WO2018014005A1 - Multiple-reporter systems for indirectly screening differentiated cells by cell type and/or cell function - Google Patents

Abstract

The disclosure provides for multiple-reporter systems that are capable of rapidly and indirectly screening cells for a certain cell type and/or cell function.

Description

MULTIPLE-REPORTER SYSTEMS FOR INDIRECTLY SCREENING DIFFERENTIATED CELLS BY CELL TYPE AND/OR CELL FUNCTION

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from U.S. Provisional Application Serial No. 62/363,196, filed July 15, 2016, the disclosures of which are incorporated herein by

reference .

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. NIH K18 DK091445 awarded by the National Institutes of Health. The government has certain rights in the invention.

TECHNICAL FIELD

[0003] The disclosure provides for multiple-reporter systems that are capable of rapidly and indirectly screening cells for a certain cell type and/or cell function.

BACKGROUND

[0004] As diabetes becomes more prevalent, the demand for effective treatment or a cure becomes more urgent. One promising avenue being explored is differentiation of stem cells into pancreatic β-like cells, which could serve as replacement tissue for transplant therapy.

SUMMARY

[0005] The disclosure provides for multiple-reporter systems that are capable of rapidly and indirectly screening cells for a certain cell type and/or cell function. The multiple-reporter systems are ideally suited to the stem cell field, where stem cells are differentiated to have a certain cell type. For example, three multiple-reporter systems are provided herein that indicate in beta-cells differentiated from stem cells, whether the insulin promoter is activated, whether glucose stimulates the rise in cytosolic calcium, and whether insulin is secreted. These multiple-reporter systems work with standard wide-field

fluorescence microscopes, and do not require the more expensive and sophisticated confocal or TIRF microscopes. Together, these multiple-reporter systems can be used for rapid fluorescence screening for glucose-stimulated insulin secretion, obviating the slower and more expensive process of ELISA screening. [ 0006] In a certain embodiment, the disclosure provides for a multiple-reporter system comprising a mammalian expression vector backbone which comprises: a cassette having a tissue specific regulatory element that drives the expression of a sequence that encodes a Cre recombinase; two reporter coding sequences, a first reporter coding sequence and a second reporter coding sequence, wherein the two reporter coding sequences further comprise polyadenylation signal sequences, and wherein the first reporter coding sequence is flanked by LoxP sites and wherein the first reporter coding sequence can be excised from the vector by the action of the Cre recombinase; and a constitutive promoter that is located upstream of the two reporter coding sequences that drives the expression of the first or second reporter coding sequence, wherein the first reporter coding sequence encodes a first reporter that has different properties than a second reporter encoded by the second reporter coding sequence. In a further embodiment, the mammalian expression vector backbone is derived from pDsRed2-Nl. In yet a further embodiment, the tissue specific regulatory element is an insulin promoter or a fragment thereof. In a particular embodiment, tissue specific regulatory element is the proximal 378- nt region of the human insulin promoter. In a further embodiment, the first reporter is fluorescent protein. Examples of fluorescent proteins include, but are not limited to, mCherry, mOrange, GFP, EGFP, AmCyanl, AsRed2, mBanana, Dendra2, DSRed2, DsRed-Express , E2- Crimson, HcRedl, PAmCherry, mPlum, mRaspberry, mStrawberry, tdTomato, Timer, ZsGreenl, ZsYellowl, and YFP. In another embodiment, the fluorescent protein is mCherry. In an alternate embodiment, the fluorescent protein is EGFP. In yet another embodiment, the second reporter is a fluorescent protein or a chimeric protein that comprises a fluorescent protein. In a further embodiment, the chimeric protein is GCaMP6. In yet a further embodiment, the multiple-reporter system is GCaMP6f dual reporter system. In an alternate embodiment, the chimeric protein comprises mOrange and C-peptide. In yet another alternate embodiment, the multiple-reporter system is mOrange dual reporter system.

[ 0007] In another embodiment the disclosure also provides for a method of determining whether a beta-cell differentiated from a stem or progenitor cell has elevated level of cytoplasmic calcium in response to glucose comprising: transfecting a beta-cell with the multiple reporter system disclosed herein; introducing glucose; measuring green fluorescence emitted from the beta-cells.

[0008] In yet another embodiment the disclosure further provides a method of determining whether a beta-cell differentiated from a stem or progenitor cell secretes insulin in response to glucose comprising: transfecting a beta-cell with the multiple reporter system disclosed herein; introducing glucose; measuring orange fluorescence emitted from the beta-cells.

[0009] The disclosure provides vectors and construct. In one embodiment, the vector comprises a sequence as set forth in SEQ ID NO:l or a sequence that is 95-99% identical to SEQ ID NO:l and functions the same as a vector of SEQ ID NO:l. In one embodiment, the vector comprises a sequence as set forth in SEQ ID NO: 6 or a sequence that is 95-99% identical to SEQ ID NO: 6 and functions the same as a vector of SEQ ID NO: 6. In one embodiment, the vector comprises a sequence as set forth in SEQ ID NO: 10 or a sequence that is 95-99% identical to SEQ ID NO: 10 and functions the same as a vector of SEQ ID NO: 10.

DESCRIPTION OF DRAWINGS

[0010] Figure 1A-B provides (A) a schematic depiction of a reporter construct of the disclosure and (B) a plasmid map of the indirect dual reporter (hlnsP) vector (see also SEQ ID NO: 10) . The hlnsP vector reports the activation of the insulin promoter.

[0011] Figure 2A-B provides a (A) plasmid map and (B) sequence of the GCaMP dual reporter vector and coding sequences contained therein (SEQ ID NOs:l-5). The GCaMP vector reports elevations in cystoplasmic calcium.

[0012] Figure 3A-B provides a (A) plasmid map and (B) sequence of the mOrage dual reporter vector and coding sequences contained therein (SEQ ID NOs:6-9) . The mOrage vector reports insulin secretion .

[0013] Figure 4A-D demonstrates CXCR4⁺/hAFCs cell morphology after transduction but prior differentiation. Before differentiation the CXCR4⁺/hAFCs cells were flat. (A) Light microscope image of Lenti- EGFP transfected cells. (B) Lenti-EGFP transfected cells exhibit green fluorescence using a fluorescent microscope. (C) Light microscope image of Lenti-PDXl transfected cells. (D) Lenti-PDXl transfected cells did not fluoresce.

[0014] Figure 5A-D demonstrates CXCR4⁺/hAFCs cell morphology after 5 stages of differentiation. After 5 stages of differentiation, the cells started to round up and form a small circular shape. (A)

Light microscope image of Lenti-EGFP transfected cells. (B) Lenti- EGFP transfected cells exhibit green fluorescence using a

fluorescent microscope. (C) Light microscope image of Lenti-PDXl transfected cells. (D) Lenti-PDXl transfected cells did not fluoresce .

[0015] Figure 6A-C demonstrates the mRNA expression of PDXl (A) , somatostatin (B) and insulin (C) . (A, B) , the human amniotic fluid-derived CXCR4⁺ cells began from state 2 of differentiation following transduction with Lenti-PDXl viral particles was tested for the expression of PDXl and somatostain. (C) The Lenti-EGFP hAFCs/CXCR4⁺ and undifferentiated hAFCs/CXCR4⁺ were tested for the expression of insulin. Human islet cells were used for insulin positive control.

DETAILED DESCRIPTION

[0016] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a biomarker" includes a plurality of such biomarkers and reference to "the metabolic feature" includes reference to one or more metabolic features and equivalents thereof known to those skilled in the art, and so forth.

[0017] Also, the use of "or" means "and/or" unless stated otherwise. Similarly, "comprise," "comprises," "comprising" "include," "includes," and "including" are interchangeable and not intended to be limiting.

[0018] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language

"consisting essentially of" or "consisting of." [ 0019] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, the exemplary methods and materials are disclosed herein.

[ 0020] All publications mentioned herein are incorporated by reference in full for the purpose of describing and disclosing methodologies that might be used in connection with the description herein. Furthermore, with respect to any term that is presented in one or more publications that is similar to, or identical with, a term that has been expressly defined in this disclosure, the definition of the term as expressly provided in this disclosure will control in all respects.

[ 0021] The disclosure provides a reporter system for analysis of cell-type or phenotypic expression of certain genes. The reporter systems and methods are useful for analysis of genetic engineering of cells, differentiation of cells (e.g., stem cells to parenchymal cells) as well as de-differentiation of cells. Although the examples below discuss the reporter system in the context of beta islet cell differentiation and development, it should be recognized that the system is applicable to a number of other cell types including, but not limited to, liver cell, skin cells (dermal, epidermal, keratinocytes ) , neuronal cells, cardiac, myocytes, renal cells, intestinal cells, endothelial cells, fibroblasts, immune cells (T-cells, NK, etc.), etc. Moreover, the disclosure provides methods of using the reporter systems to determine the bioactivity and cell type for use in various therapies.

[ 0022] Diabetes prevalence is increasing worldwide and severe comorbidities persist, despite the availability of insulin treatment. Cell replacement strategies are thus being developed to treat this metabolic disease. A key treatment step will be production of sufficient quantities of fully functional pancreatic beta- or betalike-cells suitable for replacing missing or defective beta-cells. This goal has stimulated renewed interest in understanding human islet cell biology. However, because of the difficulty and high cost associated with isolating human islets, most studies focus on in vitro characterization of immortalized human or animal cell lines as surrogates for primary beta-cells. Rodent insulinoma cell lines derived from cancers arising after radiation treatment (rat RIN, INS-1, CRI-Gl) or viral transformation (hamster HIT, bHC) have been especially useful models of beta-cell biology. These cells exhibit high levels of insulin production and glucose

responsiveness. However, both of these pancreatic beta-cell attributes are lost with time in culture and increased numbers of cell passages. Unfortunately, the generation and characterization of human insulinoma or beta-cell-derived cell lines that preserve normal glucose responsiveness has not been reported.

[ 0023] Stem and progenitor cells with the ability to differentiate into lineages of the endocrine pancreas have become a promising, renewable source of transplantable cells for type 1 diabetes mellitus therapy. Insulin-producing cells can be generated from pluripotent embryonic stem cells and induced pluripotent stem cells, using a multi-stage approach that mimics the signaling pathways necessary for embryonic pancreatic development. Fetal stem cell sources have been of great interest as a result of their broad potency and lack of ethical concerns. C-kit-selected amniotic fluid-derived stem cells (hAFSCs) are a particularly attractive therapeutic cell source because of their extensive capacity for self-renewal in culture, broad multipotency and lack of teratoma formation. AFSCs have been shown to differentiate efficiently into mesenchymal lineages, such as bone, cartilage, fat and muscle, and, when cultured under specific inductive conditions, will also differentiate into endodermal and ectodermal lineages, albeit less efficiently. AFSCs express most of the MSC-associated markers (CD29, CD44, CD73, CD90 and CD105) but also express markers typically associated with ES cells (Oct-4, Sox2, SSEA-4 and Tra-1- 60), which suggests that they lie somewhere between ES cells and adult somatic stem cells on the developmental continuum.

Additionally, hAFSCs do not express cell surface markers associated with rejection, including CD80, CD86 and CD40, and they exhibit immunomodulatory activity. In addition, induced pluripotent stem cells (iPSs) are being developed. iPSs are produced by dedifferentiating parenchymal or stromal cells to a pluripotent state using factors such as Myc, OCT5, KLF and SOX2. The iPSs can then be cultured and differentiated to other cell types including beta islet cells.

[ 0024 ] Despite significant progress in differentiating various stem cell populations into various parenchymal cells. The ability to determine the cells biological activity and characteristics has been difficult. For example, progress has been made to

differential stem cells into β islet cells, although the amount of insulin produced is typically below the levels required for sustained physiological impact. Therefore, several methods have been employed to enhance insulin production in adult stem or progenitor cell populations, including overexpression of pancreatic genes. The pancreatic master transcription factor pancreatic duodenal homeobox 1 (PDX1) is indispensable in pancreatic

development and maintenance of β-cells function. Ectopic expression of Pdxl has been shown to lead to insulin production in vitro in BM-MSCs and hepatic cells and reduced hyperglycemia upon

transplantation in STZ-treated mice. Glucose is the major nutrient regulator of pancreatic β-cell function and coordinately regulates insulin gene expression, insulin biosynthesis, and insulin secretion. Glucose controls all steps of insulin gene expression, including transcription, preRNA splicing, and mRNA stability. A3, El, and CI are the major glucose-responsive transcription control elements of the insulin gene. In addition, a more distal glucose- responsive element appears to bind a glucose-sensitive complex that is specifically present in primary islets but remains to be identified. Glucose promotes the binding of PDX-1 to the A3 site and PDX-1 transactivating potency. In addition, it is now

recognized that PDX-1 stimulation of insulin gene transcription involves recruitment of co-activators, such as p300, that affect chromatin structure through post-translational modifications of histones such as methylation and/or acetylation. The signal transduction mechanisms by which glucose increases PDX-1 binding to the insulin promoter have been studied extensively but remain controversial. Glucose appears to promote translocation and modification of a cytoplasmic, inactive 31-kDa form to the nuclear, active 46-kDa species. Although this transformation likely requires phosphorylation, the large increase in apparent molecular mass suggests that PDX-1 undergoes multiple post-translational

modifications, possibly by O-linked iV-acetylglucosamine or small ubiquitin-related modifier 1. A number of kinases were proposed to mediate PDX-1 phosphorylation, including p38 mitogen-activated protein kinase, phosphatidylinositol-3 kinase, and extracellular signal-regulated kinases.

[ 0025] A major challenge today in generating functional

parenchymal cells from stem cells such as glucose-responsive and insulin-secreting beta-like cells from human stem cells is that the majority of protocols for generating such cells do not produce glucose-responsive cells, and those that do produce functional cells need further refinement to improve the efficiency of cell production and to improve their functional properties. The ability of the cells to secrete insulin appropriately in response to glucose stimulation is critical, because these cells are being generated for replacement tissue for therapeutic transplant in the setting of severe type 1 or 2 diabetes mellitus. If the cells secrete insufficient insulin, the patient will remain diabetic; on the other hand, if they secrete too much, they may induce life- threatening hypoglycemia. To refine the protocols, the cells produced must be screened for glucose-stimulated secretion. The standard method for measuring insulin secretion is the antibody- based ELISA assay, which is extremely expensive and time-consuming.

[ 0026] The disclosure provides for one or more indirect multiple- reporter systems for identifying cells of a certain cell type, and/or detecting or determining whether the cells respond to tissue specific signaling. In such cases, the multiple-reporter systems are ideally suited for determining whether a certain specific cell type has differentiated from a more primitive cell type (e.g., a stem or progenitor cell) .

[ 0027] The disclosure provides a multi-vector system (e.g., a two or three vector system) which are capable of reporting cellular function of at least three parameters (e.g., calcium flux, pH changes, specific gene expression) . For example, the multi- reporter constructs of the disclosure can report (a) whether cells are making insulin and packaging it; (b) when the cells are stimulated with with glucose where they are responsive, e.g., cells are depolarizing and calcium is entering under glucose simulation so the reporter detect rising levels of calcium; and (c) when cells secrete insulin, a pH change occurs and the reporter provides a visual fluorescence change.

[ 0028] In one embodiment, the multi-vector reporter system comprises 3 individual vectors each containing a report construct that can be transfected or transduced via lentivirus, adenovirus or the like into a cell. When using stem cells or early-differentiated cells a lentivirus or an adenovirus vector will be used to deliver the reporter constructs.

[ 0029] As used herein a report construct refers to the reporter gene that is operably linked to promoters or other nucleic acid components that facilitate expression. A reporter construct further refers to any upstream or downstream nucleic acid

components that are intended to modulate the expression a reporter gene (e.g., Cre-Lox) . A reporter gene refers a gene that expresses a product that can be detectably measured (e.g., fluorescence, bioluminescents , colorometric measurements and precipitation, or the presence of a transcript itself, e.g., by upregulation or downregulation) . A vector as used herein refers to a

polynucleotide, typically a viral polynucleotide that contains a reporter construct and facilitates delivery, and optionally integration, of a reporter construct to a cell and/or into the genome of a cell.

[ 0030] In one embodiment, the 3 vectors can be redesigned to comprise 2 vectors with the same report contructs

[ 0031] The multi-vector report system of the disclosure can be used for screening in drug development (small molecules and biologicals ) ; for diabetes therapies and cell based therapy development; cell-based insulin replacement therapies and the like.

[ 0032] As mentioned above, one problem with development of cell- based diabetes therapy has been that beta-like cells have been developed that make insulin and secrete it but typically do not respond to glucose. Moreover, current methods to measure insulin secretion involve complicated and expensive assay. Thus, the multi- vector report constructs of the disclosure provide vectors that streamline the screening reduce costs.

[ 0033] A reporter construct of the disclosure comprises a plurality of nucleic acid elements /domains . In one embodiment, the reporter construct comprises a first promoter operably linked to a Cre recombinase coding sequence. The first promoter can be a tissue specific promoter (e.g., insulin promoter) or a promoter that is inducible or otherwise modulated by a drug or therapy or which is activated during a cell's life cycle or at a particular differentiated state. When the first promoter is activated it causes expression of the Cre recombinase. The reporter construct further comprises a second constitutive, tissue specific or inducible promoter linked to a first detectable reporter gene (e.g., fluorescent or luminescent reporter gene) . Typically the second promoter is constitutive in nature such as a CMV

promoter/enhancer element. The first detectable reporter can be any number of fluorescent reporter (e.g., mCherry, EGFP etc.) or luminescent reporters. The first fluorescent report is flanked by LoxP nucleic acid elements, such that if the first promoter is active then the first detectable reporter gene will be excised, thus eliminating its expression by recombination (i.e., Cre-Lox recombination) . The first detectable reporter gene is operably linked to a second detectable reporter gene that can be a second fluorescent gene, luminescent gene or a reporter that is sensitive to a change in cellular physiology (e.g. pH, Ca²⁺ and the like) .

[ 0034] Figure 1A depicts a schematic of a reporter construct of the disclosure. Figure 1 also depicts a result based upon the activation of the first promoter or non-activation of the first promoter. It will be apparent that if the first promoter is activated then recombination will result in the elimination of the first detectable reporter gene, thereby resulting in only the expression of the second detectable reporter gene. In contrast, when the first promoter is not activated, the Cre recombinase is not expressed, thus the first detectable reporter gene is

expressed, but the second detectable reporter gene is noexpressed because of the stop codon at the first reporter. This is

advantageous for selection and analysis because the first or second reporter are active, but not both, to make it easy to select cells that are insulin positive or insulin negative.

[ 0035] As mentioned above, the promoters can be tissue specific or inducible promoter. The report systems of the disclosure can be readily manipulated to activate transcription of a multiple- reporter system, e.g., after addition of a chemical or biological agent. This may be accomplished by use of promoters that activate transcription based upon interacting with specific activators, e.g., hormones, cytokines, glucose, etc. For example, in

therapeutic applications where the multiple-reporter systems are used to identify whether a stem cell has differentiated into gonadal tissue where specific steroids are produced or provided, use of androgen or estrogen regulated promoters may be

advantageous. Such promoters that are hormone regulatable include MMTV, MT-1, ecdysone and RuBisco. Other hormone regulated promoters such as those responsive to thyroid, pituitary and adrenal hormones may be used. Cytokine and inflammatory protein responsive promoters that could be used include K and T Kininogen (Kageyama et al . , 1987), c-fos, TNF-alpha, C-reactive protein (Arcone et al . , 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988), alpha-1 antitypsin, lipoprotein lipase (Zechner et al . , 1988), angiotensinogen (Ron et al., 1990), fibrinogen, c-jun (inducible by phorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide) , collagenase (induced by phorbol esters and retinoic acid) , metallothionein (heavy metal and glucocorticoid inducible) , Stromelysin (inducible by phorbol ester, interleukin-1 and EGF) , alpha-2 macroglobulin and alpha-1 antichymotrypsin. Tumor specific promoters such as osteocalcin, hypoxia-responsive element

(HRE) , MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may also be used to regulate gene expression in tumor cells. In addition, this list of promoters should not be construed to be exhaustive or limiting, those of skill in the art will know of other promoters that may be used in conjunction with the promoters and methods disclosed herein. Further examples of tissue specific promoters are provided in Table 1. TABLE 1. Tissue Specific Promoters

Tissue Promoter

Pancreas Insulin, Elastin, Amylase, pdr-1, pdx-1,

glucokinase

Liver Albumin, PEPCK, HBV enhancer, a fetoprotein,

apolipoprotein C, -1 antitrypsin, vitellogenin, NF-AB Transthyretin

Skeletal muscle Myosin H chain, Muscle creatine kinase,

Dystrophin, Calpain p94 Skeletal alpha-actin fast troponin

Skin Keratin K6, Keratin Kl

Lung CFTR, Human cytokeratin 18 (K18) , Pulmonary

surfactant proteins A, B and C, CC-10 PI

Smooth muscle sm22 a SM-alpha-actin

Endothelium Endothelin-1 , E-selectin, von Willebrand factor

TIE, KDR/flk-1, Melanocytes, Tyrosinase

Adipose tissue Lipoprotein lipase (Zechner et al . , 1988),

Adipsin (Spiegelman et al., 1989), acetyl-CoA carboxylase (Pape and Kim, 1989) , glycerophosphate dehydrogenase (Dani et al., 1989), adipocyte P2 (Hunt et al . , 1986)

Breast Whey Acidic Protien (WAP) (Andres et al . PNAS

84:1299-1303 1987

Blood β-globin

[ 0036] "Tissue-specific regulatory elements" are regulatory elements (e.g., promoters) that are capable of driving

transcription of a gene in one tissue while remaining largely "silent" in other tissue types. It will be understood, however, that tissue-specific promoters may have a detectable amount of "background" or "base" activity in those tissues where they are silent. The degree to which a promoter is selectively activated in a target tissue can be expressed as a selectivity ratio (activity in a target tissue/activity in a control tissue) . In this regard, a tissue specific promoter useful in the practice of the disclosure typically has a selectivity ratio of greater than about 5. But preferably would have a higher selectivity ratio (e.g., greater than about 15) .

[ 0037] It will be further understood that certain promoters, while not restricted in activity to a single tissue type, may

nevertheless show selectivity in that they may be active in one group of tissues, and less active or silent in another group. Such promoters are also termed "tissue specific", and are contemplated for use with the disclosure. Accordingly, the tissue-specific regulatory elements described herein, have applicability in regulating the expression of reporter coding sequences, response elements, enzymes, expression cassette, etc. in a highly selective manner .

[ 0038] In a certain embodiment, a multiple-reporter systems described herein comprise an expression cassette which comprises a tissue specific regulatory element (e.g., a promoter and/or an enhancer) or a fragment thereof. In further embodiment, the tissue specific regulatory elements drive expression of Cre recombinase protein when activated by a tissue specific activator.

[ 0039] In a particular embodiment, a multiple-reporter system described here comprises two or more (e.g., 2, 3, 4, 5, 6 or a range between any two of the foregoing numbers) reporter coding sequences. In another embodiment, the two or more reporter coding sequences comprise downstream polyadenylation (A) signal sequences. In a further embodiment, upstream of the two or more reporter coding sequences is a constitutively active promoter, wherein the constitutively active promoter drive can drive the expression of one or more reporter coding sequences. Examples of constitutively active promoters include, but are not limited to, CMV, SV40, EFl , PGK1, Ubc, human beta actin, CAG, GDS, HI and U6. In another embodiment, the multiple-reporter systems described here comprise one or more reporter coding sequences which are flanked by LoxP (L) sites. In a further embodiment, the multiple-reporter systems described here comprise one or more reporter coding sequences which are flanked by LoxP (L) sites, and one or more reporter coding sequences which are not flanked by LoxP (L) sites. In a particular embodiment, the disclosure provides for a first reporter coding sequence that is flanked by LoxP sites, and a second reporter coding sequence that is downstream of the first reporter coding sequence, such that the first reporter coding sequence is excised by the action of Cre recombinase protein, while the second reporter coding sequence is expressed. In yet a further embodiment, a multiple-reporter system described here comprises two or more

(e.g., 2, 3, 4, 5, 6 or a range between any two of the foregoing numbers) reporter coding sequences, wherein at least two of the reporter coding sequences are separated by intervening sequence that comprises an internal ribosomal entry site. The advantages of such a system is that the reporter coding sequences can be expressed separately even if only one messenger RNA is transcribed.

[ 0040] In a certain embodiment, the multiple-reporter systems described here comprise one or more (e.g., 1, 2, 3, 4, 5, 6, or a range between any two of the foregoing numbers) reporter coding sequences which code for fluorescent proteins that when exposed to certain wavelengths of light exhibit fluorescence. Examples of such proteins, include, but are not limited to, mCherry, mOrange, GFP, EGFP, AmCyanl, AsRed2, mBanana, Dendra2, DSRed2, DsRed- Express, E2-Crimson, HcRedl, PAmCherry, mPlum, mRaspberry, mStrawberry, tdTomato, Timer, ZsGreenl, ZsYellowl, and YFP. In a further embodiment, a multiple-reporter systems described here comprises at least two reporter coding sequences that code for fluorescent proteins that emit light at different wavelengths. In an alternate embodiment, the disclosure provides for a multiple- reporter system which comprises at least two reporter coding sequences, wherein at least one reporter coding sequence codes for a fluorescent protein and wherein at least one reporter coding sequence codes for a non-fluorescent reporter protein. In a further embodiment, the non-fluorescent protein encodes an enzyme that can convert a substrate into a product that has a

characteristic color. Examples of such non-fluorescent reporter proteins include, but are not limited to, beta-galactosidase, chloramphenicol acetyl transferase, and luciferase.

[ 0041] In a particular embodiment, for any reporter coding sequence described herein, the reporter coding sequence may further comprise a sequence which codes for at least one additional peptide

(e.g., C peptide) and/or protein (e.g., response element, cytokine, chemokine, etc.) in addition to the reporter protein, such that the resulting product is a fusion or chimeric protein. In another embodiment, a reporter coding sequence described herein further comprises a sequence that codes for a response element. In a further embodiment, a multiple-reporter system disclosed herein comprises a reporting coding sequence that encodes GCaMP.

[ 0042] In certain embodiments, the disclosure provides for one or more indirect reporter systems for identifying insulin producing

(INS⁺) beta- and non-insulin-producing (INS^") cells, for identifying an increase in cytoplasmic calcium levels in INS⁺ beta-cells, and/or for screening for INS⁺ beta cells that can or cannot secrete insulin. In a particular embodiment, the disclosure provides for one or more multiple-reporter systems that comprise a cassette which comprises a human insulin gene promoter (phlNS) that drives expression of Cre recombinase protein when activated by an insulin promoter activator, such as glucose. In a further embodiment, the disclosure further provides for a constitutive promoter which drives expression of a first reporter gene coding sequence or a second reporter gene coding sequence, wherein the first reporter coding sequence is adjacent to the second reporter coding sequence and the first reporter coding sequence is flanked by LoxP (L) sites. In a further embodiment, the first or second reporter coding sequence encodes fluorescent proteins that emit light at different wavelengths. In a particular embodiment, the first reporter coding sequence encodes mCherry.

[ 0043] In a particular embodiment, the disclosure provides for a multiple-reporter system which comprises: a cassette which

comprises a human insulin gene promoter (phlNS) that drives expression of Cre recombinase protein, and a constitutive promoter which drives expression of a first reporter gene coding sequence or a second reporter gene coding sequence, wherein the first reporter coding sequence is adjacent to the second reporter coding sequence and the first reporter coding sequence is flanked by LoxP (L) sites. In a further embodiment, the multiple-reporter system can exclude expression of the other reporter gene by the stop codon allowing for selection based upon promoter activity. [ 0044] It should be noted that the disclosure provides for multiple-reporter system that are separate and serve different functions, the disclosure also provides for multiple-reporter systems that comprise multiple functionalities that can be used to indicate multiple states of a cell or multiple cellular processes

(e.g., detecting both cytoplasmic calcium levels and insulin secretion) .

[ 0045] The disclosure provides a sequence listing (see, e.g., Figures 2B and 3B) which depict coding sequences and domains of the vectors and constructs provided herein. For example, the following domains and nucleic acid elements are provided and described:

• Insulin promoter - base pairs from about 20-445 of SEQ ID NO: 1

• Cre recombinase - base pairs from about 464-1333 of SEQ ID NO: 1

• CMV enhancer/promoter - base pairs from about 1833-2341 of SEQ ID NO: 1

• loxP domain - base pairs from about 2400-2435 of SEQ ID NO:l

• mCherry - base pairs from about 2441-3154 of SEQ ID NO:l

• gcamp6f - base pairs from about 3447-4790 of SEQ ID NO:l

• EGFP - base pairs from about 2454-3173 of SEQ ID NO: 6

[ 0046] SEQ ID NO : 1 provides a vector containing a cassette useful in the methods of the disclosure. SEQ ID NO:l comprises a Gcamp6f reporter construct that is useful for detecting changes in calcium flux. The vector includes a number of domains and described and set forth in Figure 2B. One of skill in the art will appreciate that when a loxP site is described herein, reference to Figure 2B provide the corresponding nucleic acid element etc. SEQ ID NO: 6 provides a vector containing a cassette useful in the methods of the disclosure. SEQ ID NO: 6 comprises a EGFP reporter construct that is useful for detecting changes in pH and insulin secretion. The vector includes a number of domains and described and set forth in Figure 3B. One of skill in the art will appreciate that when reference is made to EGFP, as described herein, reference to Figure 3B provide the corresponding nucleic acid sequence and polypeptide sequence etc. [ 0047] As is clearly recognized in the art minor insubstantial changes can be made to the coding sequences and/or polypeptide encoded thereby without changing the biological function of domains, nucleic acid elements and polynucleotides.

[ 0048] The sequence listing appended hereto provide exemplary polynucleotide sequences providing vectors, constructs and encoded polypeptides useful in the methods described herein. It is understood that the addition of sequences which do not alter the encoded activity of a nucleic acid molecule, such as the addition of a non-functional or non-coding sequence (e.g., polyHIS tags), is a conservative variation of the basic nucleic acid.

[ 0049] The term "polynucleotide, " "nucleic acid" or

"recombinant nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DNA) , and, where appropriate, ribonucleic acid (RNA) .

[ 0050] Those of skill in the art will recognize that, due to the degenerate nature of the genetic code, a variety of codons differing in their nucleotide sequences can be used to encode a given amino acid. A particular polynucleotide encoding a

polypeptide described above are referenced herein merely to illustrate an embodiment of the disclosure, and the disclosure includes polynucleotides of any sequence that encode a polypeptide comprising the same amino acid sequence of the polypeptides and proteins utilized in the methods of the disclosure. In similar fashion, a polypeptide can typically tolerate one or more amino acid substitutions, deletions, and insertions in its amino acid sequence without loss or significant loss of a desired activity. The disclosure includes such polypeptides with alternate amino acid sequences, and the amino acid sequences encoded by the DNA sequences shown herein merely illustrate exemplary embodiments of the disclosure.

[ 0051] The disclosure provides polynucleotides in the form of recombinant DNA expression vectors or plasmids, as described in more detail elsewhere herein. Generally, such vectors can either replicate in the cytoplasm of the host microorganism or integrate into the chromosomal DNA of the host microorganism. In either case, the vector can be a stable vector (i.e., the vector remains present over many cell divisions, even if only with selective pressure) or a transient vector (i.e., the vector is gradually lost by host microorganisms with increasing numbers of cell divisions) . The disclosure provides DNA molecules in isolated (i.e., not

necessarily pure, but existing in a preparation in an abundance and/or concentration not found in nature) and purified (i.e., substantially free of contaminating materials or substantially free of materials with which the corresponding DNA would be found in nature) form.

[ 0052] As will be understood by those of skill in the art, it can be advantageous to modify a coding sequence to enhance its expression in a particular host. The genetic code is redundant with 64 possible codons, but most organisms typically use a subset of these codons. The codons that are utilized most often in a species are called optimal codons, and those not utilized very often are classified as rare or low-usage codons. Codons can be substituted to reflect the preferred codon usage of the host, a process sometimes called "codon optimization" or "controlling for species codon bias."

[ 0053] Optimized coding sequences containing codons preferred by a particular prokaryotic or eukaryotic host (see also, Murray et al. (1989) Nucl. Acids Res. 17:477-508) can be prepared, for example, to increase the rate of translation or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, as compared with transcripts produced from a non- optimized sequence. Translation stop codons can also be modified to reflect host preference. For example, typical stop codons for S. cerevisiae and mammals are UAA and UGA, respectively. The typical stop codon for monocotyledonous plants is UGA, whereas insects and E. coli commonly use UAA as the stop codon (Dalphin et al. (1996) Nucl. Acids Res. 24: 216-218) . Methodology for optimizing a nucleotide sequence for expression in a plant is provided, for example, in U.S. Pat. No. 6,015,891, and the references cited therein .

[ 0054] A "vector" generally refers to a polynucleotide that can be propagated and/or transferred between organisms, cells, or cellular components. Vectors include viruses, bacteriophage, pro- viruses, plasmids, phagemids, transposons, and artificial

chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artificial chromosomes), and PLACs (plant artificial chromosomes), and the like, that are "episomes," that is, that replicate autonomously or can integrate into a chromosome of a host cell. A vector can also be a naked RNA polynucleotide, a naked DNA polynucleotide, a polynucleotide composed of both DNA and RNA within the same strand, a poly-lysine-conj ugated DNA or RNA, a peptide-conj ugated DNA or RNA, a liposome-conj ugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.

[ 0055] The various components of an expression vector can vary widely, depending on the intended use of the vector and the host cell(s) in which the vector is intended to replicate or drive expression. Expression vector components suitable for the

expression of genes and maintenance of vectors in E. coli, yeast, Streptomyces , and other commonly used cells are widely known and commercially available. For example, suitable promoters for inclusion in the expression vectors of the disclosure include those that function in eukaryotic or prokaryotic host microorganisms. Promoters can comprise regulatory sequences that allow for regulation of expression relative to the growth of the host microorganism or that cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus. For E. coli and certain other bacterial host cells, promoters derived from genes for biosynthetic enzymes, antibiotic-resistance conferring enzymes, and phage proteins can be used and include, for example, the galactose, lactose (lac) , maltose, tryptophan (trp) , beta- lactamase (bla) , bacteriophage lambda PL, and T5 promoters. In addition, synthetic promoters, such as the tac promoter (U.S. Pat. No. 4,551,433, which is incorporated herein by reference in its entirety), can also be used. For E. coli expression vectors, it is useful to include an E. coli origin of replication, such as from pUC, plP, pi, and pBR.

[ 0056] Thus, recombinant expression vectors contain at least one expression system, which, in turn, is composed of at least a portion of a coding sequences operably linked to a promoter and optionally termination sequences that operate to effect expression of the coding sequence in compatible host cells. The host cells are modified by transformation with the recombinant DNA expression vectors of the disclosure to contain the expression system sequences either as extrachromosomal elements or integrated into the chromosome.

[ 0057] A "conservative substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain or a substitution of a nucleotide with a different nucleotide that does not change the codon. Codon tables are well known. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains

(e.g., threonine, valine, isoleucine) and aromatic side chains

(e.g., tyrosine, phenylalanine, tryptophan, histidine) . The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) , Threonine (T) ; 2) Aspartic Acid (D) , Glutamic Acid (E) ; 3) Asparagine (N) , Glutamine

(Q) ; 4) Arginine (R) , Lysine (K) ; 5) Isoleucine (I) , Leucine (L) , Methionine (M) , Alanine (A) , Valine (V) , and 6) Phenylalanine (F) , Tyrosine (Y) , Tryptophan (W) .

[ 0058] Sequence identity, which can also be referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software

Package of the Genetics Computer Group (GCG) , University of

Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap" and "Bestfit" which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from

different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.

[0059] A typical algorithm used for comparing a molecule sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997) . Typical parameters for BLASTp are: Expectation value: 10 (default) ; Filter: seg (default) ; Cost to open a gap: 11 (default) ; Cost to extend a gap: 1 (default) ; Max. alignments: 100 (default) ; Word size: 11 (default) ; No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

[0060] The disclosure contemplates vector and construct sequence that are at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:l, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 for use in the constructs and methods of the disclosure.

[0061] In further embodiments, cells can be transfected with the one or more multiple-reporter systems disclosed herein using standard transfection or transduction methodologies. For example, a variety of DNA transfection methods, such as calcium phosphate coprecipitation, electroporation and cationic liposome-mediated transfection (e.g., lipofectamine®) can be used to introduce a multiple-reporter system into cells.

[0062] The following examples are intended to illustrate but not limit the disclosure. While they are typical of those that might be used, other procedures known to those skilled in the art may alternatively be used.

EXAMPLES

[0063] A number of embodiments have been described herein.

Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

[0064] Isolation and culture of hAFSCs . hAFSCs can be isolated as reported in De Coppi et al . {Nat. Biotechnol . , 25:100-106 (2007)). Total amniotic fluid is cultured in AminoMAX^™ II (Gibco-Invitrogen, Grand Island, NY, USA) on a coverglass for at least 2 weeks, until the cell density had reached approximately 70%. This population is then immuno-selected using a rabbit polyclonal antibody against c- kit (CD117; Santa Cruz Biotechnology, Santa Cruz, CA, USA) .

Selected hAFSCs are then expanded in Chang medium: a-MEM

(Invitrogen, Carlsbad, CA, USA) containing 15% newborn calf serum/fetal bovine serum (FBS) , 1% glutamine and 1%

penicillin/streptomycin (Invitrogen), supplemented with 18% Chang B and 2% Chang C (Irvine Scientific, Irvine, CA, USA) at 37° C in a 5% CO₂ atmosphere.

[0065] Generation of hAF-derived CXCR4⁺ cells (Stage 1) . Human amniotic fluid-derived CXCR4⁺ cells transduced with Lenti-EGFP (negative control) or Lenti-PDXl viral particles. Transduced cells are cultured for 24-48 h in RPMI with no FBS, and then in RPMI plus 0.2% FBS and activin A (100 ng/mL) for 48 hours.

[0066] Differentiation of hAF-derived CXCR4+ cells into insulin producing cells (Stages 2-5) . hAF-derived CXCR4⁺ were

differentiated into B-cells by using a modification of the protocol described in Kroon et al. (Nature Biotechnology 26(4) : 443-452 (2008) ) . hAF-derived CXCR4⁺ cells were cultured in RPMI plus 2% FBS and KGF (25-50 ng/mL) for 3 d (Stage 2), and then cultured in DMEM plus 1% B27, retinoic acid (2 μΜ) , KAAD-cyclopamine (0.25 μΜ) and Noggin (50 ng/mL) for 3 d (Stage 3) . After which, the cells were cultured in DMEM plus 1% B27, betacellulin (BTC) (10 nM) and nicotinamide (10 mM) for 2 days (stage 4) . Finally, the cells were incubated with carboxymethyllysine (CML) , 1% B27, BTC (10 nM) and nicotinamide (10 mM) for six days. In the studies presented herein, hAF-derived CXCR4⁺ cells have been utilized. hAF-derived CXCR4⁺ cells are endoderm committed cells. It was found that hAF- derived CXCR4⁺ cells could be effectively differentiated into β-like cells using the staged process disclosed herein. As seen in FIGs. 4-5, hAF-derived CXCR4⁺ cells transduced with Lenti-EGFP or Lenti- PDXl changed morphology after the differentiation protocol.

[0067] Detection of the Expression of Fluorescent proteins using Fluorescence Microscopy. Expression of fluorescent proteins is examined with an Olympus 1X70 fluorescence microscope (Olympus, Japan) or a Leica TCS SP5 confocal microscope (Leica Microsystems, Germany) . Images are analyzed and edited in MetaMorph software.

[0068] Cell Sorting. For cell sorting, cells are trypsinized after imaging, suspended in medium, and subjected to FACS sorting for GFP⁺, mCherry⁺, and/or mOrange⁺ cells. FACS is performed by using a BD FACSAria cell sorter.

[0069] RNA Isolation and qPCR. Total RNAs are isolated from the cells using Trizol ( Invitrogen) . After digestion of total RNAs with Turbo DNase (Ambion) , cDNAs are made from the total RNAs with an iScript cDNA synthesis kit (Bio-Rad) , according to the

manufacturer's instructions. The cDNA id diluted three-fold with water prior to quantitative PCR (q-PCR) analysis. Gene-specific q- PCR primer/probe sets (customized TaqMan Gene Expression Assays, Applied Biosystems) for GAPDH, PDX1, somatostatin and insulin are used for q-PCR. Reactions are performed in for each sample using TaqMan Universal PCR Master Mix with a CFX-96 system (Bio-Rad) . The q-PCR is performed using the manufacturer's instructions. For each sample, expression of marker genes is normalized to GAPDH. Data are expressed as a relative expression level.

[0070] Imttionofluorescent staining and confocal microscopy. Cells grown on sterilized, 0.4% gelatin-coated patch-clamp dishes are washed with PBS and fixed with 4% paraformaldehyde for 20 minutes at room temperature. For detection of Cre nuclear proteins, dishes are permeabilized for 10 minutes with 0.25% Nonidet P-40 (NP40) . Cells are blocked overnight at 4 °C with 5% fetal goat serum, 1% bovine serum albumin, and 0.1% Triton X-100, and incubated for 1 hour with the primary mouse monoclonal anti-Cre antibody (Sigma- Aldrich) that is diluted in the blocking solution (1:500) . Cells are then washed and incubated for 1 hour with the secondary anti- mouse Cy-2 or Cy-3 conjugated antibody (1:250; Jackson

ImmunoResearch) . Images are taken using a Leica TCS SP5 confocal microscope. EGFP, mOrange and mCherry are visualized by endogenous fluorescence .

[0071] Specificity of the proximal 378-nt region in the human insulin promoter. The complete human insulin promoter/enhancer is estimated to be ~4 kb in length. The proximal 378-nt region (2363 to +15) is highly conserved between humans and rodents. It contains important regulatory motifs previously used to make reporters for identifying INS⁺ cells. It has been previously reported that this insulin promoter functions as a tissue specific promoter (e.g., see Lee et al . , PLoS One 7(4):e35521 (2012)). This insulin promoter

(pINS) was used in the dual reporter vectors disclosed herein.

[0072] General protocol for producing the dual-color reporter systems: Using a suitable plasmid backbone, such as pDsRed2-Nl

(Clontech Cat. No. 632406), the plasmid can be modified to comprise a cassette having a portion of human Insulin promoter (hlnsP) and sequence enconding Cre recombinase and two or more reporter coding sequences in manner as taught in Lee et al. (PloS ONE 7(4) :e35521

(2012) ) , which is incorporated herein in its entirety.

[0073] "Indirect" dual-color reporter system (hlnsP dual reporter)

(see FIG. 1) . Rather than use the insulin promoter to directly drive EGFP expression ("direct"), an indirect dual-color reporter construct was utilized. The insulin promoter drove expression of Cre recombinase in the vector, which lead to excision of an mCherry coding region located between CMV promoter and EGFP coding region. The mCherry coding region is followed by a stop codon. Thus, this indirect vector system leads to mutually exclusive expression of either red or green fluorescent proteins. The green cells are insulin-producing ones, whereas the red ones are non-insulin- producing ones. By contrast, distinguishing INS⁺ from INS^" cells with a 'direct' strategy depends on identifying cells that are doubly fluorescent and often leads to ambiguous results—the relative levels of fluorescence for the two reporter colors can be highly variable (due to variability in the relative strength of the two promoters driving fluorescent protein expression, differences in the relative fluorescence intensities, and/or relative rate of degradation of the proteins) . By contrast, the 'indirect' system used herein reports all cells that have been transduced or transfected, so efficiency of transduction/trans fection is easily calculated. Regardless of which fluorescent protein is expressed, expression is under control of the same CMV promoter. Thus, successful Ins gene expression can be easily monitored by noting a discrete change in color from red to green. In addition, the approach presented herein results in separate colors arising from the same transfected cells using a single transgene construct, which is not possible with a single-color reporter.

[0074] Calcium reporter system (GCaMP6f dual reporter) (see FIG.

2 ) : When the insulin promoter is activated and Cre recombinase protein is expressed, the mCherry coding region flanked with LoxP sites is excised. The GFP-Calmodulin fusion protein 6 (GCaMP6) , a fast-response genetically encoded calcium indicator protein, is then expressed. Thus, the vector reports

cytoplasmic calcium elevation, when glucose stimulation occurs. The cytoplasmic calcium elevation will trigger insulin secretion. The green cells indicate insulin-producing and potential insulin- secreting beta cells.

[0075] Insulin-secreting exocytosis reporter system (mOrange dual reporter) (see FIG.3): When the insulin promoter is activated, Cre recombinase protein excises the EGFP coding region flanked with LoxP sites. The cells exhibit a weak orange fluorescence, which brightens only on secretion. To facilitate screening for the functional β-like cells that secrete insulin in response to glucose stimulation, the mOrange dual reporter encodes a mOrange

fluorescent protein which is dimly fluorescent in the insulin vesicle, but which fluoresces brightly when it undergoes

exocytosis. In cells with no insulin promoter activity, the EGFP coding region is not excised, so the cells exhibit green

fluorescence .

[0076] ^λ Indirect' /calcium reporter/insulin-secreting reporter system: Two or more of the following functions (1) distinguishing INS⁺ from INS^" cells, (2) reporting cytoplasmic calcium levels in INS⁺ cells, and (3) reporting insulin secretion in INS⁺ cell, can be combined in one single multi-reporter system. This single multi- reporter system would measure insulin-promoter driven activation of expression of two separate protein reporters -- the cytoplasmic calcium reporter (GCaMP) and the insulin secretion reporter

(mOrange), separated by an internal ribosomal entry site (IRES2), so that both proteins are expressed separately even though only one messenger RNA is transcribed. The insulin secretion reporter is orange in color, while the cytoplasmic calcium reporter is green, so their signals can be monitored separately and simultaneously. The system may further comprise distinguishing INS⁺ from INS^" cells by using red fluorescence as described above for the (hlnsP reporter system) . Therefore, when the insulin promoter is activated, the mOrange and GCaMP proteins are expressed instead of mCherry, which is excised.

[0077] Experiments with Lenti-EGFP or Lenti-PDXl transduced hAFCS/CXCR4⁺ before and after a differentiation protocol . Human amniotic fluid-derived CXCR4⁺ cells were transduced with Lenti-EGFP and Lent-PDXl viral particles. The Lenti-PDXl transduced human amniotic fluid-derived CXCR4⁺ cells expressed a high level of PDX1 compared to Lenti-EGFP transduced hAFCs/CXCR4+ and undifferentiated hAFCs/CXCR4⁺ (see FIG. 6A-B) . The Lenti-PDXl transduced hAFCs/CXCR4+ expressed a high level of somatostatin (see FIG. 6A) . However, there was no expression of insulin for the Lenti-PDXl transduced hAFCs/CXCR4⁺ cells (see FIG. 6C) . Lenti-EGFP-transduced hAFCs/CXCR4⁺ cells also expressed somatostatin, but less than the somatostatin expression of Lenti-PDXl transduced hAFCs/CXCR4⁺ (see FIG. 6B) .

[0078] Experiments with Lenti-PDXl transduced amniotic epithelial cells. The amniotic epithelial cells are used to test the differentiation protocol after transducing with Lenti-PDXl viral particles. The Lenti-PDXl transduced amniotic epithelial cells are further used to test the expression of insulin. The cells that produce insulin are confirmed and selected with the mOrange dual reporter to assess their functional properties. The Lenti-PDXl transduced amniotic fluid-derived stem cells are used to produce insulin, which can be used for other experiments and applications.

[0079] A number of embodiments have been described herein.

Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of this disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A multiple-reporter system comprising a mammalian expression vector backbone which comprises:

a cassette having a tissue specific or inducible regulatory element that drives the expression of a sequence that encodes a Cre recombinase ;

two reporter coding sequences,

a first reporter coding sequence and a second reporter coding sequence, wherein the two reporter coding sequences further comprise polyadenylation signal sequences, and wherein the first reporter coding sequence is flanked by LoxP sites and wherein the first reporter coding sequence can be excised from the vector by the action of the Cre recombinase; and a constitutive promoter that is located upstream of the two reporter coding sequences that drives the expression of the first and/or second reporter coding sequence,

wherein the first reporter coding sequence encodes a first reporter that has different properties than a second reporter encoded by the second reporter coding sequence.

2. The multiple-reporter system of claim 1, wherein the mammalian expression vector backbone is derived from pDsRed2-Nl.

3. The multiple-reporter system of claim 1, wherein the tissue specific regulatory element is an insulin promoter or a fragment thereof .

4. The multiple-reporter system of claim 1, wherein the tissue specific regulatory element is the proximal 378-nt region of the human insulin promoter.

5. The multiple-reporter system of claim 1, wherein the first reporter is fluorescent protein.

6. The multiple-reporter system of claim 5, wherein the

fluorescent protein is selected from the group consisting of mCherry, mOrange, GFP, EGFP, AmCyanl, AsRed2, mBanana, Dendra2, DSRed2, DsRed-Express , E2-Crimson, HcRedl, PAmCherry, mPlum, mRaspberry, mStrawberry, tdTomato, Timer, ZsGreenl, ZsYellowl, and YFP.

7. The multiple-reporter system of claim 6, wherein the fluorescent protein is mCherry.

8. The multiple-reporter system of claim 6, wherein the fluorescent protein is EGFP.

9. The multiple-reporter system of any one of claim 1-8, wherein the second reporter is a fluorescent protein or a chimeric protein that comprises a fluorescent protein.

10. The multiple-reporter system of claim 9, wherein the fluorescent protein is selected from the group consisting of mCherry, mOrange, GFP, EGFP, AmCyanl, AsRed2, mBanana, Dendra2, DSRed2, DsRed-Express , E2-Crimson, HcRedl, PAmCherry, mPlum, mRaspberry, mStrawberry, tdTomato, Timer, ZsGreenl, ZsYellowl, and YFP.

11. The multiple-reporter system of claim 9, wherein the chimeric protein is GCaMP6.

12. The multiple-reporter system of claim 11, wherein the multiple-reporter system is GCaMP6f dual reporter system.

13. The multiple-reporter system of claim 9, wherein the chimeric protein comprises mOrange and C-peptide.

14. The multiple-reporter system of claim 13, wherein the multiple-reporter system is mOrange dual reporter system.

15. A method of determining whether a beta-cell differentiated from a stem or progenitor cell has elevated level of cytoplasmic calcium in response to glucose comprising: transfecting a beta-cell with the multiple reporter system of claim 12;

introducing glucose;

measuring green fluorescence emitted from the beta-cells.

16. A method of determining whether a beta-cell differentiated from a stem or progenitor cell secretes insulin in response to glucose comprising:

transfecting a beta-cell with the multiple reporter system of claim 14;

introducing glucose;

measuring orange fluorescence emitted from the beta-cells.