Therapeutic expansion of human cells and tissues and methods for their use

Abstract

The present disclosure provides methods for expanding tissue samples from humans for the treatment of certain diseases. The methods involve isolating small amounts of relevant tissue by biopsy or other tissue collection methods, expanding such tissue ex vivo while maintaining its natural function, and reimplanting the expanded tissue to alleviate the disease in humans. One example is expanding human pancreatic islet tissue ex vivo and reimplanting the expanded cells into individuals with type 1 diabetes for the treatment and long-term remission or cure of the disease.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 436,546, filed December 31, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Certain cell types in the body are capable of continuous growth and division, including the skin, various hematopoietic bone marrow progenitors, and intestinal epithelium. For example, the human erythroid system, which is primarily composed of red blood cells (RBCs) and the bone marrow where RBCs are synthesized, produces over 100 billion RBCs per day (>2 million cells per second; Sender and Milo, 2021).

[0003] On the other hand, certain tissues in the adult human body are composed of cells that, under most circumstances, are essentially shut out of the cell division cycle. This is because certain functional cell types in the human body require tightly controlled cell division to maintain the health and well-being of the organism. Endocrine cells are one example. These cells secrete hormones into the bloodstream that are essential for normal physiological homeostasis, but they represent cells whose division is highly restricted by the organism.

[0004] Hormone-secreting tissues must be carefully regulated to prevent the overproduction of specific hormones. Many endocrine cells synthesize specific hormones not only constitutively but also in a pulsatile and reactive manner in response to physiological changes in the organism. When cells constitutively produce a specific hormone, careful control of the total number of those cells is essential to maintaining the health of the organism. If hormone-producing cells proliferate excessively due to poorly controlled cell division (e.g., sporadic mutations), persistent production of hormones such as thyroid hormone, epinephrine, or insulin can result in excessive amounts of the corresponding hormone. If untreated, hormone overproduction can lead to serious illness and even death.

[0005] Human diseases such as pheochromocytoma, growth hormone-secreting pituitary adenoma, thyroid tumors secreting T3 and T4, and insulin-secreting pancreatic tumors known as insulinomas are representative examples of cellular hyperproliferations whose corresponding secreted hormones can cause morbidity or mortality. For example, untreated insulinomas can cause excessive insulin secretion, leading to seizures and episodes of severe hypoglycemia that can result in death. However, these tumors are generally small, rarely metastasize, and are easily cured by surgical resection (Kasper et al. 2015: Harrison's Principles of Internal Medicine).

[0006] For the aforementioned reasons, long-lived organisms, such as humans and other mammals, with an average lifespan of more than 10 years, carefully regulate cell division in specific cell types within their bodies. Human cells maintain multiple levels of cell division locking mechanisms to prevent unintended excessive proliferation of specific cell types, such as endocrine cells, neurons, and glial cells. These cell division locks, or CDLs, maintain cells in the G0 phase and prevent entry into the cell cycle. One category of CDLs includes certain tumor suppressor proteins, while another category includes certain metabolic regulatory proteins. Cell types with strong CDLs include neurons, which release potent neurotransmitters such as dopamine; glial cells, such as oligodendrocytes; Schwann cells, which produce the myelin sheath that insulates and protects neurons; and endocrine cells, such as insulin-secreting beta cells and glucagon-secreting alpha cells. Summary of the Invention [Means for solving the problem]

[0007] Certain human diseases are characterized by the loss of specific cell types within vital tissues or organs within the body. In adults, these cells or tissues are CDL cells. Representative examples include type 1 diabetes, certain types of type 2 diabetes, Parkinson's disease, Waterhouse-Friedrichsen syndrome, and multiple sclerosis (Kasper et al., 2015; Gholamzad et al., 2019; Buzzard et al., 2017; Harris et al., 2020; Harris et al., 2020).

[0008] In some cases, it may be desirable to treat the underlying disease by replacing lost tissues and associated cells, a process broadly known as regenerative medicine. However, a major challenge to doing this is the difficulty of growing many living tissues (and the cell types they contain) either in vivo (inside the body) or ex vivo (outside the body).

[0009] Skin is one organ that can be effectively grown outside the body, and remarkable therapeutic effects have been achieved in severe skin diseases, such as burn patients and patients with rare, life-threatening genetic mutations in skin cell development. In one case study, a 7-year-old child with life-threatening junctional epidermolysis bullosa was effectively treated by transducing his own human primary keratinocytes with a Moloney murine leukemia virus promoter construct incorporating the complete wild-type human LAMB3 cDNA (Hirsch et al., 2017). This was a groundbreaking development despite the fact that human keratinocytes are easy to culture outside the body.

[0010] Other human tissue types, such as cardiomyocytes, are more difficult to culture in vitro. This has led researchers to turn to stem cell-based approaches. Groundbreaking work by Yamanaka and colleagues in Japan demonstrated that mature, differentiated cells can be reprogrammed to an embryonic-like state and then redifferentiated into a variety of mature cell types with properties similar to those of mature wild-type cells (Takahashi and Yamanaka, 2006; Yamanaka, 2020). Over the past two decades, extensive research has been conducted in the field of induced pluripotent stem cells (iPSCs) to realize the potential of regenerative medicine, but as discussed below, results have been mixed. What is currently clear is that the stem cell reprogramming process appears to resemble the developmental pathway that occurs during human embryogenesis, but there are important unresolved differences that could affect the long-term safety and efficacy of iPSC-based therapies.

[0011] For example, to generate sufficient numbers of cardiomyocytes (myocytes) to replace cardiac tissue damaged by myocardial infarction, researchers have adopted a stem cell-based approach and developed a method to generate large numbers of myocyte-like cells. Using iPSCs, researchers attempted to generate large numbers of myocyte-like cells and subsequently apply them to repair scar tissue formed by hypoxia after myocardial infarction. When transplanted into primate hearts, these stem cell-derived myocytes demonstrated difficulty in normal function. The stem cell-derived myocytes did not exhibit synchronized contractions with existing cardiomyocytes and were unable to adapt to normal electrical rhythms. The electrocardiograms (ECGs) of retransplanted primates showed obvious abnormalities, leading to life-threatening arrhythmias (EbioMedicine, 2018).

[0012] Both embryonic stem cells and induced pluripotent stem cells can be differentiated into a wide variety of cell types indistinguishable from their fully differentiated human counterparts. Gene expression analysis of fully differentiated human adult cells derived from specific tissues, such as cardiomyocytes or insulin-producing beta cells of the pancreas, and similar cell types derived from laboratory-differentiated stem cells reveals numerous gene expression differences (Yamanaka, S., 2020). These findings raise questions about the long-term safety of stem cell-based therapies intended for therapeutic reimplantation into humans (Kushner et al., 2014). Indeed, numerous cases have been reported in which tumors developed after the introduction of stem cell-derived tissues into experimental animals. Teratomas are aggressive, undifferentiated tumors that have been shown to form in mammals from all three primitive embryonic tissue layers (ectoderm, mesoderm, and endoderm). This indicates that iPSCs pose serious long-term safety risks in human clinical applications ( Blum et al., 2009 ; Cunningham et al. 2012 ; Fong et al., 2010 ; Kum et al., 2012 ; Lee et al., 2009 ; Mutter et al., 1987 ; Prokhorova et al., 2009 ).

[0013] The aforementioned problems and safety issues associated with stem cell use could be avoided if a technology could be established to selectively release the cell division locking mechanism that prevents specific adult tissues and cell types, such as human beta cells, from significantly growing or dividing. The present disclosure is the first to demonstrate a technology that selects therapeutically useful, fully differentiated, non-dividing cells (or cell populations) and temporarily releases the cell division locking state, thereby allowing a controlled period of cell division until a sufficient number of cells / tissues are generated for therapeutic use. This is achieved by inducing a temporary steady-state thermodynamic change in cellular energy utilization, resulting in cell cycle entry, without the use of recombinant DNA techniques or genome editing technologies such as CRISPR / CAS-9. The latter must be avoided for the purpose of reimplantation and to maintain the genomic integrity of otherwise healthy human cell types that are relatively abundant in the body. Therefore, for patient safety, it is desirable to avoid the use of recombinant DNA or genome editing technologies. However, temporary modulation of RNA expression levels using gene silencing methods such as small interfering RNA (siRNA) is useful and a preferred embodiment.

[0014] At the heart of this disclosure is the temporary release of the normal regulation of cell division that limits the in vitro growth of specific human tissues and cell types. By temporarily releasing the cell division lock (CDL) that prevents critical tissue-specific cells, such as endocrine or neuronal cells, from entering the cell cycle in a controlled, reversible manner, the technology described herein enables the in vitro expansion of such cell types while maintaining key phenotypic characteristics as mature, fully differentiated cells. This approach is fundamentally different from existing approaches using embryonic stem cells (ES cells) or induced pluripotent stem cells (iPSCs), which require cells to first be grown to sufficient numbers in an undifferentiated state and then redifferentiated to possess characteristics that approximate the cell type required for therapy. Figure 1 provides a diagram outlining the differences between conventional stem cell-based therapeutic approaches and the disclosed method.

[0015] Thus, this disclosure allows researchers to expand selected cell types ex vivo to therapeutic quantities sufficient for future reimplantation into humans, while avoiding the need to use stem cell-based approaches to achieve sufficient cell growth. This approach avoids the safety issues, gene expression variability, and tumorigenesis risk associated with stem cell use (Ohnishi et al., 2014; Yasuda et al., 2018).

[0016] Unless otherwise defined, terms used in this specification should be interpreted in the sense commonly used in the art. The main terms used in this specification and their definitions are listed below.

[0017] As used herein, the term "tissue" refers to a group or layer of similarly specialized cells that collectively perform a particular function.

[0018] The terms "compound" and "agent" are used interchangeably herein and refer to both chemical and biological agents.

[0019] As used herein, the term "chemical agent" refers to a substance with a molecular weight of less than 2000 atomic mass units (daltons). Such substances are also called "small molecules."

[0020] As used herein, the term "biological agent" refers to molecules, including proteins and polynucleotides, having a molecular weight of 2,000 atomic mass units (amu or daltons) or more and 990,000 amu or less.

[0021] As used herein, the terms "active agent" and "agonist" are used interchangeably.

[0022] As used herein, the terms "inhibitor" and "antagonist" are used interchangeably.

[0023] As used herein, the term "expression-inhibiting agent" encompasses compounds that inhibit gene expression by any mechanism, including the inhibition of the production of functional mRNA or the promotion of mRNA degradation.

[0024] As used herein, "inhibiting" a substance means Binding to proteins in cells, tissues, blood, or the body and reducing their activity; Attenuating or eliminating the function of a protein; Decreasing the amount or level of a protein, and / or It refers to the inhibition of the expression or production of a protein.

[0025] Unless otherwise defined, the terms "inhibitor" and "antagonist" are used herein as synonyms. The inhibitory effect of an inhibitor may be partial or complete. As used herein, the terms "bind," "binding," and "binding to" have their usual meanings in the field of biochemistry (e.g., describing interactions between substances such as enzyme-substrate, protein-DNA, receptor-ligand, etc.). As used herein, the term "binding to" is synonymous with "interacting with" in the context of discussing the relationship between a substance and its target protein or nucleic acid.

[0026] As used herein, the term "antibody" refers to a protein or immunoglobulin raised against an antigen and capable of "specifically binding" to that antigen. As used herein, an antibody that "specifically binds" interacts exclusively with the epitope of the antigen that induced its synthesis, or with a structurally similar epitope. An antibody that "specifically binds" to an epitope is one that, under appropriate conditions, interacts with that epitope even in the presence of multiple potential binding targets.

[0027] As used herein, the term "antigen" refers to a protein or peptide target bearing an epitope to which an antibody specifically binds.

[0028] As used herein, the term "fragment" refers to a portion of a polypeptide or polynucleotide. In one embodiment, the fragment retains the activity of the polypeptide or polynucleotide.

[0029] According to the present disclosure, cells obtained from a mammal, or a mammal in need thereof, are administered a therapeutically effective amount of one or more compounds / substances that modulate / activate or regulate / inhibit a survival target (VT) protein or a protein that degrades VT, respectively. As used herein, the term "mammal" includes, but is not limited to, humans, laboratory animals, household pets, and farm animals.

[0030] As used herein, the phrase "effective amount" refers to an amount of a compound, material, cell, group of cells, or composition comprising a compound of the present disclosure, which is effective to produce a desired effect (e.g., increased expression of a survival target, increased amount of a survival target, and / or decreased activity of a protein or nucleic acid sequence that inhibits a survival target) in at least some cell subpopulations (e.g., pancreatic cells) within an animal.

[0031] As used herein, the phrase "therapeutically effective amount" means an amount of a compound, material, cell, group of cells, or composition comprising a compound of the present disclosure that is sufficient to produce a desired therapeutic effect in at least one cell subpopulation (e.g., pancreatic cells) in an animal body, with a reasonable benefit-to-risk ratio, including any appropriate side effects, suitable for any medical treatment.

[0032] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more.

[0033] "Adequate" or "suitable" conditions for an event are those that do not prevent the event from occurring, and thus allow, enhance, facilitate, and / or contribute to the event.

[0034] As used herein, the term "provide" in reference to a composition, cell, group of cells, antibody, nucleic acid, or small molecule compound means to manufacture, purchase, or otherwise obtain the composition, antibody, nucleic acid, or small molecule in question.

[0035] As used in this specification and claims, the term "or" is generally used in its meaning including "and / or" unless otherwise defined. The term "and / or" means one or all of the listed elements or a combination of any two or more of the listed elements. Although "and / or" may be used in this specification, this does not imply that "or" does not mean "and / or" elsewhere.

[0036] The words "preferred" and "preferably" refer to embodiments of the present disclosure that may offer certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the present disclosure.

[0037] As used herein, the terms "have," "has," "having," "include," "includes," "including," "comprise," "comprises," "comprising," or similar terms are used in an open and inclusive sense and generally mean "including, but not limited to."

[0038]

[0023] When embodiments are described herein using terms such as "have," "has," "include," "includes," "including," "comprise," "comprises," or similar terms, it is understood that other similar embodiments are also provided that are described using the terms "consisting of" and / or "consisting essentially of." The term "consisting of" means including and limited to the elements that follow the phrase "consisting of." That is, "consisting of" indicates that the listed elements are essential, and that no other elements may be present. The term "consisting essentially of" includes the elements listed after the phrase, and may include other unlisted elements so long as they do not interfere with or contribute to the specified activity or function of the disclosed listed elements.

[0039] References throughout this specification to "one embodiment," "an embodiment," "particular embodiment," or "some embodiments" mean that the particular feature, configuration, composition, or characteristic described in connection with that embodiment is included in at least one embodiment of the present disclosure. Thus, the use of such phrases in different places throughout this specification does not necessarily refer to the same embodiment of the present disclosure. Furthermore, particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0040] Throughout this disclosure, various aspects of the present disclosure may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the size of the range.

[0041] For clarity, certain embodiments may be described separately herein, and in particular, any combination of compatible features described in connection with one or more embodiments herein is included, unless it is expressly specified that a feature of a particular embodiment is incompatible with a feature of another embodiment.

[0042] For any method disclosed herein that includes separate steps, the steps may be performed in any feasible order, and any combination of two or more steps may be performed simultaneously, if desired.

[0043] The following detailed description of the exemplary embodiments of the present disclosure is best read in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0044] [Figure 1]This is a schematic diagram illustrating the difference between expanding cell populations using stem cells and using fully differentiated mature cells. The stem cell approach involves a three-step process: (1) dedifferentiating mature cells into induced pluripotent stem cells (iPSCs), which eliminate the mature cell phenotype; (2) expanding the iPSCs until sufficient quantities are obtained; and (3) redifferentiating the iPSCs into structurally and functionally similar, but not identical, mature differentiated cells. In contrast, our new method involves transiently and reversibly inducing iPSCs to enter the cell cycle and propagating them while maintaining their fully differentiated phenotype. [Figure 2] 32D cells expressing insulin receptor and insulin receptor substrate-2 (IRS-2) show improved viability after treatment with insulin (INS) or an IRS activator (HP-508). 32DIR-IRS-2 cells treated with IRS activators show similar growth rates to INS-treated cells, but their viability after 72 hours of culture is higher than that of INS-treated cells (Housey and Balash, 2018; Housey and Balash, 2023). INS-treated cells exhibited numerous intracytoplasmic inclusions, reflecting increased cellular stress and reduced viability. In contrast, IRS activator-treated cells maintained normal cell morphology (circled) in several regions. While INS-treated cells typically had a viability of 60% or less, IRS activator-treated cells achieved a viability of over 90% even after 72 hours (data not shown). Viability was measured using an automated cell counter using trypan blue exclusion. [Figure 3A]Growth curves of human islet tissue cultured in vitro in the presence of IRS activators and growth promoters are shown. Growth curves of human islet tissue containing beta cells in the presence of an IRS-2 activator (HP-508) combined with cyclic treatment with the indicated siRNAs are shown. Tissue growth was measured by digital imaging using a Zeiss CD-7 automated microscope. Total tissue surface area was calculated using ZEN software. Under these conditions, human islet tissue, with a long diameter of less than 300 micrometers, typically develops into tissue masses with a vertical height (thickness) of 15-30 micrometers. TP53 grows in the presence of siRNA that inhibits the expression of the human tumor suppressor protein P53 (Figure 4). STK4 grows in the presence of siRNA that inhibits the expression of the serine / threonine kinase 4 gene (Figure 4). VGLL4 grows in the presence of siRNA that inhibits the expression of the human degenerative ancestry member 4 gene (Figure 4). TSC-1 grows in the presence of siRNA that inhibits the expression of the human tuberous sclerosis 1 gene (Figure 4). [Figure 3B] Same as above. [Figure 4A] Examples of siRNAs that were synthesized and tested are shown. Each siRNA is shown in two formats. At the top, each strand of the duplex is shown from 5' to 3', and the presence of an "r" before the nucleotide A, U, G, or C indicates a ribonucleotide. The absence of an "r" before the nucleotide A, T, G, or C indicates a deoxyribonucleotide. At the bottom, the two strands of the hybridized duplex are shown. [Figure 4B] Same as above. [Figure 5] Insulin production by cultured human islets of Langerhans. Islets produce insulin continuously throughout the growth cycle. [Figure 6]A representative human islet-like tissue mass (approximately 1000 micrometers (μm) × 400 μm × 170 μm) grown from a small number of human donor cells (<10) using IRS activators is shown. The representative human islet tissue mass shown in the figure was maintained under culture conditions for over a year. Abbreviations are as follows: it - islet tissue, v - vascular processes. The longest diameter of the tissue mass exceeds 1000 μm. Blood vessel-like vascular processes are observed throughout the tissue mass. These structures appear during the middle and late stages of growth. As shown in Figure 3, the addition of growth-promoting agents (also referred to herein as mitogens) can shorten the time it takes to grow a tissue mass large enough to support retransplantation to just 120–180 days. DETAILED DESCRIPTION OF THE INVENTION

[0045] The drawings are not to scale. Like numbers used in the figures refer to like components, steps, etc. However, it will be understood that the use of a number to refer to a component in a given figure is not intended to limit the component in another figure labeled with the same number. Furthermore, the use of a different number to refer to a component does not exclude the differently numbered component from being the same or similar to the other numbered component.

[0046] The present disclosure includes a method for expanding patient-derived cells ex vivo. The method can include providing a tissue sample or similar specific cells (cells of interest) from a subject, such as a tissue sample obtained by biopsy or a cell population of interest obtained by enrichment or purification. The tissue contains similar specific cells that together perform a specific function. The tissue is obtained from an individual with a disease pathology that involves tissue loss. Examples of diseases include, but are not limited to, type 1 diabetes (T1D) and Parkinson's disease. In one embodiment, the disease is T1D and the subject has a relative or absolute insulin deficiency. The similar specific cells are cells of interest for ex vivo expansion. When the disease is T1D, examples of tissue and similar specific cells include, but are not limited to, pancreatic tissue, islets of Langerhans, and beta cells. When the disease is Parkinson's disease, examples of tissue and similar specific cells include, but are not limited to, substantia nigra tissue and dopaminergic neurons.

[0047] A tissue containing the desired cells of interest is established in culture. In one embodiment, this can include establishing cells (e.g., tissue sample and / or cells of interest) in culture. This can be accomplished using a known rich medium, such as Dulbecco's modified Eagle's medium supplemented with 10% human serum. Other media types are also available (Yao and Asayama, 2017). Higher concentrations of serum may be used if necessary. If the relevant cell type can be cultured in vitro, it is not necessary to identify a survival target and identify a compound that activates, stabilizes, or reduces the degradation of that survival target. The terms "viable" and "survival" refer to the maintenance of a cell type in culture without a significant decrease in total cell number, although net growth in cell number is not required. Figure 2 shows a representative example of the effect of the compound HP-508 on the viability of human beta cells in culture.

[0048] Propagation of cells (e.g., tissue samples and / or cells of interest) involves culturing the desired cell type in the presence of a compound that activates a target protein with a cellular function essential for maintaining cell viability under laboratory culture conditions. The target protein or nucleic acid sequence is referred to as a survival target (VT). An example of a survival target in some cells is the IRS-2 protein. Cells expressing the IRS-2 protein include islet cells (e.g., beta cells) and substantia nigra (e.g., dopaminergic neurons). In other embodiments, survival targets for specific tissues or cells can be identified by selecting target proteins or nucleic acid sequences (e.g., miRNAs) from the human genome (e.g., REF GenBank). This can be achieved using a variety of standard techniques, but one of the most common methods involves targeted gene knockout studies using mice or other primates. For most cell types, a survival target is not necessary, as it has been well documented that most human and other mammalian cell types remain viable in in vitro culture even in the absence of growth / proliferation. In rare cases where transplanted islet tissue containing viable beta cells, such as human beta cells, cannot survive indefinitely in culture, appropriate survival targets can be identified by systematic gene knockout studies using individual genes expressed in the cell type of interest. Gene expression analysis and generation of knockout mice are standard techniques in the art. In such cases, identification of suitable survival targets is determined by the loss of the corresponding cell type of interest during embryonic or early postnatal development in knockout mice, where the absence of the gene encoding the VT protein leads to progressive loss of the relevant cells (Withers et al., 1998; Withers et al., 1999; Hennige et al., 2003). Example 1 provides such an approach.

[0049] In these embodiments that identify a survival target, the method further includes selecting or designing a compound known to activate, stabilize, or inhibit degradation of the VT. Such compounds are referred to herein as survival target enhancers (VTEs). Compounds may be used if they are known to activate VT. Alternatively, if such compounds do not exist, they may be created, for example, by using antisense oligonucleotides or small interfering ribonucleic acid (siRNA) targeting genes known to cause VT degradation. The net effect of such treatment is to increase the relative intracellular abundance of VT. VTEs that activate, stabilize, or inhibit degradation of the survival target IRS-2 protein include IRS activators. Non-limiting examples of IRS activators include HP-508 (Housey and Balash, 2018; Housey and Balash, 2023).

[0050] Proliferating cells (e.g., tissue samples and / or cells of interest) involves culturing them in the presence of a substance that promotes cell division. In one embodiment, the substance inhibits the expression of cell division locking (CDL) proteins. Inhibiting CDL protein expression triggers the initiation and maintenance of cell growth. Using a substance that promotes cell division minimizes the time cells must grow ex vivo before being reimplanted into a patient. Maintaining the phenotypic characteristics of the selected cell type (e.g., hormone secretion, neurotransmitter release, etc.) is important. Keeping these phenotypic characteristics to a minimum during ex vivo culture is necessary to obtain a therapeutically effective cell type suitable for reimplantation. Suitable CDL proteins include, but are not limited to, proteins that regulate metabolic status and tumor suppressor proteins (tumor suppressors). Specific examples include, but are not limited to, siRNAs designed to target proteins encoded by any of the following genes:These siRNAs are synthesized individually and tested for their ability to promote growth of cells of interest: activin genes (all mutants), myosin (all mutants), ANX7, APC, ARF, ATM, ATR, BCL2, BECLIN1, BIM, BLM, BMPR, BRCA1, BRCA2, BUB3, α-Catenin, CBFA2 / AML1 / RUNX1, CDH1 (E-CAD, E-Cadherin), CDKN1A, CDKN1B, CDKN2A, CDKN2C, CHK 1, EXT1, EXT2, FBXW7(CDC4), FEN1, FHIT, FST, H2AX, HIPK2, HRPT2, INPP4B, Integrin, LIG4, LKB1, MAD2, MEN1, MEN2, MKNK2, MLH1, M SH2, MSH6, MUTYH, NBS1, NF1, NF2, NKX3.1(NKX3A), P15, P53, PLK4, PMS1, PMS2, PTC, PTCH, PTEN, PTPN1, RB, RB1, RECQL4, Ribosomal Protein Gene (L35, L37A, RPS19, S8), RNASEL, SDH, SMAD2, SMAD3, SMAD4 / DPC4, STK4, SU(FU), TEAD1, TGFH, TGFβR, TSC-1, TSC-2, VGLL4, VHL, WRN, WT1, and TP53. Specific examples include TP53, STK4, VGLL4, TSC-1, CDKN1B, and CDKN2A (Macleod, 2000; Payne and Kemp, 2005). Optionally, the cells of interest can be screened to identify siRNAs, RNA sequences, or RNA analogs capable of suppressing CDL protein expression. This allows the cells of interest to temporarily re-enter the cell cycle and proliferate for a sufficient period of time to obtain a sufficient cell mass for therapy.

[0051] Those skilled in the art will recognize that there are a variety of known methods that can be used to inhibit the expression of CDL proteins, including, but not limited to:

[0052] Gene silencing using short interfering RNA (siRNA) and related approaches using RNA interference (RNAi) are preferred embodiments (Housey and Balash, 2019). It is now well established that RNAi plays an important role in post-transcriptional gene silencing via molecules such as siRNA. siRNAs are approximately 19-22 nucleotide (nt) double-stranded RNA (dsRNA) molecules that have the ability to suppress or silence the translation of messenger RNAs (mRNAs) in a sequence-specific manner (Walton et al., 2010; Sibley et al., 2010).

[0053] Polynucleotides can be used to suppress the expression of specific genes. Such inhibitory polynucleotides include RNA interference (RNAi) mediated by double-stranded small interfering RNA (siRNA), which silences genes with extremely high specificity. siRNA contains a sequence complementary to a protein-coding messenger RNA (mRNA) and leads to mRNA degradation. Those skilled in the art can design and synthesize siRNA molecules capable of suppressing CDL protein expression. siRNA molecules for suppressing CDL protein expression are commercially available (e.g., Dharmacon, Lafayette, Colo.). Automated synthesis of nucleic acids has been established and includes modifications at numerous positions on the nucleotide and ribose / deoxyribose ring systems (Sibley et al., 2010; Walton et al., 2010).

[0054] Another type of inhibitory nucleotide includes antisense RNA, which is a single strand complementary to a protein-encoding mRNA and blocks its translation into protein by hybridizing with the mRNA. The siRNA used in the methods herein has the ability to suppress the expression of CDL protein. RNA interference methods represent a useful approach in molecular targeted therapy. Therefore, in another embodiment, siRNA or other RNAi methods are utilized. The siRNA is synthesized and functionally tested to suppress the expression of CDL protein in a therapeutically effective manner in mammals. Oligonucleotide synthesis methods for producing siRNA have been established. Forms of RNAi that can be used to suppress CDL protein levels in mammals include, but are not limited to, RNA-DNA chimeras, tandem hairpin RNAs, tandem siRNAs, tRNA-shRNAs, etc. (Sibley et al., 2010).

[0055] In one embodiment, polynucleotides useful herein include double-stranded RNA (dsRNA) polynucleotides. The sequence of a polynucleotide, as used herein, includes a sense strand and a single strand of 16 to 30 nucleotides, e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The sense strand is substantially identical, preferably completely identical, to a target mRNA (e.g., an mRNA encoding a CDL protein). As used herein, the term "identical" means that the nucleotide sequence of the sense strand completely matches a portion of the nucleotide sequence of the target mRNA. As used herein, the term "substantially identical" means that the sequence of the sense strand differs from the sequence of the target mRNA by one, two, or three nucleotides (preferably one nucleotide), with the remaining nucleotides being identical to the mRNA sequence. The one, two, or three nucleotides in the sense strand are referred to as non-complementary nucleotides. When polynucleotide comprises the sense strand that is substantially identical to target mRNA, 1, 2 or 3 non-complementary nucleotides are preferably located at the center of sense strand.For example, when sense strand is 21 nucleotides long, non-complementary nucleotides are usually located at nucleotide 9, 10, 11 or 12, preferably nucleotide 10 or 11.In this specification, the other strand of dsRNA polynucleotide is called antisense strand, and is complementary to sense strand.

[0056] The sense and antisense strands of a dsRNA polynucleotide may be covalently linked by a spacer, typically consisting of nucleotides. Such polynucleotides are known in the art as short hairpin RNAs (shRNAs). Base pairing between the sense and antisense strands causes the spacer region to form a loop. The number of nucleotides constituting the loop varies, with loops of 3 to 23 nucleotides reported (Sui et al., Proc. Nat'l. Acad. Sci. USA, 99, 5515-5520 (2002) and Jacque et al., Nature, 418, 435-438 (2002)).

[0057] In one embodiment, the polynucleotide useful herein comprises a single-stranded RNA (ssRNA) polynucleotide. The sequence of the polynucleotide comprises a single strand having at least 16 nucleotides, referred to herein as the antisense strand. The antisense strand is substantially complementary, preferably completely complementary, to a target mRNA (e.g., an mRNA encoding a CDL protein). In one embodiment, the polynucleotide for reducing the expression of a coding region in a cell comprises substantially the entire coding region, or in some cases the entire coding region. The antisense strand is substantially complementary, preferably completely complementary, to a target coding region or target mRNA. As used herein, the term "substantially complementary" means that at least 1, 2, or 3 nucleotides of the antisense strand are not complementary to the nucleotide sequence of the target mRNA.

[0058] The polynucleotides of the present disclosure are preferably biologically active. Biologically active polynucleotides cause post-transcriptional suppression (also known as silencing) of the expression of a target coding region. Without intending to be bound by theory, when the polynucleotides of the present invention are introduced into a cell, they hybridize to the target mRNA and signal an intracellular endonuclease to cleave the target mRNA. As a result, expression of the polypeptide encoded by the mRNA is suppressed. Whether expression of the target coding region is suppressed can be determined, for example, by measuring a decrease in the amount of target mRNA in the cell, a decrease in the amount of the polypeptide encoded by the mRNA, or a decrease in the activity of the polypeptide encoded by the mRNA.

[0059] Polynucleotides of the present disclosure may contain additional nucleotides, for example, the sense strand may include additional nucleotides at the 5' end, 3' end, or both ends, as long as the additional nucleotides are identical to the corresponding target mRNA and the overall length of the sense strand does not exceed 30 nucleotides.

[0060] Polynucleotides may be modified. Such modifications are useful for increasing the stability of polynucleotides under certain circumstances. Modifications may involve the sugar, base, backbone, or any combination thereof of the nucleic acid. Modifications may be synthetic, naturally occurring, or non-naturally occurring. Polynucleotides may contain modifications in one or more nucleic acids within the polynucleotide. Examples of backbone modifications include, but are not limited to, phosphonoacetate, thiophosphonoacetate, phosphorothioate, phosphorodithioate, phosphoramidate, methylphosphonate, chiral methylphosphonate, 2-O-methylribonucleotide, peptide nucleic acid, and the like. Examples of nucleobase modifications include, but are not limited to, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouridine, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), or 6-azapyrimidine, or 6-alkylpyrimidine (e.g., 6-methyluridine), or propyne modifications. Examples of nucleic acid sugar modifications include, but are not limited to, 2'-sugar modifications such as 2'-O-methyl nucleotides, 2'-deoxy-2'-fluoro nucleotides, 2'-deoxy-2'-fluoroarabino, 2'-O-methoxyethyl nucleotides, 2'-O-trifluoromethyl nucleotides, 2'-O-ethyl-trifluoromethoxy nucleotides, 2'-O-difluoromethoxy-ethoxy nucleotides, 2'-deoxy nucleotides, etc. Polynucleotides can be commercially synthesized to contain such modifications (e.g., Dharmacon Inc., Lafayette, Colo.).

[0061] Those skilled in the art will recognize that the methods may include ensuring that the tissue and / or cells of interest maintain the appropriate phenotypic characteristics of the selected cell type (e.g., hormone secretion, neurotransmitter release, etc.) necessary to obtain a therapeutic cell population for reimplantation. For example, if the tissue and / or cells of interest are derived from pancreatic tissue (e.g., islets of Langerhans), sustained production of insulin is desirable, and if the tissue and / or cells of interest are derived from substantia nigra tissue (e.g., dopaminergic neurons), sustained production of dopamine is desirable.

[0062] Once a therapeutically effective amount of cells / tissue is obtained, the cells are reimplanted at the appropriate site. For example, in the case of beta cells for the treatment of diabetes, cells may be injected directly into the liver via the portal vein (McEachron et al., 2018). In the case of dopamine-secreting neurons for the treatment of Parkinson's disease, cells may be reimplanted into the brain by inserting a catheter into the substantia nigra using standard neurosurgical procedures with real-time imaging, followed by injection of cells suspended in an isotonic buffer. The number of cells reimplanted may vary depending on the patient's disease state and the extent of tissue loss. In some embodiments, at least 100,000 cells, at least 200,000 cells, at least 300,000 cells, at least 400,000 cells, at least 500,000 cells, or at least 600,000 cells are administered. In some embodiments, no more than 100,000,000,000 cells are administered.

[0063] Master stocks of tissues and / or cells of interest can be expanded and cryopreserved, allowing for the potential therapeutic benefit of reimplanting additional functional tissue if disease recurs years or even decades later. This approach could achieve sustained long-term remission or functional cure for certain diseases, such as T1D and Parkinson's disease.

[0064] Optionally, the method may further comprise monitoring the status of the re-implanted tissue. If the re-implanted tissue comprises beta cells, the method may further comprise determining whether the subject's T1D-related condition has improved, such as whether their blood glucose control has been restored. If the re-implanted tissue comprises dopaminergic neurons, the method may further comprise determining whether the subject's Parkinson's disease-related condition has improved, such as whether their dopamine production has increased. Alternatively, elevated levels of brain-derived neurotrophic factor can also be monitored (see Example 2 below).

[0065] Type 1 diabetes (T1D, formerly known as early-onset diabetes) develops when a patient's immune system mistakenly attacks the beta cell population of the pancreatic islets of Langerhans. T1D is therefore an autoimmune disease. The current standard of care for T1D centers around continuous insulin administration, of which various types are available.

[0066] Numerous cell therapy approaches for T1D have been attempted using islets of Langerhans from various sources, including bovine, porcine, and human cadaveric donors, but have met with little success. The longest-lasting approach to date is the transplantation of human islets isolated from cadaveric donors. Because these donors are not genetically identical to the recipient, allogeneic transplant recipients must continue to take immunosuppressant medications to reduce immune-mediated graft rejection. Even when successful, such transplants typically last only 3–5 years, after which the transplant tissue ceases to function, and the recipient must either return to daily insulin therapy or receive a second transplant from a different allogeneic donor.

[0067] Because allogeneic transplantation has significant life-threatening limitations, the FDA (U.S. Food and Drug Administration) has designated all forms of allogeneic transplantation as an experimental procedure. More information on potential side effects and adverse events associated with allogeneic transplantation can be found on the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) website (see Pancreatic Islet Transplantation: https: / / www.niddk.nih.gov / health-information / diabetes / overview / insulin-medicines-treatments / pancreatic-islet-transplantation). As noted, the most serious side effects and adverse events associated with allogeneic transplantation are related to the chronic immunosuppressive treatment required to avoid graft-versus-host disease and immune rejection by the recipient, which results in functional loss of the transplanted islets. Chronic immunosuppression is required even if the donor islets are derived from induced pluripotent stem cells rather than from a cadaveric donor pancreas. Recipients may develop antibodies against the donor cells, complicating efforts to find a suitable organ donor for future (and almost always necessary) retransplantation.

[0068] Most importantly, the long-term use of chronic immunosuppressants can cause many serious side effects. These immunosuppressants are essential to prevent the recipient from destroying the donor islets, regardless of their origin (iPSC or cadaveric donor). According to the NIDDK website, potential side effects associated with long-term immunosuppressant use include increased risk of infection, increased incidence of cancer, gastrointestinal side effects (e.g., vomiting, nausea, diarrhea), headache, tremors, high blood pressure, hypercholesterolemia and hypertriglyceridemia, and kidney damage (https: / / www.niddk.nih.gov / health-information / diabetes / overview / insulin-medicines-treatments / pancreatic-islet-transplantation).

[0069] Despite the best efforts of many laboratories, it has been shown that it is not possible to maintain human islet tissue isolated from cadaveric donors in vitro for more than a few months, during which time no significant growth occurs and insulin synthesis and release gradually declines. Therefore, cellular approaches to replace insulin-producing beta cells have relied on either allogeneic transplantation, performed after isolation from cadaveric donors, or the use of insulin-producing cells differentiated from iPSCs. Both of these approaches still require the use of immunosuppressants, and the iPSC approach presents additional long-term safety concerns, as discussed above. [Example]

[0070] The present disclosure is illustrated by the following examples, and it should be understood that the specific examples, materials, amounts, and procedures are to be broadly interpreted in accordance with the scope and spirit of the disclosure set forth herein.

[0071] Example 1 Treatment of type 1 diabetes by in vitro replication of human insulin-producing beta cells Human islets of Langerhans (including beta cells) maintained in tissue or organ culture do not proliferate to sufficient numbers to provide therapeutic benefit for retransplantation. Therefore, to maintain cells in culture without continued loss, it is necessary to identify and activate survival targets, such as those mentioned above. Mouse gene knockout experiments have shown that insulin receptor substrate-2 (IRS-2) contributes to maintaining cell viability in beta cells present in the pancreatic islets of Langerhans (Hennige et al., 2003; Kuznetsova et al., 2016). Therefore, animals with both IRS-2 alleles knocked out develop diabetes shortly after birth, and transgene replacement of IRS-2 reverses the diabetes. Therefore, IRS-2 fulfills the criteria for a survival target (VT).

[0072] These studies used a compound known to activate IRS-2 (designated HP-508). HP-508 is a representative IRS-2 activator (Housey and Balash, 2018; Housey and Balash, 2023). As described herein, HP-508 is classified as a survival target enhancer (VTE).

[0073] Individuals with T1D may wish to expand their remaining beta cells ex vivo and re-implant with autologous genetically identical tissue containing sufficient beta cells to resolve diabetes and normalize blood glucose levels. The individual will simultaneously act as both donor and recipient, receiving their own genetically identical beta cells upon completion of the treatment process.

[0074] Most patients with T1D have been shown to have residual functional beta cells in their pancreases. The presence of these functional beta cells can be confirmed with a simple blood test that detects C-peptide, a by-product of endogenous insulin production by beta cells. To determine whether a patient has residual beta cells in their pancreas, a blood sample is drawn and C-peptide levels are measured. If a patient's C-peptide level is 5% or higher of the normal range (approximately 0.04 ng / ml or higher), functional beta cells are present and the patient is a candidate for this treatment.

[0075] Next, a surgical biopsy specimen is taken from the pancreas. Typically, the tail of the pancreas is removed, and approximately 20–25% of the total pancreatic tissue is harvested. This allows for the patient to maintain normal pancreatic exocrine function by preserving 75–80% of the remaining pancreatic tissue. Depending on the patient's C-peptide production level, a smaller biopsy sample may be sufficient.

[0076] Figures 2-6 show the results of human islets obtained from cadaveric donor pancreases by surgical resection. After resection, the pancreatic tissue was processed according to a standard islet tissue isolation procedure. This procedure has been previously published for allogeneic donor islet transplantation and allogeneic transplantation for patients with chronic pancreatitis (McEachron and Bellin, 2018).

[0077] After islet isolation, the total number of isolated islets is determined using standard methods (see Chetboun et al., 2023 and references therein). The isolated islets are then expanded under the following culture conditions:

[0078] Islets of Langerhans isolated from biopsy samples are first counted, and the initial total number is recorded. The tissue is then transferred to tissue culture medium supplemented with the IRS-2 activator HP-508. The tissue is then allowed to acclimate to in vitro culture conditions for periods ranging from one hour to several days. The presence of HP-508 (referred to as VTE for this purpose) serves to maintain the viability of the islet tissue, while periodic subculture under specific conditions selectively removes fibroblast-like cells that contaminate the biopsy sample and proliferate at a significantly faster rate than human islet tissue. The ability of HP-508 to improve cell viability is demonstrated in Figure 2.

[0079] Islet tissue maintained in the presence of IRS activators survives for over a year and exhibits a slow but sustainable growth pattern (Figure 3). In our laboratory, human islet tissue has been maintained under these conditions for over three years and has demonstrated sustained, slow growth. However, without the addition of the cell proliferation promoters described herein, the growth rate is insufficient to allow retransplantation within 12 months.

[0080] After culture adaptation is complete, a second substance is added to the culture medium to promote islet tissue cell growth. As shown in Figure 3, after adding the substance that enhances islet tissue cell growth, the total tissue surface area increases approximately fourfold over a 64-day period. (Measuring tissue surface area proves useful for estimating total wet tissue mass.) This corresponds to a tissue doubling time of approximately 32 days. While still a slow rate compared to rapidly dividing cell lines, this growth rate is sufficient to produce a total islet tissue mass equivalent to at least 400,000 islet equivalents within 6–7 months, even when the initial islet mass immediately after biopsy is only 5,000 islet equivalents. Allogeneic islet transplantation typically uses 5,000–10,000 islet equivalents per kg of body weight, which corresponds to approximately 350,000–700,000 islet equivalents in a 70 kg individual (McEachron and Bellin, 2018). If more islets are obtained from the initial biopsy, the minimum required growth period is shortened.

[0081] Substances that promote cell growth in combination with IRS activators include activators of cyclophosphamide-dependent kinases that regulate cell cycle division, and substances that block proteins that block cell cycle division under normal conditions, including proteins that control metabolic status and proteins that prevent tumor formation (tumor suppressors).

[0082] As shown in Figure 5, by combining an IRS activator with a cell growth promoter, large enough islet tissue masses were formed, and even blood vessel-like structures spontaneously developed as the islets grew and expanded.

[0083] The present disclosure obviates the need for long-term use of immunosuppressive drugs, thereby avoiding the numerous side effects associated with their use. Furthermore, unlike iPSC-mediated approaches, there is no need to dedifferentiate selected cells into iPSCs and then redifferentiate them into insulin-producing "beta-like cells." This avoids the generation of hybrid cell types whose long-term stability after reinfusion into the patient is uncertain.

[0084] Example 2 Regeneration of dopaminergic neurons isolated from human substantia nigra tissue Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra, a deep region of the human brain located just above the basal ganglia. Dopaminergic neurons in the substantia nigra have been shown to require IRS-2 for their survival (Xie et al., 2021; Ramalingam et al., 2017). Loss of IRS2 function results in a loss of dopaminergic neurons.

[0085] The compound HP-508 has been shown to activate IRS2, which acts as a VTE. Neurons were cultured in a medium containing HP-508 in an amount sufficient to maintain healthy cells.

[0086] PD patients lose dopaminergic neurons present in the substantia nigra. A portion of healthy dopaminergic neurons is harvested from the patient via neurosurgical biopsy and expanded ex vivo. At the time of the biopsy, an indwelling catheter is inserted into the substantia nigra and maintained therein during the period of neuronal growth and proliferation. The harvested cells are expanded ex vivo, and the expanded cells are then re-implanted. Re-implantation of functional dopaminergic neurons helps ameliorate past symptoms. In this example, as in Example 1, individuals are both donors and recipients of dopamine cells. Thus, these cells are syngeneic or autologous (genetically identical) to the surrounding tissue, reducing the likelihood of an autoimmune response in the patient.

[0087] After culture adaptation is complete, a second substance is added to the culture medium to promote cell growth of neuronal tissue. The appropriate amount of neurons required for retransplantation is determined by the physician based on the degree of substantia nigra tissue loss observed through imaging studies. Furthermore, brain-derived neurotrophic factor (BDNF) has been shown to be reduced in PD patients. Therefore, administering a therapeutically effective amount of ex vivo expanded dopaminergic substantia nigra neurons restores BDNF blood levels to normal levels (Palasz et al., 2020). Blood samples can be measured using an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody directed against a known BDNF antigen or its fragments to determine when a sufficient number of cells have been retransplanted. Furthermore, the indwelling catheter may be maintained long enough to retransplant additional cells until biomarkers associated with normal function are achieved.

[0088] Example 3 Treatment of sensorineural hearing loss by ex vivo expansion of human cochlear hair cells Cells located in the cochlea of ​​the inner ear are essential for normal hearing. These cells contain hair follicles and are essential for converting sound and vibration into electrical signals in spiral ganglion neurons and transmitting these signals to the brain. Sensorineural hearing loss is a major form of hearing loss worldwide, and the current mainstay of treatment is hearing aids. It has not been possible to grow fully differentiated inner ear hair cells in vitro, and stem cell approaches are currently being tested, but challenges remain, as discussed herein (Qi et al., 2023).

[0089] Using the disclosed method, an ENT surgeon obtains a biopsy of inner ear hair cells and expands them ex vivo as described in Examples 1 and 2. Following ex vivo expansion, a therapeutically effective amount of the cells needed to replace lost cochlear hair cells is reinjected in an amount sufficient to regrow the inner cochlear wall as functional hair cells. This approach restores hearing without relying on external hearing aids.

[0090] Those skilled in the art will readily recognize that such an approach is adaptable to other tissue retransplantation applications, such as multiple sclerosis, where irregenerative oligodendrocyte and Schwann cell demyelination occurs, specific motor neuron loss in spinal cord injury, and other human diseases where progressive loss of specific functional cell types occurs.

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[0092] The complete disclosures of all patents, patent applications, and publications cited herein, as well as electronically available materials (e.g., nucleotide sequence data submissions to GenBank and RefSeq, etc., amino acid sequence data submissions to SwissProt, PIR, PRF, PDB, etc., and translations from annotated coding regions in GenBank and RefSeq), are hereby incorporated by reference in their entirety. Supplementary materials cited in publications (e.g., supplementary tables, figures, materials and methods, and / or experimental data) are also hereby incorporated by reference in their entirety. In the event of a discrepancy between the disclosure of this application and the disclosure of any document incorporated by reference, the disclosure of this application governs. The foregoing detailed description and examples are provided for ease of understanding only and should not be read as unnecessary limitations. The present disclosure is not limited to the exact details shown and described herein, as variations obvious to those skilled in the art will be within the scope of the disclosure as defined by the claims.

[0093] Unless otherwise indicated, all numbers expressing quantities of ingredients, molecular weights, and the like used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless otherwise indicated, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the recitation of equivalents to the scope of the claims, each numerical parameter should be construed in light of at least the number of reported significant digits and by applying ordinary rounding techniques.

[0094] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, all numerical values ​​inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0095] All headings herein are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so designated.

Claims

1. 1. A method for growing tissue or cells in vitro, comprising: (a) providing a biopsy specimen of said tissue from an individual having a disease, said disease resulting in loss of said tissue; (b) expanding the tissue or cells in vitro to obtain expanded tissue or cells, wherein the expansion comprises culturing the tissue in the presence of a compound that activates an insulin receptor substrate (IRS) protein and a substance that enhances cell division; (c) reimplanting a therapeutically effective amount of said expanded tissue or cells into said individual.

2. 10. The method of claim 1, wherein the disease comprises type 1 diabetes or Parkinson's disease.

3. The method of claim 1, wherein the substance comprises an siRNA that reduces the expression of TP53, STK4, VGLL4, or TSC-1.

4. 10. The method of claim 1, wherein the therapeutically effective amount of the expanded tissue comprises a mass equivalent to at least 400,000 cells.

5. The method of claim 1 , wherein the individual is a human.

6. 1. A method of treating a subject having diabetes, comprising: (a) providing a biopsy specimen of pancreatic tissue from an individual with diabetes; (b) isolating the islets of Langerhans contained in said biopsy specimen; (c) expanding the islets of Langerhans in vitro to obtain expanded islets of Langerhans, wherein the expansion comprises culturing the islets of Langerhans in the presence of a compound that activates an insulin receptor substrate (IRS) protein and a substance that enhances cell division; (d) retransplanting a therapeutically effective amount of the expanded islets of Langerhans into the individual.

7. The method of claim 6, wherein the substance comprises an siRNA that reduces the expression of TP53, STK4, VGLL4, or TSC-1.

8. The method of claim 6, wherein the therapeutically effective amount of the expanded islets of Langerhans comprises a mass equivalent to at least 400,000 islets of Langerhans.

9. 7. The method of claim 6, wherein the reimplantation comprises portal vein injection, omental injection, or a combination thereof.

10. 7. The method of claim 6, wherein the method further comprises determining whether glycemic control has been restored in the subject.

11. The method of claim 6 , wherein the subject is a human.

12. 1. A method for expanding human insulin-producing tissue in vitro, comprising: (a) providing a biopsy specimen of pancreatic tissue from a diabetic subject having a relative or absolute deficiency of insulin; (b) isolating the islets of Langerhans contained in said biopsy specimen; (c) expanding the islets of Langerhans to obtain expanded islets of Langerhans, wherein the expansion comprises culturing the tissue in the presence of a compound that activates insulin receptor substrate (IRS) protein and a substance that enhances cell division; (d) confirming that the proliferated islets of Langerhans continue to produce insulin.

13. The method of claim 12, wherein the substance comprises an siRNA that reduces the expression of TP53, STK4, VGLL4, or TSC-1.

14. 13. The method of claim 12, wherein the therapeutically effective amount of the expanded islets of Langerhans comprises a mass equivalent to at least 400,000 islets of Langerhans.

15. The method of claim 12, further comprising retransplanting the expanded islets of Langerhans.

16. 16. The method of claim 15, wherein the reimplantation comprises portal vein injection, omental injection, or a combination thereof.

17. 13. The method of claim 12, wherein the biopsy specimen is obtained from a human.

18. 1. A method of treating a subject having Parkinson's disease, comprising: (a) providing a biopsy specimen of the substantia nigra region of the brain of said subject, said biopsy specimen comprising dopaminergic neurons; (b) expanding the substantia nigra tissue in vitro to obtain expanded substantia nigra tissue, wherein the expansion comprises culturing the tissue in the presence of a compound that activates an insulin receptor substrate (IRS) protein and a substance that enhances cell division; (c) reimplanting a therapeutically effective amount of said proliferated tissue into said individual.

19. The method of claim 18, wherein the substance comprises an siRNA that reduces the expression of TP53, STK4, VGLL4, or TSC-1.

20. 20. The method of claim 18, wherein the therapeutically effective amount of the expanded tissue comprises a mass equivalent to at least 400,000 cells.

21. 20. The method of claim 18, wherein the reimplantation comprises transcranial injection, intracranial injection, or a combination thereof.

22. 20. The method of claim 18, further comprising determining whether substantia nigra function has recovered in the subject.

23. 20. The method of claim 18, wherein the biopsy specimen is obtained from a human.

24. 1. A method for growing human dopaminergic substantia nigra neural tissue in vitro, comprising: (a) providing a biopsy specimen of the substantia nigra containing dopaminergic neurons from a subject with Parkinson's disease; (b) expanding the biopsy specimen of the substantia nigra in vitro to obtain expanded substantia nigra cells, said expansion comprising culturing the biopsy specimen in the presence of a compound that activates an insulin receptor substrate (IRS) protein and a substance that enhances cell division; (c) confirming that the proliferated substantia nigra tissue continues to produce dopamine.

25. 25. The method of claim 24, wherein the material comprises a mass equivalent to at least 400,000 cells.

26. 25. The method of claim 24, wherein the biopsy specimen is obtained from a human.

27. 25. The method of claim 24, further comprising reimplanting a therapeutically effective amount of the expanded cells into the individual.

28. 28. The method of claim 27, wherein the reimplantation comprises transcranial injection, intracranial injection, or a combination thereof.

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