acute bladder infection, chronic bladder infection, genetic predisposed UTI , genetic predisposed bladder infection, acute cystitis, acute pyelonephritis, acute prostatitis, chronic prostatitis, UTI with gross hematuria, UTI associated with nephrolithiasis, UTI associated with neurogenic bladder, and the like) , overactive bladder, urinary stones, kidney stones, bladder stones, abnormal urinary tract shape or function, constipation, menopause, hormone dysfunction, pregnancy, childbirth, pelvic floor prolapse, aging, hysterectomy, weight grain, obesity, enlarged prostate, cancer, prostate cancer, urinary tract obstruction, neurological disorders (including, e.g.,
  • Ul may be associated with, caused by, or exacerbated by consumption or ingestion of certain foods, beverages, medications, or chemicals including but not limited to, e.g. , alcohol, alpha antagonists (e.g. , nasal decongestants, pseudoephedrine, etc.), alpha blockers (e.g. , doxazosin, prazosin, tamsulosin, terazosin, etc.), ACE inhibtors (e.g. , benazepril, captopril, etc.) , antidepressants (e.g. , amitriptyline, desipramine, nortriptyline, etc.), antihistamines (e.g.
  • alpha antagonists e.g. , nasal decongestants, pseudoephedrine, etc.
  • alpha blockers e.g. , doxazosin, prazosin, tamsulosin, terazosin, etc.
  • chlorpheniramine, diphenhydramine, etc. antipsychotics
  • antipsychotics e.g. , haloperidol, risperidone thioridazine, thiothixene, etc.
  • caffeine e.g. , coffee, cola, tea, nonprescription headache remedies, etc.
  • calcium channel blockers e.g. , diltiazem, verapamil, etc.
  • diuretics e.g. , furosemide, thiazides, etc.
  • opioids e.g. , morphine, etc.
  • sedatives e.g. , diazepam, flurazepam, lorazepam, etc. and the like.
  • a subject in need of treatment according to the methods described herein may be a subject consuming or ingesting or having been prescribed, including chronic consumption, chronic ingestion, or chronically medicated with, one or more of the foods, beverages, medications, or chemicals that cause or exacerbate U l .
  • the treatment methods described herein include the alleviation or reduction or prevention of one or more symptoms of Ul .
  • Symptoms of Ul will vary, may be infrequent, occasional, frequent, or constant, and in some cases may include but are not limited to, e.g. , leaking a small amount of urine, leaking a moderate amount of urine, leaking a large amount of urine, urine leaking caused by exerting pressure on the bladder (e.g.
  • Ul e.g., urgency Ul, idiopathic urge incontinence, etc.
  • Ul may be a result of a primary condition (e.g., overactive bladder syndrome, etc.) resulting in the subject urinating more than eight times per day or more than once at night.
  • the treatment methods described herein include the alleviation or reduction or prevention of one or more symptoms of overactive bladder syndrome (also described as in some instances as unstable bladder, spastic bladder, overactive bladder, reflex incontinence, etc.).
  • overactive bladder syndrome may be associated with Ul or may be a primary condition leading to Ul or exacerbated by Ul or may be a secondary condition.
  • symptoms of overactive bladder syndrome which the treatment methods described herein may alleviate or reduce or prevent include but are not limited to: loss of urine for no apparent reason while suddenly feeling the need or urge to urinate; inappropriate bladder contractions; nocturnal bladder emptying; bladder emptying after drinking a small amount of liquid (e.g., water); bladder emptying when hearing running water; bladder emptying when touching water; etc.
  • Overactive bladder syndrome may have a neurological and/or muscular etiology, including but not limited to e.g., damage to the nerves of the bladder or other area of the nervous system affecting the bladder and/or damage to the muscles of the bladder.
  • overactive bladder may be a result of neurological disease including but not limited to, e.g., multiple sclerosis, Parkinson's disease, Alzheimer's disease, etc.
  • overactive active bladder syndrome may be a result of stroke, cancer, (e.g., brain tumors, bladder cancer, etc.) or other injury (e.g., brain injury, spinal injury, injury to the bladder, etc.).
  • overactive bladder may be a result of injury to the bladder, including but not limited to, e.g., surgical injury to the bladder muscles.
  • the individual to be treated is an individual with Fl.
  • Fl Fecal Incontinence
  • Fl includes any form of Fl, including but not limited to, e.g., Fl caused by damage to the excretory tract by diarrhea, Fl caused by constipation, Fl caused by hemorrhoids, Fl caused by rectal prolapse, Fl caused by rectocele, Fl caused by damage to the excretory tract by surgery (e.g., rectal surgery), Fl caused by inflammatory bowel disease, Fl caused by Crohn's disease, Fl caused by trauma (e.g., childbirth), urgency Fl, Fl as a result of diabetes, anatomical Fl (e.g., Fl caused by an anatomical malformation including but not limited to congenital malformations, e.g., Spina bifida, etc.), Fl as a result of cancer (e.g., colorectal cancer, anal cancer, etc.) and Fl as a result of any other primary condition.
  • anatomical Fl e.g., Fl caused by an an
  • the individual has recently undergone treatment for Fl (e.g., Fl corrective surgery, etc.).
  • Fl e.g., Fl corrective surgery, etc.
  • Any and all forms of Fl, whether treated or untreated, or Fl resulting from any primary condition, whether treated or untreated, are suitable Fl conditions to be treated by the subject methods described herein.
  • Symptoms of Fl and methods of measuring and/or diagnosing Fl are known in the art and include, e.g. , anal manometry, anal ultra sound, MRI , defecography, sigmoidoscopy, colonoscopy, anal EMG, and those described in Whitehead et al. , 2009 Aug; 137(2):512-7, 517.e1 -2, the disclosure of which is incorporated herein by reference in its entirety.
  • the methods of treatment described herein include administering a therapeutically effective amount of a homogenous population of smooth muscle precursor cells to a subject in need thereof in order to treat the subject for a smooth muscle cell dysfunction or deficiency.
  • the smooth muscle cell dysfunction or deficiency is incontinence (e.g. , Ul or Fl) and the effective amount is a dose effective (i.e. a therapeutically effective dose) to obtain a desired pharmacologic and/or physiologic effect in the subject with incontinence including, e.g. , reducing the symptoms of U l and/or Fl .
  • the smooth muscle cell dysfunction or deficiency is overactive bladder syndrome and the effective amount is a dose effective (i.e. a therapeutically effective dose) to obtain a desired pharmacologic and/or physiologic effect in the subject with overactive bladder syndrome including, e.g. , reducing the symptoms of overactive bladder syndrome.
  • the health and physical condition of the individual to be treated e.g. , age, the taxonomic group of individual to be treated (e.g. , human, non-human primate, primate, etc.), the degree of resolution desired (e.g. , the amount of alleviation or reduction of symptoms), the formulation of the cell composition, the treating clinician's assessment of the medical situation, and other relevant factors.
  • age e.g. , human, non-human primate, primate, etc.
  • the degree of resolution desired e.g. , the amount of alleviation or reduction of symptoms
  • the formulation of the cell composition e.g. , the treating clinician's assessment of the medical situation, and other relevant factors.
  • a “therapeutically effective dose” or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e. , achieve therapeutic efficacy) or reduce, alleviate, or prevent symptoms to a desired extent as determined by the patient or the clinician.
  • a therapeutically effective dose can be administered in one or more administrations.
  • a therapeutically effective dose of cells e.g. , smooth muscle precursor cells, and the like
  • compositions e.g. , smooth muscle precursor cell and differentiated smooth muscle cell compositions
  • smooth muscle dysfunction smooth muscle deficiency, incontinence, Ul , Fl , etc.
  • smooth muscle deficiency smooth muscle deficiency
  • incontinence Ul , Fl , etc.
  • a therapeutically effective dose of cells is 1 x10 3 or more cells (e.g. , 5x10 3 or more, 1 x10 4 cells, 5x10 4 or more, 1 x10 s or more, 5x10 s or more, 1 x 10 6 or more, 2x10 6 or more, 5x10 6 or more, 1 x10 7 cells, 5x10 7 or more, 1 x10 8 or more, 5x10 8 or more, 1 x 10 9 or more, 5x10 9 or more, or 1 x10 10 or more) .
  • a therapeutically effective dose of cells is in a range of from 1 x10 3 cells to 1 x10 10 cells (e.g, from 5x10 3 cells to 1 x10 10 cells, from 1 x10 4 cells to 1 x10 10 cells, from 5x10 4 cells to 1x10 10 cells, from 1 x10 s cells to 1 x10 10 cells, from 5x10 s cells to 1 x10 10 cells, from 1 x10 6 cells to 1 x10 10 cells, from 5x10 s cells to 1 x10 10 cells, from 1 x10 7 cells to 1 x10 10 cells, from 5x10 7 cells to 1 x10 10 cells, from 1 x10 8 cells to 1 x10 10 cells, from 5x10 8 cells to 1 x10 10 , from 5x10 3 cells to 5x10 9 cells, from 1 x10 4 cells to 5x10 9 cells, from 5x10 4 cells to 5x10 9 cells, from 1 x10 s cells to 5x10 9 cells, from 1 x10
  • the concentration of cells (e.g. , smooth muscle precursor cells, etc.) to be administered is in a range of from 1 x 10 s cells/ml to 1 x 10 9 cells/ml (e.g. , from 1 x 10 s cells/ml to 1 x 10 8 cells/ml, from 5 x 10 s cells/ml to 1 x 10 8 cells/ml, from 5 x 10 s cells/ml to 5 x 10 7 cells/ml, from 1 x 10 6 cells/ml to 1 x 10 8 cells/ml, from 1 x 10 6 cells/ml to 5 x 10 7 cells/ml, from 1 x 10 6 cells/ml to 1 x 10 7 cells/ml, from 1 x 10 6 cells/ml to 6 x 10 6 cells/ml, or from 2 x 10 6 cells/ml to 8 x 10 6 cells/ml).
  • 1 x 10 s cells/ml to 1 x 10 9 cells/ml e.g
  • the concentration of cells to be administered is 1 x 1 0 s cells/ml or more (e.g. , 1 x 10 s cells/ml or more, 2 x 10 s cells/ml or more, 3 x 10 s cells/ml or more, 4 x 1 0 s cells/ml or more, 5 x 10 s cells/ml or more, 6 x 10 s cells/ml or more, 7 x 10 s cells/ml or more, 8 x 10 s cells/ml or more, 9 x 10 s cells/ml or more, 1 x 10 6 cells/ml or more, 2 x 10 6 cells/ml or more, 3 x 10 6 cells/ml or more, 4 x 10 6 cells/ml or more, 5 x 10 6 cells/ml or more, 6 x 10 6 cells/ml or more, 7 x 10 6 cells/ml or more, or 8 x 10 6 cells/ml or more).
  • an effective dose of the cells described herein may be coadministered with one or more additional agents.
  • an effective dose of smooth muscle precursor cells from a homogenous population of smooth muscle precursor cells derived according to the methods described herein may be co-administered with one or more additional agents.
  • Additional agents useful in such co-administration include agents that improve the overall effectiveness of the effective dose of cells or decrease the dose of cells necessary to achieve an effect essentially equal to administration of an effective dose of the cells without the additional agent.
  • Non-limiting examples of additional agents that may be co-administered with smooth muscle precursors derived according to the methods described herein include: differentiated smooth muscle cells, conventional agents for treating Ul , non-smooth muscle progenitor cells, pro-survival factors, pro-engraftment factors, functional mobilization agents, and the like. Additional agents useful in co-administration also include agents useful in treating or preventing conditions associated with incontinence, e.g. , Ul (e.g. , agents useful in treating or preventing UTI), such agents are described in, e.g. , Kodner & Gupton (2010) Am Fam Physician. 82(6) :638-643, the disclosure of which is incorporated herein by reference.
  • subject may be treated through co-administration of cells as described herein and a conventional treatment for a smooth muscle disorder or dysfunction including, e.g. , the administration of one or more conventional agents for treating a particular smooth muscle disorder or dysfunction.
  • agents for treating Ul agents known in the art that prevent or inhibit Ul or symptoms of U l or conditions or symptoms of conditions related to Ul including but not limited to e.g. , or anticholinergic drugs (e.g. , darifenacin, fesoterodine, oxybutynin, solifenacin, tolterodine, trospium, and the like), beta-3 adrenergic agonists (e.g.
  • tricyclic antidepressants e.g. , imipramine hydrochloride and the like
  • antispasmodic agents botox, estrogen, capsaicin, resiniferatoxin, alpha-blockers (e.g. , doxazosin mesylate, prazosin hydrochloride, terazosin hydrochloride, and the like) , and combinations thereof.
  • agents for treating Fl agents known in the art that prevent or inhibit Fl or symptoms of Fl or conditions or symptoms of conditions related to Fl including but not limited to e.g. , those medication for Ul that can also be used to treat Fl , antidiarrheal medications (e.g. , loperamide, diphenoxylate, etc.), bulk laxatives (.e.g, Citrucel, Metamucil, etc.), and the like.
  • antidiarrheal medications e.g. , loperamide, diphenoxylate, etc.
  • bulk laxatives .e.g, Citrucel, Metamucil, etc.
  • non-smooth muscle progenitor cell is meant a progenitor cell that has not been lineage restricted to the smooth muscle cell lineage or a fate-restricted cell that is of a lineage other than that of the smooth muscle cell lineage.
  • non-smooth muscle progenitor cell include other stem cells or pluripotent or totipotent progenitors including but not limited to e.g. , mesenchymal stem cells, adipose derived stem cells, hematopoietic stem cells, muscle satellite cells, embryonic stem cells, undifferentiated induced pluripotent stem (iPS) cells, and the like.
  • differentiated cell types of a non-smooth muscle cell lineage including e.g. , skeletal muscle-derived cells, immune cell types, and the like.
  • pro-survival factors a factor or agent that may be added to the medium, culture media, delivery excipient, or storage solution that promotes the survival of a desired cell type.
  • pro-survival factors may be general pro-survival factors that generally promote the survival of most cell types or may be specific pro-survival factors that only promote the survival of certain specific cell types.
  • pro-survival factors of the subject disclosure include but are not limited to, e.g., Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g., low does, i.e., sub- immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g., insulin-like growth factor-1 (IGF-1)), extra cellular matrix (ECM) components, hydrogels, matrigel, collagen, gelatin, agarose, alginate, poly(ethylene glycol), hyaluronic acid, etc.
  • ROCK Rho-associated kinase
  • pinacidil e.g., pinacidil, allopurinol, uricase
  • cyclosporine e.g., low does, i.e., sub- immunosuppressive dose, cyclosporine
  • pro-engraftment factors is meant a factor or agent that may be added to the administered dose or the delivery excipient or the cell storage solution that, upon delivery of the cells into a subject for treatment, increase the engraftment of the administered cells into the tissue targeted for engraftment and therapy.
  • pro-engraftment factors include factors that physically retain the administered cells at the delivery site, e.g., the injection site in the case of direct injection to the affected area, including but not limited to, e.g., gels, polymers, and highly viscous liquids that have physical properties that prevent the administered cells from freely diffusing.
  • gels, polymers, and highly viscous liquids include but are not limited to e.g., ECM components, hydrogels, matrigel, collagen, gelatin, agarose, alginate, poly(ethylene glycol), and the like.
  • co-administration and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits.
  • the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time.
  • the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms.
  • a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent.
  • the cells may be introduced by injection, catheter, intravenous perfusion, or the like.
  • the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time, being capable of use upon thawing. Once thawed, the cells may be expanded by use of growth factors and/or feeder cells or in feeder-free conditions associated with progenitor cell proliferation and differentiation. In some instances, the cells may be administered fresh such that the cells are expanded and differentiated and administer without being frozen.
  • the cells e.g. , smooth muscle precursor cells, etc.
  • compositions smooth muscle precursor cell compositions
  • a pharmaceutical composition comprising an isotonic excipient or buffer or media prepared under sufficiently sterile conditions for human administration.
  • Cell Therapy Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge University Press, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister & P. Law, Churchill Livingstone, 2000.
  • composition may also comprise or be accompanied with one or more other ingredients that facilitate the engraftment or functional mobilization of the cells. Suitable ingredients include matrix proteins that support or promote adhesion of the cells, or complementary cell types.
  • Cells of the subject methods may be autologously derived.
  • autologously derived it is meant that the cells are derived from the subject that is to be treated with the cells.
  • the cells may be derived from a tissue sample obtained from the subject including but not limited to, e.g. , a blood sample (e.g. , a peripheral blood sample), a skin sample, a bone marrow sample, a urine sample, and the like.
  • the sample from which cells are derived may be a biopsy or swab, e.g.
  • a biopsy or swab collected to diagnose, monitor, or otherwise evaluate the subject e.g. , diagnose the subject for a smooth muscle dysfunction or deficiency, e.g. , incontinence or a related condition, or for cell collection.
  • the autologous sample from which the cells are derived may be a previously collected and stored sample, e.g. , a banked tissue sample, from the subject to be treated, including but not limited to e.g.
  • banked cardiac tissue or cells banked musculoskeletal tissue or cells, banked reproductive tissue or cells, banked skin tissue or cells, banked bone tissue or cells, banked bone marrow tissue or cells, banked vascular tissue or cells, banked umbilical cord blood tissue or cells, and the like.
  • cells of the subject methods may be non- autologously derived.
  • non-autologously derived it is meant that the cells are not derived from the subject that is to be treated with the cells.
  • non-autologously derived cells may be xeno-derived (i.e. , derived from a non-human animal) or allo-derived (i.e. derived from a human donor other than the subject to be treated) .
  • Non-autologously derived cells or tissue may be derived from any convenient source of cells or tissue collected by any convenient means.
  • autologously derived or non-autologously derived cells may be determined according to the discretion of the subject's clinician and may depend on, e.g. , the health, age, genetic predisposition or other physical state of the subject.
  • autologous cells may be preferred, including, e.g. , to decrease the risk or immune rejection of the transplanted cells.
  • non-autologous cells may be preferred, including, e.g. , when the subject has a genetic defect that affects smooth muscle production.
  • Methods of derivation of pluripotent progenitor cells from an autologous or non- autologous tissue useful in the methods described herein include but are not limited to, e.g. , methods of embryonic stem cell derivation and methods of induced pluripotent stem cell derivation.
  • methods as described herein may be performed using non- autologous pluripotent progenitor cells previously derived including, e.g. , those publically or available or commercially available (e.g. , from Biotime, I nc. , Alameda, CA).
  • methods as described herein may be performed using newly derived non- autologous pluripotent progenitor cells or newly derived autologous pluripotent progenitor cells including but not limited to, e.g. , newly derived embryonic stem cells (ESC) (including, e.g. , those derived under xeno-free conditions as described in, e.g. , Lei et al. (2007) Cell Research, 17:682-688) and newly derived induced pluripotent stem cells (iPS).
  • ESC embryonic stem cells
  • iPS newly derived induced pluripotent stem cells
  • pluripotent progenitor cells e.g. , iPS cells
  • pluripotent progenitor cells useful in the methods described herein are derived by reprogramming and are genetically unmodified, including e.g. , those derived by integration-free reprogramming methods, including but not limited to those described in Goh et al.
  • the pluripotent progenitor cells are derived, using methods described herein, from cells collected from the urine, e.g. , as described in Xue et al, (2013) PLoS One 5;8(8):e70573, the disclosure of which is incorporated herein by reference.
  • derivation of pluripotent progenitor cells from an autologous or non-autologous tissue may be achieved through the use of integration-free programming , e.g. , through the use of episomal reprogramming methods. Such methods may vary depending on the cell to be reprogrammed and the desired reprogrammed state of the resulting cell.
  • episomal reprogramming methods make use of one or more episomal vectors, including but not limited to, e.g. , those available from commercial vendors (e.g. , Life Technologies, Carlsbad, CA).
  • the derived or obtained pluripotent progenitor cells are prepared, dissociated, maintained and/or expanded in culture prior to being differentiated and/or lineage restricted as described herein.
  • the pluripotent progenitor cells are dissociated, e.g. , to generate a single-cell suspension.
  • the dissociation of the pluripotent progenitors is chemical, molecular (e.g. , enzyme mediated), or mechanical dissociation.
  • Methods of chemical, molecular, and/or enzyme mediated dissociation will vary and in some instances may include but are not limited to the use of, e.g. , trypsin, TrypLE ExpressTM, TrypLE SelectTM, Accutase®, StemPro® (Life Technologies, Inc. , Grand Island, NY), calcium and magnesium free media, low calcium and magnesium medium, and the like.
  • the dissociation media may further include pro-survival factors including but not limited to, e.g. , Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g. , low does, i.e. , sub-immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g. , insulin-like growth factor- 1 (IGF-1 )), Thiazovivin, etc.
  • pro-survival factors including but not limited to, e.g. , Rho-associated kinase (ROCK) inhibitor, pinacidil, allopurinol, uricase, cyclosporine (e.g. , low does, i.e. , sub-immunosuppressive dose, cyclosporine), ZVAD-fmk, pro-survival cytokines (e.g
  • methods of culturing pluripotent stem cells include xeno-free culture conditions wherein, e.g. , human cells are not cultured with any reagents derived from non-human animals.
  • methods culturing of pluripotent stem cells include feeder-free culture conditions, wherein the pluripotent stem cells are cultured under conditions that do not require feeder cells and/or in feeder cell free medium, including e.g. , commercially available feeder-free mediums, such as, e.g. , those available from STEMCELL Technologies, Inc. (Vancouver, BC).
  • methods culturing of pluripotent stem cells include culture conditions that include supplemental serum, including e.g.
  • the pluripotent progenitor cell media includes one or more pro-survival factors, e.g. , including those described herein.
  • pro-survival factors e.g. , including those described herein.
  • General methods of culturing human pluripotent progenitor cells are described in, e.g. , Freshney et al. (2007) Culture of human stem cells, Wiley-lnterscience, Hoboken, NJ and Borowski et al. (2012) Basic pluripotent stem cell culture protocols, StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook, the disclosures of which are incorporated herein by reference.
  • cells e.g. , pluripotent progenitor cells and/or cells being reprogrammed to pluripotent progenitor cells
  • Such culture conditions may, in some instances, include one or more culture agents and/or combinations of agents and culture media to serve such a purpose wherein such agents and culture media, and components thereof, include but not limited to, e.g. , basic fibroblast growth factor (bFGF), HA- 100, N2B27, PD0325901 , CHIR99021 , A-83-01 , hLIF, Essential, mTeSR, and the like.
  • bFGF basic fibroblast growth factor
  • the pluripotent progenitor cells used according to the methods described herein may be genetically unmodified.
  • genetically unmodified is meant that essentially no modification of the genome of the cells transplanted into the subject has been performed.
  • transient genetic modification is performed at some point during the derivation of the cells but essentially no genetic modification persists in the cells that are eventually transplanted into the subject (i.e. the cells are essentially indistinguishable before the transient genetic modification and after the course of the transient modification) .
  • the genome of the cells is not transiently or stably modified, e.g. , where the cells are manipulated, e.g. , pluripotent progenitors are derived or cells are transformed, without genetic modification (e.g. , modification of the nucleotide sequence of the genome) of the cells.
  • the cells used according to the methods described herein may be genetically modified.
  • genetically modified is meant that at least one nucleotide is added to, changed within, or deleted from of the genome of the cell.
  • the genetic modification may be an insertion of a heterologous sequence, e.g. , a sequence that encodes a tag, a label sequence, a reporter, a selectable marker, a gene encoding a protein from a species different from that of the host cell, etc.
  • the genetic modification corrects a defect or a mutation within the cell, e.g.
  • the genetic modification deletes or renders inoperable an endogenous gene of the host cell.
  • the genetic modification enhances an endogenous gene of the host cell.
  • the genetic modification represents a change that enhances survival, control of proliferation, and the like.
  • Cells may be genetically altered by transfection or transduction with a suitable vector, homologous recombination, or other appropriate technique, so that they express a heterologous sequence or have altered expression of an endogenous gene.
  • aspects of the disclosure include methods for generating homogeneous populations of smooth muscle precursor cells and homogeneous populations of differentiated smooth muscle cells from cultured pluripotent progenitor cells.
  • smooth muscle precursor cells and differentiated smooth muscle cells are generated through the culture of pluripotent progenitor cells in smooth muscle cell differentiation media (SMCDM), i.e., media that promotes differentiation of the progenitors into the smooth muscle cell lineage.
  • SMCDM smooth muscle cell differentiation media
  • SMCDM may vary and in some instances may include one or more smooth muscle lineage differentiation factors, including but not limited to e.g., myogenic growth factors, retinoic acid, all-trans retinoic acid (atRA), dimethyl sulfoxide (DMSO), dibutyryl-cAMP, platelet derived growth factor (PDGF), platelet derived growth factor-BB (PDGF-BB), transforming growth factor-betal (TGF-beta1), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), fetal bovine serum (FBS) (e.g., ranging from 5% to 10%, including, e.g., 5%, 7.5% and 10%), Dulbecco's modified Eagle's medium (DMEM), gelatin,
  • the methods described herein may make use of commercially available SMCDM or media supplements including but not limited to, e.g., Smooth Muscle
  • SMDS Differentiation Supplement
  • Cell Applications Inc., San Diego, CA
  • Smooth Muscle Cell Medium ScienCell Research Laboratories, Carlsbad, CA
  • SMCDM media supplements, and techniques described in, e.g., Xie et al. (201 1) Arteriosclerosis, Thrombosis, and Vascular Biology, 31 : 1485-1494; Gong et al. (2009) Tissue Eng Part A, 15(2):319-30; Shoae-Hassani et al. (2013) BJU Int., 1 12(6):854-63, the disclosures of which are incorporated herein by reference.
  • lineage restriction and/or differentiation of pluripotent progenitor cells to smooth muscle lineage and/or growth of smooth muscle cell progenitors and/or terminal differentiation of smooth muscle cells may make use of chemically defined medium (CDM).
  • CDM chemically defined medium
  • the components of such CDM will vary depending on various factors including but not limited to, e.g., the desired resultant cell type following treatment with CDM and/or the starting cell type to be treated with CDM, etc.
  • Components useful in various CDM may, in some instances, include but are not limited to, e.g., RPMI 1640 + 1 mM GlutaMax, NEAA, ⁇ - Mercapoethanol, ITS, antibiotics (e.g., penicillin/streptomycin), Activin A, BMP4, bFGF, VEGF, PDGF-BB, combinations thereof and the like.
  • lineage restriction and/or differentiation of pluripotent progenitor cells to smooth muscle lineage cells may include generating cells that are competent to form smooth muscle cells, i.e., generating smooth-muscle-cell competent progenitors including e.g., smooth-muscle-cell competent vascular progenitors.
  • Smooth-muscle-cell competent progenitors include those cells that through further culture or introduction to an in vivo environment are capable of further lineage restriction and/or differentiation into smooth muscle cells and/or terminally differentiated smooth muscle.
  • Further culture of smooth- muscle-cell competent progenitors may include culture of smooth-muscle-cell competent progenitors in a smooth muscle cell differentiation medium or a cell culture medium smooth muscle cell differentiation supplement including but not limited to e.g., those described herein and those commercially available from ThermoFisher Scientific (Waltham, MA) , Cell Applications, Inc. (San Diego, CA, ), American Type Culture Collection (ATCC) (Manassas, VA), and the like.
  • the derivation of a particular cell type may include the culture of the cell in a single chemically defined medium for a designated period of time.
  • the derivation of a particular cell type may include the culture of the cell in a series of different chemically defined mediums including but not limited to e.g. , two or more different chemically defined mediums, three or more different chemically defined mediums, four or more different chemically defined mediums, etc.
  • the derivation of a particular cell type in a series of different chemically defined mediums includes at least a first culture period in a first medium and a second culture period in a second medium where the culture periods may be the same or different.
  • Useful culture periods in a particular chemically defined medium for the derivation of a particular cell type or types will vary and may range from an hour to days or weeks including but not limited to e.g. , from one hour to one week, one hour to two weeks, one hour to three weeks, one hour to two days, one hour to one day, one day to six days, one day to five days, one day to four days, one day to three days, one day to two days, two days to one week, three days to one week, four days to one week, five days to one week, six days to one week, two days to two weeks, three days to two weeks, four days to two weeks, five days to two weeks, six days to two weeks, two days to three days, three days to four days, four days to five days, five days to six days, two days to eight days, three days to nine days, four days to ten days, five days to eleven days, three days to five days, three days to six days, three days to seven days, three days to eight days, three days to nine days, four days to ten
  • tissue culture substrate and differentiated smooth muscle cells are generated with the use of a tissue culture substrate.
  • a tissue culture substrate may aid in the differentiation or lineage restriction of pluripotent progenitors, e.g., into a smooth muscle cell lineage, providing for improved differentiation or lineage restriction of the pluripotent progenitors (e.g., increased rate of differentiation of the desired cell type (i.e.
  • tissue culture substrate may aid in the removal of identified cell types.
  • tissue culture substrates facilitate the removal of individual cells or groups of cells, e.g., through the dissociation of the tissue culture substrate or a portion of the tissue culture substrate from the tissue culture vessel.
  • Any convenient method of dissociating the tissue culture substrate from the tissue culture vessel may be used, and in some instances, may be performed using a laser, e.g., through the use of laser cutting methods including, e.g., laser microdissection.
  • Dissociation of the tissue culture substrate from the tissue culture vessel may be performed manually, may be computer-assisted, or may be automated.
  • tissue culture substrates and tissue culture coatings may be three dimensional such that the cultured cells grow on and within the substrate.
  • tissue culture substrates and tissue culture coatings may be thin and essentially two dimensional such that the cultured cells grow on the substrate or coating.
  • the particular dimensions of such tissue culture substrates will vary and in some instance may, when applied to the tissue culture vessel, range from 10 to 500 microns in thickness, including but not limited to, e.g., 10 to 50, 50 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 350, 350 to 400, 400 to 450, 450 to 500, etc.
  • tissue culture substrates and/or tissue culture coatings that find use in the methods and systems of the subject disclosure include but are not limited to: matrigel, collagen matrix (e.g., 3D collagen matrix), collagen hydrogels, poly-L-lactide scaffolds, polyethylene glycol, polymer membranes (e.g., silicone polymer membranes), and the like.
  • matrigel collagen matrix (e.g., 3D collagen matrix), collagen hydrogels, poly-L-lactide scaffolds, polyethylene glycol, polymer membranes (e.g., silicone polymer membranes), and the like.
  • Three-dimensional (3D) tissue culture scaffolds may be naturally or artificially derived, in whole or in part.
  • a naturally derived 3D tissue culture scaffold may be made, in whole or in part, of a naturally derived matrix including e.g., those matrices fabricated from human, animal or plant tissue.
  • Such natural matrices potentially display biocompatibility and biological activity properties.
  • Suitable naturally derived matrices will vary and may include e.g., collagen matrices including e.g., mineralized collagen, collagen type I, collagen type II, collagen type III, collagen type IV, collagen type V, collagen type VI, collagen type VII, collagen type VIII, collagen type IX, collagen type X, collagen type XI, collagen type XII, collagen type XIII, collagen type XIV, collagen type XV, collagen type XVI, collagen type XVII, collagen type XVIII, collagen type XIX, and the like and combinations thereof.
  • Suitable naturally derived matrices also include polysaccharides matrices including e.g., alginate, a polysaccharide isolated from seaweed, matrices.
  • synthetic matrices may find use in 3D tissue culture scaffolds.
  • a variety of synthetic biodegradable polymers can be utilized to fabricate three-dimensional scaffolds. In general, these materials are utilized as structural elements in the scaffold.
  • Non- limiting exemplary synthetic matrices include nylon (polyamides), dacron (polyesters), polystyrene, polypropylene, polyacrylates, polyvinyl compounds (e.g., polyvinylchloride), polycarbonate (PVC), polytetrafluorethylene (PTFE; TEFLON), thermanox (TPX), nitrocellulose, cotton, polyglycolic acid (PGA), cat gut sutures, isolated or synthetic cellulose, isolated or synthetic gelatin, isolated or synthetic dextran, etc.
  • Such materials may be woven into a mesh, for example, to form the 3D matrix.
  • Certain materials, such as nylon, polystyrene, etc. are poor substrates for cellular attachment alone and thus may be used as a component of the scaffold to which cellular attachment is not desired or may be modified to improve their cellular attachment properties, e.g., by pre-treatment or functionalization prior to culture, where attachment is desired.
  • 3D tissue culture scaffolds may vary in degradability any may be biodegradable or may be non-biodegradability. Selection of degradability properties will depend on a number of factors including e.g., how long the matrix is need to establish desired cell characteristics, whether the matrix will be introduced into a living subject, e.g., as part of a cell
  • a scaffold whether single or multi-layered may provide nanopatterning cues, e.g., composed of collagen, to direct cellular orientation and/or differentiation influencing the functional outcome of the cultured cells.
  • a scaffold may be a single, multi-layered, e.g., bi-layered, tri-layered, etc. aligned, random, semi-random, etc.
  • a scaffold may be a decellularized tissue, including e.g., where the decellularized tissue is derived from the tissue to be repaired e.g., a bladder, a sphincter, etc.
  • 3D tissue culture scaffolds will vary and may depend on, e.g., the type of cells and/or tissue being derived, the shape of the tissue being replaced, the shape of the defect being repaired, the particular culture technique or chamber employed, the particular surgical technique used to introduce the produced cells, etc.
  • Useful 3D tissue culture scaffold shapes include but are not limited to e.g., flat/planar (including e.g., flat/planar bi-layered, flat/planar tri-layered, etc.) with a considerable thickness including but not limited to flat/planar square, flat/planar rectangle, flat/planar triangle, flat/planar polygon, flat/planar circle, flat/planar ellipse, convex square, convex rectangle, convex triangle, convex polygon, convex circle, convex ellipse, concave square, concave rectangle, concave triangle, concave polygon, concave circle, concave ellipse, rectangular solid (cuboid), cube, pyramid, truncated pyramid (e.g.
  • 3D scaffolds of either the same or different shapes, may be combined in and such combinations may vary.
  • concentric tube shaped scaffolds having different diameters may be combined, e.g. , into a multi-layered scaffold, or a tube shaped scaffold may be combined concentrically with a cylindrical scaffold, e.g. , where the inner diameter of the tube essentially conforms to the outer diameter of the cylinder to form a "circumferentially wrapped" cylinder scaffold.
  • such three dimensional scaffolds find use in the design of geometrically defined tissue culture substrates including e.g. , where the specific three- dimensional design of the tissue culture substrate significantly impacts one or more cell culture characteristics.
  • a tissue culture substrate may be geometrically designed or geometrically patterned to promote uniform cell culture characteristics, uniform cell growth characteristics, or uniform cell differentiation characteristics.
  • such geometrically designed or geometrically patterned tissue culture substrates provide for more uniform differentiation of pluripotent progenitor cells into a desired cell type, e.g. , smooth muscle precursor cells or differentiated smooth muscle cells, as compared to traditional non- patterned cell culture methods, e.g. , methods in which cells are allowed to randomly attach essentially anywhere on the entire tissue culture surface, e.g. , uncoated surface or uniformly coated surface.
  • more uniform differentiation is meant that the differentiation rate of a desired cell type from a culture of pluripotent progenitors on a patterned substrate is greater or more efficient than the differentiation rate of a desired cell type from a culture of pluripotent progenitors on a non-patterned substrate or that fewer undesired cell types are produced on the patterned substrate as compared to a non-patterned substrate of cells cultured under essentially the same conditions.
  • a geometrically patterned tissue culture substrate may be patterned into an array.
  • Such patterned arrays include one or more repeating tissue culture substrate geometric features that, as a whole, produce a plurality of identical tissue culture substrate elements arranged in a pattern.
  • the design of such patterned arrays will vary in multiple parameters including but not limited to, e.g. , the number of elements in the array, the shape of the elements in the array, the distance between the elements in the array, the density of the elements in the array, the shape of the overall array, the size of the overall array, etc.
  • the shape of the elements of subject arrays include: circular, oval, rectangular, triangular, linear, etc.
  • the number of elements in the subject arrays will vary and may range from, e.g.
  • 4 to 10 6 including but not limited to e.g. , 4 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 10 to 50, 10 to 100, 50 to 100, 20 to 200, 100 to 200, 100 to 300, 100 to 400, 100 to 500, 200 to 400, 200 to 500, 100 to 1000, 200 to 2000, 500 to 1000, 500 to 2000, 1000 to 5000, 1000 to 10 4 , 1000 to 10 s , 1000 to 10 6 , 10 4 to 10 s , 10 4 to 10 6 , 10 s to 10 6 , etc.
  • the elements of a subject array may be arranged in columns and rows.
  • the size of the overall array is configured to fit within a standard tissue culture vessel, including but not limited to, e.g. , a standard tissue culture flask, a standard tissue culture plate, a standard tissue culture plate, a standard tissue culture chamber (e.g. , the chamber of a tissue culture chamber slide), a standard tissue culture well (e.g. , a well of a standard tissue culture multi-well plate), and the like.
  • a standard tissue culture vessel including but not limited to, e.g. , a standard tissue culture flask, a standard tissue culture plate, a standard tissue culture plate, a standard tissue culture chamber (e.g. , the chamber of a tissue culture chamber slide), a standard tissue culture well (e.g. , a well of a standard tissue culture multi-well plate), and the like.
  • Any convenient method for generating and/or patterning an array for use in the methods and systems as described herein may be employed, including but not limited to, e.g. , the production of tissue culture
  • Patterned arrays of the subject disclosure may be configured to control certain cell culture conditions including but not limited to, e.g. , the number of attached cells, the number of attached colonies, attached cell density, attached colony density, the number of nearest cell neighbors, the number of nearest colony neighbors, cell shape, colony shape, cell migration, and the like.
  • the array may be produced and/or patterned as described in, e.g. , Myers et al. (2013) Integr. Biol. , 5: 1495, the disclosure of which is incorporated herein by reference.
  • the patterning of a tissue culture substrate of the subject disclosure provides for culture conditions the promote the uniform differentiation of a desired cell type, e.g. , smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • a desired cell type e.g. , smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • the patterning of a tissue culture substrate of the subject disclosure provides for cell density that promotes the uniform differentiation of smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • the cell density of the plated pluripotent progenitor cells that promotes the uniform growth and differentiation of smooth muscle precursor cells and/or differentiated smooth muscle cells may vary and in some instances may range from 50 to 500,000 pluripotent progenitor cells per 35mm tissue culture plate, including but not limited to e.g.
  • the distance between elements of the patterned array that promotes the uniform growth and differentiation of smooth muscle precursor cells and/or differentiated smooth muscle cells may vary and in some instances may range from 0.1 mm to 5mm, including but not limited to e.g.
  • the shape and size of the elements of an array that promotes the uniform growth and differentiation of smooth muscle precursor cells and/or differentiated smooth muscle cells may vary and in some instances the elements may be circular and the diameter of the circular elements may range from 50 to 500 microns, including but not limited to e.g. , 50 to 75, 75 to 100, 100 to 125, 125 to 150, 150 to 175, 175 to 200, 200 to
  • the patterned array is configured such that the cell density of the plated pluripotent progenitor cells is between 50,000 to 100,000 cells per 35mm tissue culture plate.
  • the tissue culture substrate elements arrayed in a 35mm tissue culture plate are circular and 350 micron in diameter.
  • the tissue culture substrate in a 35mm tissue culture plate is 250 microns thick.
  • aspects of the disclosure include methods of making determinations about cell type by non-invasive monitoring of cellular behaviors, including e.g. , constant non-invasive live- cell monitoring.
  • Such monitoring allow for the assessment of various observed cellular behavior parameters including but not limited to, e.g. , cluster formation, cell migration , cell velocity and direction, mitosis rate, apoptotic rate, nuclear/cytoplasm ratio, cell density, cell size, cell shape, number of neighboring cells, sequential progression in differentiation timeline, and the like and combinations thereof.
  • a determination of cell type may be based upon the cluster formation behavior of a particular cell or group of cells, e.g.
  • a determination of cell type may be based upon a single observed cellular behavior parameter. In other instances, a determination of cell type may be based upon two or more observed cellular behavior parameters, including but not limited to e.g. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 different observed cellular behavior parameters. For example, in some instances a determination of cell type may be based upon observations of mitosis (e.g. , including the number of times a particular cell undergoes mitosis in a predetermined amount of time), cell velocity (e.g.
  • the parameters described herein may be initial values, e.g. , values determined at the start of analysis (i.e. values determined at "time zero"). In other instances, the parameters described herein may be values determined constantly over the entire analysis period.
  • the non-invasive methods of cell identification as described herein do not make use of cellular dyes or labels or other chemicals traditionally added to cell cultures to make assessments of cells.
  • the methods of cell identification allow for dye-free identification of desired cell types, e.g. , smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • the methods described herein and the cultures used to produce the cells used in the methods described herein are essentially free of cytoplasmic dyes and/or nucleic acid dyes and/or cell viability dyes including but not limited to, e.g. , DNA dyes, DNA intercalating dyes, vial dyes, propidium iodide, calcein, Hoechst dyes, and the like.
  • Methods of non-invasive monitoring of cellular behaviors used herein involve the collection of images of cells at constant or frequent time-points and processing of the collected images to generate data for the computation of cellular behavior parameters for individual cells, e.g. , each cell, within the culture.
  • collection of the images for non-invasive monitoring includes the use of light microscopy including but not limited to, e.g. , bright field microscopy, phase microscopy, phase contrast microscopy, quantitative phase imaging, time-lapse microscopy, differential interference contrast (DIC) microscopy, interference reflection microscopy, fluorescent microscopy, combinations thereof, and the like.
  • light microscopy including but not limited to, e.g. , bright field microscopy, phase microscopy, phase contrast microscopy, quantitative phase imaging, time-lapse microscopy, differential interference contrast (DIC) microscopy, interference reflection microscopy, fluorescent microscopy, combinations thereof, and the like.
  • DIC differential interference contrast
  • processing of the images includes computer-assisted or automated cell segmentation or determination of cell boundaries, e.g. , to generate a binary mask, and cell tracking and computation of cellular features within segmented images.
  • Methods of cell tracking and computation of cellular features may vary and in some instances include but are not limited to boundary error correction, nearest temporal neighbor cell detection, computation of cell features based on Fourier harmonics, computation of cell features based on spherical harmonics, and the like.
  • Monitoring of cellular behavior parameters may be qualitative or quantitative.
  • after the generation of cellular behavior parameter data such data is post-processed including e.g. , the generation of individual raw data or summary data for each cell or each cell's behavioral characteristics or each cell's environment characteristics (e.g.
  • Such initial, raw, processed, or post-processed data may be saved in a computer readable form on computer readable media, e.g. , on a local drive (e.g. , a hard-drive, a flash drive, etc.) , on a remote drive (e.g. a remote drive accessed via the internet) , on a disk (e.g. , a CD or DVD), etc.
  • a local drive e.g. , a hard-drive, a flash drive, etc.
  • a remote drive e.g. a remote drive accessed via the internet
  • a disk e.g. , a CD or DVD
  • Such data may be correlated and/or stored with cell location information, also stored in computer readable format, such that individual cells or populations of cells may be identified based on their behavioral parameters and their corresponding position may be referenced for post hoc re-identification of the subject cell or population of cells, e.g. , such that the cell can be further monitored or manipulated, e.g. , removed or isolated.
  • General methods for image processing including image capture, segmentation, quantification , cell tracking, etc. , are available in, e.g. , Russ JC (1999) The image processing handbook, 3 rd ed. , CRC Press, Boca Raton, FL; Chen et al. (2012) Com put Math Methods Med, 2012: 101536, the disclosures of which are incorporated herein by reference.
  • the methods of non-invasive monitoring of cellular behaviors as described herein are utilized to identify aberrant cells growing in a culture of pluripotent progenitor cells, smooth muscle precursor cells, or differentiated smooth muscle cells.
  • aberrant cells generally refers to any unwanted cell-type growing within a culture of desired cells or growing with a culture of cells to be differentiated into desired cells.
  • aberrant cells are cells that could potentially cause harm to a patient or subject if those aberrant cells were administered to the subject or patient.
  • Aberrant cells that may cause harm to a subject will vary and in some instances include but are not limited to, e.g.
  • Aberrant cells may be detected based on a measured or predetermined deviation of a particular behavioral parameter from that expected of the desired cell type or from that expected of normal cells, e.g. , of a particular cell type, or from that of the average cell of the cell culture population or a subpopulation of the cell culture. For example, in some instances one or more behavioral parameters of a particular cell may be compared to a reference standard or a control group of cells or the norms of the culture as a whole or a subpopulation of the culture to determine whether the particular cell is aberrant.
  • the behavioral characteristics of a known population of aberrant cells are measured in order to generate an aberrant reference standard to which individual cells may be compared.
  • the behavioral parameters of a known sample of karyotypically abnormal cells are measured and such measurements are used as a reference to identify karyotypically abnormal cells and karyotypically normal cells in a culture of desired cells as described herein.
  • identification of aberrant cells is automated. Non-invasive monitoring of cell culture, e.g., as described herein for the identification of particular cell-types and/or aberrant cells, etc., may be performed according to the specific cell derivation and/or purification and/or isolation method being employed and, as such, may vary.
  • non-invasive monitoring may be performed throughout the duration of a cell culture, e.g., including throughout the entire time during which desired cell types are derived including but not limited to, e.g., throughout the entire expansion of pluripotent progenitors, throughout the entire lineage restriction of smooth muscle progenitors, throughout the entire differentiation of smooth muscle progenitors, throughout the entire differentiation of smooth muscle cells, and the like.
  • desired cell types including but not limited to, e.g., throughout the entire expansion of pluripotent progenitors, throughout the entire lineage restriction of smooth muscle progenitors, throughout the entire differentiation of smooth muscle progenitors, throughout the entire differentiation of smooth muscle cells, and the like.
  • the terms "monitoring throughout” or "imaging throughout” a particular cell culture process may encompass necessary breaks in monitoring or imaging, e.g., necessitated by routine procedures of cell culture during which monitoring or imaging may not be possible or practical, e.g., during media changes or cell passaging.
  • non-invasive monitoring of cell culture may be performed for only a portion of the culture of the desired cell types or the purified cell population.
  • cells derived according to the methods as described herein may only be imaged for one or more finite periods during the culture or derivation of the cells including but not limited to, e.g., one or more time periods during the expansion of the cells, one or more time periods during the lineage restriction of the cells, one or more time periods during the differentiation of the cells and combinations thereof, etc.
  • the number of time periods during the derivation of a particular desired cell type or population of cells may vary depending on various factors including but not limited to, e.g., the desired cell type, the desired purity of the cell population, the desired amount of cells, the time necessary for culture of the desired cell type, the particular culture conditions, etc.
  • the number of imaging periods performed during the derivation of a particular cell type or during a particular phase of derivation may range from one to 20 or more, including but not limited to, e.g., 1 to 15, 1 to 10, 1 to 5, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, more than 20, etc.
  • the frequency of imaging periods during the derivation of a particular cell type or a particular cell population will vary, e.g., depending on the number of imaging periods, the length of imaging periods, etc.
  • the frequency of imaging periods may range from weekly to hourly or more, including but not limited to, e.g., weekly, twice weekly, three times a week, every two days, every other day, daily, twice daily, three times daily, four times daily, every five hours, every four hours, every three hours, every two hours, hourly, and the like.
  • the length of imaging periods will vary, e.g., depending on the particular cellular feature to be detected. For example, where a particular cellular feature to be detected may be rapidly detected a shorter imaging period may be employed and where a particular cellular feature to be detected may not be rapidly detected a longer imaging period may be employed. As such, in some instances, the length of an imaging period may range from one second or less to many hours or days and may, as previously described, last for the duration of the derivation of a particular cell type or cell population.
  • an imaging period may range from one second to 2 days or more, including but one limited to, e.g., 1 to 5 seconds, 1 to 10 seconds, 1 to 30 seconds, 1 to 60 seconds, 30 to 60 seconds, 1 to 2 minutes, 1 to 3 minutes, 1 to 4 minutes, 1 to 5 minutes, 1 to 10 minutes, 5 to 10 minutes, 10 to 20 minutes, 10 to 30 minutes, 10 to 40 minutes, 10 to 50 minutes, 10 to 60 minutes, 30 to 60 minutes, 1 hour to 2 hours, 1 hour to 3 hours, 1 hour to 4 hours, 2 hours to 6 hours, 4 hours to 8 hours, 4 hours to 12 hours, 6 hours to 12 hours, 12 hours to 24 hours, 6 hours to 24 day, 6 hours to 48 hours, 12 hours to 48 hours, 24 hours to 48 hours, and the like.
  • a cell derivation method as described herein may include one or more optimized monitoring windows, e.g., alone or as part of a monitoring schedule, during which cultured cells are imaged to detect a particular cellular feature.
  • aspects of the disclosure include methods of removing and/or isolating identified cells in order to generate a homogenous population of a desired cell type, e.g., a homogenous population of smooth muscle precursors or a homogenous population of differentiated smooth muscle cells.
  • cells identified as aberrant cells are removed from the culture individually or in groups.
  • removal of cells from the culture may include ablation, wherein the cell removed is destroyed and/or discarded, and/or non-destructive removal, wherein the cell removed is not destroyed and/or not damaged and remains viable and capable of performing cellular and physiological functions.
  • removal of aberrant cells by non-destructive means may be desired, e.g., where such cells may be further analyzed or where ablation of such cells may release undesired factors or undesired cell debris into the culture media.
  • a homogenous population of a desired cell type may be achieved through the ablation of undesired cells, e.g. , aberrant cells.
  • a homogenous population of a desired cell type may be achieved through the removal and collection (i.e. isolation) of essentially only the desired cell type.
  • microfluidic cell extraction laser cell extraction, laser cell ablation, laser microdissection, ultra-sonic cell ablation, chemical dissociation, or enzymatic dissociation, physical dissociation, etc.
  • aberrant cells are removed without disturbing the remaining culture or without disturbing desired cell types.
  • cells are removed through removal of the substrate on which the cells are adhered, e.g. , a tissue culture substrate or an element of a tissue culture substrate array as described herein.
  • aspects of the disclosure include methods of collecting a homogenous population of desired cells, e.g. , for the preparation of a cell therapy or pharmacological composition to be used in the therapeutic methods described herein.
  • collecting of a homogenous population of desired cells e.g. , a homogenous population of smooth muscle precursor cells
  • collecting of a homogenous population of desired cells is achieved by dissociating and collecting essentially all of the cells of a homogenous culture of desired cells, e.g. , a culture of cells where undesired cells have been previously removed.
  • collecting of a homogenous population of desired cells e.g. , a homogenous population of smooth muscle precursor cells, is achieved by selectively dissociating the desired cells, e.g.
  • concentrating and/or isolating the cells may be optionally employed, including but not limited to, e.g. , centrifugation, FACS sorting, magnetic cell sorting, filtering, re-culturing, and the like, and combinations thereof.
  • Systems of the subject disclosure may include a cell production system, e.g. , for the production of a homogenous population of smooth muscle precursor cells and/or a homogenous population of differentiated smooth muscle cells from pluripotent progenitor cells.
  • the cell production system includes a cell culture chamber or cell culture vessel for the culture of desired cell types.
  • Such cell culture chambers may be configured for the expansion of pluripotent progenitor cells and for the differentiation and/or lineage restriction of such pluripotent progenitor cells into desired cell types, e.g. , smooth muscle precursor cells and/or differentiated smooth muscle cells.
  • the cell culture chamber is also configured for the expansion of smooth muscle progenitor cells and/or differentiated smooth muscle cells.
  • the cell culture chamber or cell culture vessel may be an open culture system, including but not limited to e.g. , tissue culture dishes, tissue culture plates, tissue culture multi-well plates, tissue culture flasks, etc.
  • the cell culture chamber or cell culture vessel may be a closed culture system, including e.g. , a bioreactor, a stacked tissue culture vessel (e.g. , CellSTACK Culture Chambers available from Corning, Inc. Corning, NY).
  • culture media and or other factors or agents may be exchanged in and out of the cell culture chamber through the use of one or more pumps (e.g. , syringe pumps, peristaltic pumps, etc.) or gravity flow devices.
  • the culture system may allow for the sterile exchange of culture media, e.g.
  • the cell culture system may be configured to control certain environmental conditions, including but not limited to e.g. , temperature, humidity, light exposure, air composition (e.g. , oxygen levels, carbon dioxide levels, etc.) to achieve the conditions necessary for expansion and/or differentiation of desired cell types.
  • the cell culture chamber may include a cell culture vessel that includes one or more patterned cell culture substrates or one or more arrays of patterned cell culture substrates as described herein.
  • the cell culture chamber may be configured for the production of cells for clinical use, e.g. , according to current good manufacturing practice (cGMP) compliant cell culture practices, including the methods and configurations described in e.g. , Fekete et al. PLoS ONE (2012) 7(8) : e43255; Pham et al. (2014) J Trans Med 12:56; Gastens et al. (2007) Cell Transplant 16(7) :685-96; Fernandes et al. (2013) Stem Cell Bioprocessinq: For Cellular Therapy, Diagnostics and Drug Development, Burlington, Oxford: Elsevier Science:
  • cGMP current good manufacturing practice
  • the cell production system includes a non-invasive imaging component functionally integrated with the cell culture component for the monitoring of cells during expansion and/or differentiation.
  • the imaging component includes a microscope with one or more objectives in relationship to the cell culture chamber to allow for the imaging, e.g. , constant imaging, of the cultured cells by light microscopy methods.
  • the microscopy component may include one or more filters, polarizers, prisms, lenses, collimators, objectives, grates, masks, light sources, mirrors (e.g. , plane mirrors, dichroic mirrors, etc.), image capture devices (e.g. , digital cameras, CMOS cameras, CCD cameras, etc.), etc.
  • the cell production system is configured to allow for constant monitoring of the expanding and/or differentiating cells according to the methods for monitoring cells as described herein.
  • the cell culture chamber is wholly or partially optically permissive (e.g. , constructed out of optically permissive or optically clear materials and/or components including but not limited to, e.g. , glass, optically clear plastics, quartz, etc.) to allow for light microscopy based monitoring of the expanding and/or differentiating cells.
  • the cell culture chamber may be enclosed in an imaging chamber, e.g. , a chamber that essentially excludes ambient light to allow for light microscopy based imaging of the expanding and/or differentiating cells.
  • the imaging chamber may also encase one or more optical components of the microscope including but not limited to, e.g. , an objective, a light source, an image capture device (e.g. , a digital camera) , etc.
  • the microscope is entirely encased within the imaging chamber. In other instances, the microscope is partially or totally outside of the imaging chamber.
  • the cell production system includes a "data processing unit”, e.g. , any hardware and/or software combination that will perform the functions required of it, for receiving and processing the imaging data acquired by the non-invasive imaging
  • any data processing unit herein may be a programmable digital microprocessor such as available in the form of an electronic controller, mainframe, server or personal computer (desktop or portable).
  • suitable programming can be communicated from a remote location to the data processing unit, or previously saved in a computer program product (such as a portable or fixed computer readable storage medium, whether magnetic, optical or solid state device based) .
  • the cell production system may further include a "memory” that is capable of storing information such that it is accessible and retrievable at a later time or date by a computer. Any convenient data storage structure may be chosen, based on the means used to access the stored information.
  • the information may be stored in a "permanent memory” (i.e. memory that is not erased by termination of the electrical supply to a computer or processor) or "non-permanent memory".
  • Computer hard-drive, CD-ROM , floppy disk, portable flash drive and DVD are all examples of permanent memory.
  • Random Access Memory (RAM) is an example of non-permanent memory.
  • a file in permanent memory may be editable and re-writable.
  • the memory may store a "module" for execution by the data processing unit, wherein the module is configured to transform the imaging data set from an image to a set of parameters of one or more individual cells or one or more populations of cells.
  • the image data set may include one or more images of a particular cell, wherein, where multiple images are used, the images may be captured over time, e.g ., as a time-series.
  • Processing of the image data set may include the transformation of a time-lapse image data set into one or more cellular behavior parameters for the measurement, qualitative assessment, or quantitative assessment of the cell behaviors of one or more cells, e.g. , to make a determination of whether a particular cell or group of cells belong to a particular cell type.
  • the module processes the data and returns an assessment or as to whether a particular cell is of a designated cell type, including e.g. , whether a cell is of an aberrant cell type.
  • the cell production system may further include a cell removal component for the removal or isolation of assessed and/or identified cell types.
  • the cell removal component may be any appropriate device that allows for the removal of identified cells from the culture with minimal or essentially no disturbance to the other cells of the culture.
  • the cell removal component includes a device for the ablation of cells, including but not limited to, e.g. , a laser ablation device, an ultrasonic ablation device, etc.
  • the cell removal component includes a device for the removal of cells through the use of fluid flow including but not limited to, e.g. , a suction device, a capillary device, a laminar flow device, a microfluidics device, etc.
  • the cell removal component administers to the cell or cells to be removed agents that dissociate the cell or cells including but not limited to e.g. , chemical dissociation agents, enzymatic dissociation agents, and the like.
  • the cell removal component includes a cell culture substrate cutting device for the cutting of a cell culture substrate to which the cells to be removed are adhered, including but not limited to e.g. , a laser cutting device (e.g. , a laser microdissection device, etc.) , a physical cutting device (e.g. , a micro-knife, a micro-diamond knife, a blade (e.g.
  • the cell removal component includes one or more of the cell removal devises operably combined.
  • Such combination devices include, e.g. , a cell removal component configured to dissociate or cut an identified cell or cells from a culture and remove the identified cell and/or cell debris from the cell culture media following the dissociation and/or cutting.
  • a system for treating a subject with a smooth muscle cell dysfunction or deficiency includes a cell injection system for delivering cells in a carrier, with or without optional adjuvants, to a desired injection site, including diseased tissue, adjacent to diseased tissue, and/or within, on or near a dysfunctioning organ.
  • Such systems utilize known injection devices (e.g., including but not limited to needles, bent needles, cannulas, syringes, pumps, infusion devices, diffusion devices, etc.) and techniques (e.g., including but not limited to urethral injection, urethral sphincter injection, transurethral injection, periurethral injection, device-guided injection, anal injection, anal sphincter injection, internal anal sphincter injection, external anal sphincter injection, rectal injection, pelvic floor injection, etc.) which may or may not include a cystoscope or proctoscope/colonoscope.
  • injection devices e.g., including but not limited to needles, bent needles, cannulas, syringes, pumps, infusion devices, diffusion devices, etc.
  • techniques e.g., including but not limited to urethral injection, urethral sphincter injection, transurethral injection, periurethral injection
  • a device or technique used for the delivery of a urethral bulking agent or an ano-rectal bulking agent may be configured or adapted for use in a cell delivery system for use in delivering cells derived according to the methods described herein.
  • cell injection systems used in delivering cells to organs and/or tissues other than those of the urinary tract or excretory tract may be adapted or configured for delivering cells according to the methods described herein to subjects in need thereof.
  • systems of the subject disclosure may include a number of additional components, such as data output devices, e.g., monitors and/or speakers, data input devices, e.g., interface ports, keyboards, etc., fluid handling components, power sources, controllers, etc.
  • data output devices e.g., monitors and/or speakers
  • data input devices e.g., interface ports, keyboards, etc.
  • fluid handling components e.g., power sources, controllers, etc.
  • compositions and kits for use in the subject methods include any combination of components for performing the subject methods.
  • a composition can include, but is not limited to and does not require, the following: cell dissociation agents and/or media, cell reprogramming agents and/or media, pluripotent progenitor cells, cell culture agents and/or media, cell differentiation agents and/or media; lineage restriction agents and/or media; differentiated smooth muscle cells, conventional agents for treating incontinence, non-smooth muscle progenitor cells, pro-survival factors, pro-engraftment factors, functional mobilization agents and any combination thereof.
  • a kit can include, but is not limited to and does not require, the following: any of the above described composition components, a sample collection container, a sample collection device (e.g., a sample collection container that includes a sample enrichment mechanism including, e.g., a filter), a tissue collection device (e.g., a biopsy device), a tissue dissociation device, a cell culture vessel, a cell production system; and any combination thereof.
  • a kit can include, but is not limited to and does not require, a cell delivery system and/or a cell injection system configured for delivery of cells derived according to the methods described herein.
  • kits may include a cell injection system configured for injection of delivery of cells into a desired area of the subject in order to effectively treat the subject for a smooth muscle cell dysfunction or deficiency, e.g. , through delivery of cells to the urethral sphincter or anal sphincter.
  • kits may include a cell delivery or injection system, as described herein, including individual components of such systems in assembled or unassembled form.
  • cells derived according to the methods described herein may be "preloaded" into a cell injection or delivery system such that the system is provided in a "ready-to-use" configuration.
  • a cell injection or delivery system may be provided in an "unloaded” configuration such that cells derived according to the methods described herein must be loaded into the system, with any desired carrier or vehicle, prior to use.
  • the subject kits may further include (in certain embodiments) instructions for practicing the subject methods.
  • These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.
  • One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g. , a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like.
  • Yet another form of these instructions is a computer readable medium, e.g. , diskette, compact disk (CD) , flash drive, and the like, on which the information has been recorded.
  • Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.
  • RT room temperature
  • base pairs (bp) kilobases (kb); picoliters (pi); seconds (s or sec); minutes (m or min) ; hours (h or hr); days (d) ; weeks (wk or wks) ; nanoliters (nl); microliters (ul) ; milliliters (ml); liters (L) ; nanograms (ng); micrograms (ug); milligrams (mg); grams ((g), in the context of mass) ; kilograms (kg); equivalents of the force of gravity ((g), in the context of centrifugation); nanomolar (nM); micromolar (uM), millimolar (mM); molar (M); amino acids (aa); kilobases (kb); base pairs (bp); nucleotides (nt); intramuscular (i.m.); intraperitoneal (i.p.); subcutaneous (s.c); and the like.
  • Undifferentiated pluripotent stem cell lines hESC lines H9 (46, XX) and human adult- derived iPSC lines HUF43 (46, XX) were maintained in feeder free conditions with mTeSRI (Stem Cell Technologies, Vancouver, BC, Canada).
  • mTeSRI Stem Cell Technologies, Vancouver, BC, Canada.
  • colonies were pretreated with Rock inhibitor (Y-27632, Cellagen Technology) for 30mins and then exposed to Accutase ((Innovative Cell Technologies, Inc, San Diego, CA) supplemented with Rock inhibitor for 2-3mins.
  • Accutase was diluted ten-fold with Calcium - Magnesium free PBS and the suspension was gently flushed several times by 1 ml pipette tip.
  • the suspension was centrifuged at 100rpm for 5mins and the cells were counted and seeded in mTeSR supplemented with Thiazovivin (Santa Cruz Biotechnology). Cells were incubated for 30mins at 370C in 5% C02. After changing the medium to plain mTeSR, the cells were taken for imaging. Medium was changed every 24hours.
  • hESCs were first exposed to mTeSR for 24hours and then were exposed to DMEM medium (Invitrogen) supplemented with 20% fetal bovine serum (Invitrogen). Medium was changed every 24hours.
  • the Biostation IM-Q was used to image single cells isolated from hESCs and iPSCs.
  • the Biostation IM-Q is a compact cell incubation and monitoring system that maintains a stable incubation environment (37°C, 5% C0 2, 100% humidity). Forty eight fields of view were acquired every 10mins for 96 hours. Media changes were done every 24hrs using the perfusion system on the Biostation. Acquired images were processed using the CMT algorithms.
  • a microfluidic probe was built by mounting two syringe pumps (ULTRA MICRO PUMP 3, WPI Inc., USA) with a 500 ⁇ _ (for withdrawal) glass syringe (cat. # 7640-01 , Hamilton, USA) and a 250 ⁇ _ (for injection) glass syringe (cat. # 7639-01 , Hamilton, USA) onto an XYZ micromanipulator (MD4-M3-L, WPI Inc. USA).
  • the 500 ⁇ _ syringe was connected to a 100 ⁇ internal diameter fused silica capillary (TSP100170, Polymicro Technologies, UK) while the 250 ⁇ _ syringe was connected to a 50 ⁇ internal diameter fused silica capillary (TSP050150, Polymicro Technologies, UK) using needles 25 gauge needles (cat. #7732-05, Hamilton, USA) and Micro Tight Union Peek connectors (P- 720, Upchurch Scientific, USA).
  • the two fused silica capillaries were routed trough a 90% angle through a gauge 16 needle that was bent. This keeps the two capillaries into conformal contact and makes them come into the petri dish at a right angle to the media surface.
  • the probe is initially calibrated by first loading a blue food dye into the injection syringe. Then all the capillaries and tubes are primed with media. The probe is mounted on an inverted phase contrast microscope such that the two capillaries are centered with the microscope objective. A cell free medium loaded petri dish is placed on the microscope. The probe is lowered into the dish such that the capillary tips are approximately 30-100 ⁇ above the dish surface and fully submerged into media. The injection and withdrawal flow rates are adjusted until a clear area of influence is seen demarcated by the dye (see Figure 5B). The dye is removed from the syringe and accutase is loaded after the syringe is thoroughly washed with sterile di-H 2 0.
  • test dish was replaced with the cultured cell's dish.
  • the cells of interest are identified and positioned in the center of the field of view.
  • the probe is lowered into the dish and positioned right above the cell of interest, such that the cell is right below the injection capillary.
  • the injection and withdrawal flow are initiated and the cell is monitored for dislodgement. As soon as the cell is released from the dish surface the flow is stopped.
  • the injection capillary used to withdraw the loose cell by reversing the flow in the corresponding syringe pump. As soon as the cell is removed the flow is stopped.
  • the probe is removed from the pertri dish and placed into 5 ⁇ _ of 2x RT- qPCR mixture. 5 ⁇ _ is ejected from the injection capillary into the RT-qPCR mixture this would include the extracted cell.
  • the RT-qPCR reaction can be run the corresponding primers and probes for gene expression analysis.
  • CMT Cell Movement Tracker
  • phase contrast images were analyzed to find cell boundaries. From this, a binary mask, an image solely constructed out of black and white pixels, is created to differentiate regions where cells are located from the background of the image. This step is the most crucial step in CMT program because proper cell boundary detection is crucial for the collection of important data about cell morphology and migration patterns.
  • CMT also offers options to manually edit the boundaries of cells in case cell or cell-cluster morphology is highly valued in a given experiment.
  • CMT computes cell shape information such as elliptic Fourier harmonics and spherical harmonics, which can be used to model cell behavior quantitatively. Error correction tools are provided for cell tracking errors or manually detecting mitotic events.
  • CMT provides a very user-friendly interface to save old data, so that edits can be made on a given data set over a period of several days. Additionally, after computing individual cell features, the program can export the data along with some important outputs about each cell's environment (such as the number of cell neighbors, eccentricity of the cells, and minimum number of neighbors). Using the mitotic detection feature, the program can construct a generation mapping based on mitotic events over the course of a time-lapse video.
  • Trisomy of chromosome 12 is often seen with continuous culture of pluripotent stem cells and in certain cancer cells (see, e.g., Seol et al., 2008). This abnormality can be detected through certain dynamic differences between normal karyotypic cells and the one with trisomy.
  • H9s H9s
  • Cell Moment Tracker (CMT, Figure 4) Using customized semi-automated tracking software, Cell Moment Tracker (CMT, Figure 4) , it was observed that along with survival, there were distinct changes between cell cycle lengths depending upon the seeding density of the hESCs. Cells seeded at higher density, had shorter cell cycle times and hence had higher mitotic rates compared to those seeded at mid and low density ( Figure 1 b) . Furthermore, it was also observed that cells seeded at low densities extended more appendages towards neighboring cells, increasing both their cellular area and size. Cells at high density were more compact and aggregated efficiently with neighbors, thus enhancing colony formation. These behaviors distinguish pluripotent stem cells from other cell types and differentiating hESCs as further detailed below.
  • Type 1 cells show organized and directed migration pattern as compared to the Type 2 cells that display random and selection based movements (Figure 1 e, panels i and ii) and have a higher rate of velocity (Figure 1 h).
  • Type 3 cells demonstrated random migratory patterns and traveled a large distance ( Figure 1 e, panel iii) .
  • E-cadherin expression was most robust and membrane bound in Type 1 and Type 2 cells whereas in Type 3, it was cytoplasmic and indicative of poor survivability (Figure 1f).
  • Type 1 cells also showed higher mitotic index as compared to Type 2 and Type 3 cells ( Figure 1 g).
  • Quantifying behavior of individual cells is beneficial in building reliable methods to probe the fundamental biology and predict cellular responses of pluripotent stem cells and their differentiated progeny.
  • variability in internal states leads to remarkable heterogeneity in the behavior of isogenic cells exposed to a uniform stimulus (see, e.g. , Snijder et al. , 201 1 ).
  • a method for analysis that is noninvasive and able to identify subtle morphological and behavioral differences, including shape and movement pattern differences, using a time- lapse imaging system.
  • This system was adapted to screen and purify smooth muscle precursor cells and smooth muscle cells (SMCs) derived from pluripotent stem cells (PSCs) in a regenerative therapy for urinary incontinence (Ul).
  • SMCs smooth muscle precursor cells and smooth muscle cells
  • PSCs pluripotent stem cells
  • stem cells that are more pure and uniform than conventional methods are cultivated, which is achieved by controlling initial distributions of the cells.
  • the stem cells are differentiated starting with a defined spatial distribution and during this process the every cell is imaged and followed with auditable data via time-lapse quantitative imaging. Artificial intelligence is used to identify any unwanted cells and eliminate such cells with laser-based technologies.
  • the differentiated target cell population is created that is more pure than traditional methods by controlling the starting culture conditions.
  • traditional in vitro stem cell culturing colonies or single cells are placed randomly in a dish producing colonies of all different sizes and distances apart. Differentiation occurs from the edges of the colonies, and thus the speed of differentiation is different for all the cells in the colonies and the efficiency when differentiating those cells into bladder smooth muscle cells is low.
  • FIG. 7 shows such stenciling the cells are uniform at the end of the process, unlike the results when cells are randomly plated.
  • QPI Quantitative Phase Imaging
  • QPI time-lapse quantitative phase imaging
  • Time-dependent QPI data is correlated with molecular measurements of single targeted cells in vitro to determine imaging signatures that provide information about the cells in an unprecedented non-destructive and label-free method.
  • Such non-destructive and label-free molecular measurements are crucial to quality control in stem cell treatments.
  • Cellular social behavior, cell cycle time, cell velocity and direction of motion, 3D shape change over time, and nuclear-cytoplasm density ratio, a comparison of protein density in the nucleus vs. cytoplasm are uniquely tracked from QPI to distinguish PSCs from differentiated cell types and to identify abnormal or cancerous cells as demonstrated in Figure 10.
  • QPI signatures over time predict stem cell fate.
  • Effective cells are harvested and tumor-causing PSCs are eliminated by pattern recognition via automated texture analysis ( Figure 1 1) identification of the areas of wanted and unwanted cells. Unwanted and potentially dangerous cells are separated from desired cell types using laser microdissection, a method where cells are grown on a polymer membrane and a laser cuts out defined areas, identified with textures analysis, of the membrane to harvest or remove certain cell populations. This method was evaluated using fluorescent dyes ( Figure 11) and achieved 90% purity when the cells were identified with fluorescence.
  • FIGS 12A-B directly measure the efficacy of transplanted PMSCs in comparison to untreated (Pure controls, healthy animals without surgical manipulation) and sham treated (Sham-U, surgically manipulated animals not receiving cell transplantation) control rats as well as rats receiving transplants of adult bladder smooth muscle cells (B- SMC).
  • Urethral sphincter function was measured 5 weeks following treatment in two independent studies performed with ( Figure 12A) and without ( Figure 12B) collagen, which is the current human clinical equivalent. Both studies show restored urethral function to normal level in the PSMC treatment groups. The studies together also demonstrate the relatively high variability and thus low reliability of B-SMC transplantation.
  • Human smooth muscle progenitor cells generated according to the methods described herein from either H9 cells or iPS cells, were tagged by nucleofection with a luciferase reporter for in vivo tracking and survival studies in mouse. 1 .1 million tagged- human-pSMC were injected into the hindlegs of SCID mice with 50 ⁇ of a 50% matrigel delivery composition. Luminescence of tagged injected pSMC derived from either H9 or iPSC was detected following injection. At Days 0 and 3 ( Figure 15) strong signal in the injected hindleg was evident.
  • H&E staining did not detect teratoma in any of 80 sections through the transplantation site (example H&E stained sections are provided in Figure 20).
  • Dual immunofluorescence analysis for a marker of the human derived cells and the smooth muscle marker Smoothelin clearly showed the presence of the injected cells within the skeletal muscle of the dissected and sectioned mouse hindleg at 10X ( Figure 21) and 20X ( Figure 22) magnification as compared to negative control.
  • EiPSCs human induced pluripotent stem cells
  • hiPSC clones exhibited morphology similar to hESCs, were alkaline phosphatase positive and expressed pluripotency associated antigens: OCT4, SSEA4, TRA1 -60 and TRA 1 -81 , while being negative for SSEA1 (Figure. 23C).
  • the hiPSC clones were able to differentiate into cell types of all three germ layers both spontaneously in vitro by embryoid body formation and in vivo by teratoma formation (Figure. 23C-D) .
  • Gene-expression analysis showed that both undifferentiated and differentiated hiPSCs had a similar expression profile to undifferentiated and differentiated hESCs respectively ( Figure 23D).
  • hESCs and hiPSCs Differentiate to Vascular Progenitors under Xeno-Free Culture Conditions
  • H9s, luciferase-tagged H9s and HuF5 iPSC lines were differentiated to CD31 +/CD34+ vascular progenitors in xeno-free conditions. Protocols to generate CD31 +/CD34+ vascular progenitors using CDM supplemented with specific growth factors were devised.
  • Figure 24A shows a schematic of the differentiation protocol of hPSCs into vascular progenitors and, subsequently, into Smooth Muscle Cells (SMCs) .
  • SMCs Smooth Muscle Cells
  • Vascular progenitors undergo directed differentiation to SMCs
  • the homogeneous vascular progenitor populations were further differentiated to smooth muscle cells in smooth muscle growth media supplemented with PDGF-BB (10ng/ml) for 14 days (Figure 24A).
  • the cells became elongated and developed spindle- shaped, fibroblast-like morphology.
  • the expression of markers consistent with the SMC phenotype such as alpha-smooth muscle actin (aSMA) , calponin (CNN 1 ), transgelin (TAGLN 1 ), and desmin (DES) were confirmed via immunofluorescent staining (Figure 25A).
  • SMA alpha-smooth muscle actin
  • CNN 1 calponin
  • TGLN 1 transgelin
  • desmin desmin
  • hPSC-SMCs Gene expression patterns in hPSC-SMCs were similar to human aortic smooth muscle cells (HA SMCs) and bladder smooth muscle cells (BD SMCs) (Figure 25B). All generated SMC populations were karyotypically normal suggesting line stability ( Figure 25D).
  • the generated SMCs are highly expandable, proliferating for up to 8 passages in vitro. A significant fraction of these cells expressed Ki67 co-stained with TAGLN 1 , indicating that the SMCs are actively proliferating (Figure 25A) .
  • Figure 25A To compare the proliferative potential of SMCs derived from H9s, tagged-H9s, and all iPSCs lines the potential yield was calculated based on their split ratio (Figure 25C) . Starting with 1 M hPSCs, 3x10 s vascular progenitors were obtained with an efficiency of 30-40%. Furthermore, these VPCs were successfully expanded approximately 50-fold up to passage 8 ( ⁇ 50x10 6 cells) . The cells showed senescence beyond passage 8.
  • the proliferative SMCs can differentiate to mature, functional SMCs in vitro
  • Terminal differentiation was classified by expression of terminal SMC markers and the functional ability of the cells to contract. Immunoflourescent staining showed expression of two terminal SMC markers: myosin heavy-chain (MHC) and elastin (ELN), which were ubiquitously, expressed in all SMC populations (Figure 26A).
  • MHC myosin heavy-chain
  • EPN elastin
  • the functional ability of the cells was determined by observing carbachol (100 ⁇ ) induced contraction (Figure 26B).
  • H9, tagged H9, and iPSC terminal SMCs showed contraction in a tonic fashion similar to constant contraction manifested by HA SMCs. Approximately 79.39% of H9 SMCs contracted over the ten minute interval of carbachol exposure, and, of the cells that contracted, an average change in cell surface area of 19.89% was recorded (Figure 26C). In comparison, approximately 85.48% of HA SMCs contracted, with the average change in cell surface area being 20.29% ( Figure 26C).
  • Primary fibroblast cell lines were derived from patient skin biopsies obtained with written, informed patient consent and Stanford Review Board (IRB) approval.
  • the CAF line was obtained from a 50-year-old healthy female, the ID line was obtained from a 46-year-old patient, and the AM line was obtained from a 70-year-old patient.
  • the procedure for generation of iPSCs was adapted from Invitrogen (Pub#MAN0007034).
  • fibroblast cells were transfected with episomal vectors (Invitrogen) using the Neon Transfection System (Invitrogen), and plated on pre-coated vitronectin plates overnight in fibroblast media supplemented with basic fibroblast growth factor (bFGF) (Peprotech) and HA-100 (Santa Cruz).
  • bFGF basic fibroblast growth factor
  • N2B27 Invitrogen
  • bFGF fetal growth factor
  • a cocktail of small molecules consisting of PD0325901 (Stemgent), CHIR99021 (Stemgent), A-83-01 (Stemgent), HA-100 (SantaCruz), and hLIF (Life Technologies), and medium was changed every other day for 14days.
  • the media was switched to Essential8 (E8) and the cells were cultured for an additional 6-15 days for colony selection. Selected colonies were manually picked and transferred to 12- or 24-well plates pre-coated with vitronectin or matrigel for expansion in E8 or mTeSR media.
  • iPSCs were incubated with collagenase IV (200 U/ml; Gibco) at 37°C for 3-5 minutes, washed with culture medium, and scraped off the dish with a cell lifter. Colonies were further dissociated by pipetting, and split at 1 :3 ratio and transferred to a new dish. For manual passaging, individual colonies were cut using a glass tool, scraped off the plate with a cell-lifter, and transferred to a new dish. Between 5-10 passages each clone was analyzed for the presence of the episomal vectors using primers for the OriP and EBNA-1 vector regions (Invitrogen, Pub#MAN0007034).
  • Huf5 control iPSC line was previously reprogrammed by induced expression of transcription factors OCT4, KLF4, SOX2 and c-MYC using retroviral gene insertion.
  • H9 hESCs were purchased from WiCell Research Institute and luciferase-tagged H9 hESCs were acquired from Dr. Wu's laboratory at Stanford University.
  • iPSCs were dissociated using collagenase IV (200U/ml) and seeded in ultra-low attachment plates (Corning) in differentiation medium containing DMEM-F12 supplemented with 20% FBS (Gibco), 0.1 mM nonessential amino acids (Gibco), and 0.1 mM ⁇ -mercaptoethanol (Millipore). After 8 days of culturing in suspension, the EBs were transferred to gelatin-coated dish for attachment and expanded over 10-14 days. After culturing, cells were tested for germ layer markers via immunocytochemistry and RNA analysis.
  • iPSCs were harvested by brief collagenase IV treatment, suspended in 300 ⁇ mTeSR medium and injected subcutaneously/ intramuscularly/or in renal capsule into SCID mice (Charles River Laboratories International, Inc.) as described previously (see, e.g., Byrne, et al, (2009) PLoS One, 4:e71 18-e71 18, the disclosure of which is incorporated herein by reference in its entirety).
  • Visible tumors were dissected 6-8 weeks post-transplantation and fixed overnight with 4% paraformaldehyde/PBS solution. The tissues were paraffin embedded, sectioned and stained with hematoxylin and eosin. The presence of tissue representatives of all three germ layers was detected by light microscope.
  • Alkaline Phosphatase staining was performed in the dark at room temperature for 30 min using the Vector Red Alkaline Phosphatase Substrate Kit I (Vector Laboratories), according to the manufacturer's protocol.
  • immunocytochemistry cells were fixed in 4% paraformaldehyde/PBS for 20 minutes, washed twice with 0.1 % Tween-20/PBS, and blocked with 4% goat or donkey serum in PBS for 30min— all procedures were done at room temperature. After fixation, the cells were permeabilized with 1 % Triton-X/PBS for 30 minutes at room temperature for nuclear or cytoplasmic staining.
  • cells were incubated with primary antibody at room temperature for 1 hour or overnight at 4°C.
  • the cells were washed with 0.1 % Tween-20/PBS three times before secondary antibody was added and incubated for 1 hour at room temperature.
  • the cells were rinsed with 0.1 % Tween-20/PBS three times and counterstained with DAPI ( ⁇ g/ml).
  • hPSCs Human pluripotent stem cells
  • iPSCs were maintained under feeder free conditions and cultured in mTeSR or E8 media.
  • hPSCs Human pluripotent stem cells
  • iPSCs were maintained under feeder free conditions and cultured in mTeSR or E8 media.
  • cells were passaged with collagenase at a concentration of 5.5x10 ⁇ 4 cells/cm 2 and cultured overnight.
  • CDM Chemically Defined Media
  • Activin A 50ng/ml
  • R&D Activin A
  • BMP4 50ng/ml
  • Peprotech Peprotech
  • CDM was supplemented with bFGF (50ng/ml; Peprotech) and human vascular endothelial growth factor (VEGF) (40ng/ml; Peprotech) with media changes every 24hrs.
  • bFGF 50ng/ml
  • VEGF human vascular endothelial growth factor
  • FACS fluorescence activated cell sorting
  • ROCk inhibitor Prior to dissociation, ROCk inhibitor was added to the culture medium and cells were placed at 37°C for 30 minutes. Cells were dissociated using Acctuase at 37°C for 5-10min, pipetted to obtain a single cell suspension, and filtered with a 70 ⁇ cell strainer before centrifugation (1000 rpms for 5min). Cells were re-suspended in 1 ml fetal bovine serum (FBS) containing ROCk inhibitor and placed at 37°C for 30min for recuperation.
  • FBS fetal bovine serum
  • the cells were filtered again with a 70 ⁇ cell-strainer, centrifuged (l OOOrpms for 5min), and re-suspended in FACS buffer (PBS + 2mM EDTA + 0.5% FBS) at a cell concentration of 10 ⁇ 7 cells/ml.
  • FACS buffer PBS + 2mM EDTA + 0.5% FBS
  • Cells were blocked using mouse IgG (200 ⁇ g/ml; Invitrogen) for 10 min at 4°C and stained for FITC-CD31 (BD Biosciences) and PerCP-Cy5.5-CD34 (BD Biosciences) at 4°C for 30 min.
  • Stained cells were washed with 2 ml FACs buffer, centrifuged (1000 rpms for 10 min), and re-suspended in 300 ⁇ for analysis/gating and in 1 - 2 ml for sorting.
  • Cells were analyzed and sorted on a BD FACSAria III cell sorter with BD FACSDiva Software. After sorting, the purified cells were plated for SMC differentiation on collagen IV coated plates (BD Biosciences) in CDM + ROCK inhibitor for 24hours.
  • Muscle Growth Medium Media 231 [Invitrogen] + SMGS [Invitrogen]
  • PDGF-BB 10ng/ml; Invitrogen
  • the pSMCs were cultured in Smooth Muscle Differentiation Medium (Media 231 + SMDS [Invitrogen]) for 10 days with media changes every other day. Both the pSMCs and the terminally differentiated populations were characterized for expression of SMC markers, proliferative potential, contractile ability, and karyotypic stability.
  • Terminally differentiated cells were cultured in a 35mm plate coated with mouse collagen IV or gelatin (0.25% ; Sigma). Images were collected using the Nikon Biostation IM .
  • the cells were washed once with PBS then incubated at 37°C for 1 hour in FluoroBrite DMEM media (Invitrogen) containing Fluo-4 AM cell permeant (5 ⁇ ; Invitrogen) for loading. After incubation, the medium was changed with Flurobrite DMEM only and images were collected every minute for 15 minutes. Next, the media was exchanged for Fluorobrite DMEM containing carbachol (1 00 ⁇ ) and images were collected for an additional 15 minutes. For contraction only, the cells were imaged in culture media for 15 minutes prior to addition of carbochol, and for 15 minutes after addition of carbachol. Contraction was quantified by comparing change in cell surface area over time.
  • pSMCs smooth muscle precursor cells derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery+saline injection) , bladder SMC (surgery+human bladder SMC injection) and treatment (surgery+pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed-iPSC-derived pSMC, or viral reprogrammed-iPSC-derived pSMC).
  • pSMCs (2x 10 6 cells/rat) were injected peri-urethrally 3 weeks post-surgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks post injection.
  • Human bladder smooth cells (passage 3, LONZA, Allendale, NY) were used as terminally differentiated cell treatment for comparison with pSMC treatment.
  • Human smooth muscle precursor cells (pSMCs) were differentiated from human embryonic stem cells (luciferase (Flue) tagged H9 line retrovirus reprogrammed pluripotent stem cells (iPSCs), and episomal vector reprogrammed pluripotent stem cells (Epi-iPSCs).
  • pSMCs were derived from pluripotent cells essentially as described above for Example 5.
  • Implantation of human smooth muscle precursor cells was performed as follows. The animals are anesthesized with 1 -3% isoflurane. To examine human pSMC survival in vivo, one million luciferase tagged pSMCs in 50 ⁇ of Matrigel (50%) are injected directly into the adductor longus muscles of five SCID mice and imaged for three months. To examine the effect of pSMCs on the restoration of urethral function in SUI rats, cells suspended in 50 ⁇ _ of smooth muscle growth supplement (SMGS, Life Technologies) are injected periurethrally three weeks after urethrolysis. We used a commercial, terminally differentiated human smooth muscle cell line (passage 3, LONZA, Allendale, NY) for comparison with the pSMC treatment. A total of 2 ⁇ 10 6 cells suspended in the respective medium are injected into each urethra. The sham treatment stress urinary incontinence (SUI) rat (urethrolysis plus ovariectomy) is injected with saline only
  • H9-pSMC human embryonic stem cells H9 line
  • iPSC-pSMC human iPSC
  • Epi-iPSC-pSMC Epi-iPSC lines
  • rats Five weeks after injection, rats are anesthetized for LPP measurements. A suprapubic incision was made and the bladder exteriorized from the abdominal cavity. A small incision was made in the dome of bladder and a polyethylene catheter (PE-50 tube with a flared tip) (Becton Dickinson, Sparks, MD) inserted into the bladder. The catheter was connected via a 3-way stopcock to a syringe for filling with physiological saline and to a pressure transducer (TSD 104A, BIOPAC Systems Inc.) for monitoring bladder pressure.
  • PE-50 tube with a flared tip Becton Dickinson, Sparks, MD
  • LPP value of the H9-pSMC treatment group was higher than that of saline sham group, although the difference was not significant.
  • the group treated with human smooth muscle cells showed significant improvement in LPP compared to the sham saline group.
  • E-cadherin/TRA 160 dual positive single cells show clonal propagation.
  • E-cadherin a transmembrane protein that plays an important role in cell adhesion, is known to have an important function in maintaining pluripotency of hPSCs and in reprogramming of fibroblasts into iPSCs.
  • ECAD E-cadherin
  • ECAD-TRA160+ cells demonstrated nuclear OCT4 along with Ecadherin localized either in the cytoplasm or in the nucleus ( Figure 27a) and were dependent on neighbors to form pluripotent colonies, whereas ECAD+TRA160- cells failed to form any colonies. Dual positive cells also demonstrated greater numbers of mitotic events than the other two types of cells ( Figure 27d) .
  • ECAD+TRA160+, ECAD-TRA160+ and ECAD+TRA160- cells were compared with an emphasis on migration speed, it was observed that these three phenotypes reflected migratory behavior of Type 1 , 2 and 3 cells, respectively.
  • FACS buffer PBS + 0.5% FBS (BSA) + 2 mM EDTA
  • BSA 0.5% FBS
  • EDTA 2 mM EDTA
  • hPSCs can retain pluripotency and interact when cultured in somatic cell culture media.
  • Basic fibroblast growth factor can support feeder-independent growth of hESCs at high concentrations. Removal or reduced concentrations of bFGF results in loss of pluripotency.
  • mTeSRI media was used to culture hPSCs. This media contains 100 ng/ml of bFGF, whereas, somatic cell growth media such as SMGS (Smooth Muscle cell growth supplement) contains only 2 ng/ml bFGF.
  • SMGS Smooth Muscle cell growth supplement
  • I ndeed, hPSCs at Day 8 demonstrated strong expression of nuclear OCT4 and
  • the cellular dynamics during early differentiation events were examined by transferring cells to differentiation media and tracking morphological and behavioral changes for 72 hours.
  • Early differentiation was compared to spontaneous differentiation of hESCs to fibroblasts, a common fate observed in routine culturing of hESCs and following exposure to 20% fetal bovine serum. That early differentiating cells behaved differently than hESCs was observed. Rather than aggregating to form a tight colony, the cells accumulated cytoplasm, thereby increasing cell surface area and displayed altered mitotic parameters and increased cell cycle times.

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Abstract

L'invention concerne des procédés pour générer une population homogène de cellules progénitrices des muscles lisses à partir de cellules progénitrices pluripotentes et pour traiter un sujet présentant un défaut, une déficience ou une maladie des muscles lisses grâce à l'utilisation de telles cellules. L'invention concerne également des procédés pour traiter un sujet souffrant d'une déficience, d'un défaut et/ou d'une maladie des muscles lisses par une thérapie cellulaire faisant appel à des populations homogènes de précurseurs des muscles lisses et/ou de cellules différenciées des muscles lisses dérivées selon les procédés décrits dans la description. Certains aspects du traitement comprennent des procédés consistant à générer des populations homogènes de cellules précurseurs des muscles lisses et/ou des cellules différenciées des muscles lisses à partir de cellules progénitrices pluripotentes faisant appel à une identification automatique non invasive de types cellulaires sur base de paramètres de comportement cellulaire de cellules progénitrices pluripotentes cultivées et à l'élimination et/ou à l'isolement des types cellulaires identifiés. L'invention concerne également des systèmes, des compositions et des kits pour la mise en oeuvre des procédés selon l'invention.
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WO2019215090A1 (fr) 2018-05-08 2019-11-14 Universität Zürich Procédé de génération sans xeno d'une population de hmpc
WO2022261241A1 (fr) * 2021-06-08 2022-12-15 Insitro, Inc. Prédiction de pluripotence cellulaire à l'aide d'images de contraste

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019215090A1 (fr) 2018-05-08 2019-11-14 Universität Zürich Procédé de génération sans xeno d'une population de hmpc
WO2022261241A1 (fr) * 2021-06-08 2022-12-15 Insitro, Inc. Prédiction de pluripotence cellulaire à l'aide d'images de contraste

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