FIELD

The present disclosure relates generally to the field of cell biology, stem cells, and cellular differentiation. More specifically, this disclosure provides methods for generating pancreatic cells, methods of identifying cells for cell-based therapy, and related methods of use for treating diabetes.

BACKGROUND

The following discussion is provided to aid the reader in understanding the disclosure and is not admitted to describe or constitute prior art thereto.

Diabetes and Insulin

Diabetes mellitus (i.e., diabetes) is a disease in which the body's ability to produce or respond to the hormone insulin is impaired, resulting in abnormal metabolism of carbohydrates and elevated levels of glucose in the blood and urine. The disease is subdivided into several sub-types, described alternatively as Type diabetes mellitus, insulin-dependent diabetes mellitus (IDDM), mature onset diabetes of the young (MODY), latent adult diabetes (LADA), brittle diabetes, lean diabetes, Type 1.5, Type 2, Type 3, obesity-related diabetes, gestational diabetes, and other nomenclature accepted by the field.

In general, a subject with insulin-dependent diabetes is required to administer exogenous insulin to sufficiently lower blood glucose. A non-insulin-dependent subject may sufficiently lower blood glucose with pharmaceutical intervention including classes of drugs that enhance sensitivity to insulin, or excretion of glucose. A subject with insulin-dependent diabetes may benefit from a cell replacement therapy in which insulin-producing cells are implanted to the subject whether that disease is labeled as type 1, MODY, LADA, brittle, lean, Type 1.5, Type 2, Type 3, obesity related diabetes or any combination thereof.

Type I diabetes is usually diagnosed in children and young adults, and was previously known as juvenile diabetes. Only 5-10% of people with diabetes have this form of the disease. Mature onset diabetes is the most common form of the disease, and it arises due to the impairment or destruction of insulin-producing beta cells, development of insulin resistance, or both impairment of insulin-producing beta cells and development of insulin resistance. Diabetes can arise in non-obese adults and children due to a combination of genetic and environmental factors. In obese adults and children, the pancreas may attempt to make extra insulin in order to control blood glucose, but over time it is unable to keep up and maintain blood glucose at normal levels. The body may also become less sensitive to the insulin that is produced. Prolonged over-activity of the insulin-secreting beta cells may lead to beta-cell dysfunction and death.

Diabetes symptoms vary depending on how much a subject's blood glucose fluctuates. Some people, especially those with prediabetes or non-insulin dependent diabetes, may not experience symptoms initially. In Type I diabetes, symptoms tend to come on quickly and are more severe.

Some of the signs and symptoms of Type I and Type II diabetes include, but are not limited to, increased thirst; frequent urination; extreme hunger; unexplained weight loss; presence of ketones in the urine (ketones are a byproduct of the breakdown of muscle and fat that happens when there is not enough available insulin); fatigue; irritability; blurred vision; slow-healing sores; frequent infections, such as gums or skin infections and vaginal infections.

Cell-Based Therapies For Treating Diabetes

Insulin-dependent diabetes patients can potentially be cured with transplantation of new insulin producing cells, but this approach has been limited to date because these cells are hard to obtain in sufficient quantity and quality. See e.g. Pagliuca F W, et al.Cell,154(2):428-439 (2014). It has, therefore, long been a goal of biomedical research to generate insulin producing beta cells from human stem cells in a more efficient and predictable manner. Id. To achieve this goal, protocols must be established that generate uniform beta cell populations that produce insulin when exposed to glucose. However, such a protocol has remained elusive. Numerous research groups have proposed various protocols that have produced varied results using different cell lines. This inconsistent production of functional beta cells increases the total cellular dose required in order to achieve therapeutic benefit, thus increasing the cost of a potential therapy and limiting clinical applicability due to variable results.

Most established protocols for generating insulin-producing beta cells from human stem yields highly variable cell populations. See e.g. Pagliuca F W, et al.Cell,154(2):428-439 (2014). Indeed, it has been common practice in the field to tailor a given differentiation protocol to a specific cell line, thus preventing any standardization for differentiation within the art. Approaches to improve beta cell yield from various differentiation protocols has typically involved testing numerous combinations of factors that influence differentiation pathways in an iterative, trial-and-error type of approach. See e.g. Pagliuca F W, et al. Cell, 154(2):428-439 (2014); Rezenia A. et al.Nat. Biotech.,32:1121-33 (2014); Schulz TC et al. Plos One, 7:e37004 (2012). Chetty S. et al.Nat. Methods,10:553-556 (2012). Therefore, a particular protocol for making beta cells from one starting stem cell population may be ineffective for differentiating a different starting population.

Moreover, inconsistency within the resulting differentiated cell populations has impacted the clinical application of any proposed therapy, as researchers in this field have struggled to obtain differentiated populations with consistently high percentages of insulin-producing cells. This not only detracts from the potential efficacy of cell-based therapy to treat diabetes, but also raises concerns regarding the tumorigenic potential of cell transplants containing a population of heterologous cells. Additionally, low reproducibility between cell batches directly affects the cost of producing the cells and limits the translation of this protocol to the clinic.

Some of this variability in the employment of distinct differentiation protocols can be traced to the starting cell line. For example, it is shown that pluripotent cell lines can vary widely in their ability to differentiate to certain lineages. See, Bock C, et al.Cell,144:439-452 (2011); Lim H, et al.J. Vis. Exp., (90):e51755 (2014); Osafune K, et al. Nat. Biotechnol., 26:313-315 (2008). This varying capacity of individual human stem cells in their response to currently utilized differentiation protocols has proved to be a difficult obstacle to overcome, as researchers had no indication of the potential for a given cell line or starting cell to eventually yield therapeutic cells.

In addition, guidance provided from prior art may in fact be counter-productive when using a cell line with a slightly different genetic background. For example, a consistent feature of established protocols to generate insulin-producing cells from pluripotent stem cells is to limit the exposure to retinoic acid and cyclopamine. See Nostro et al.Stem Cell Reports,4:1-14 (2015). Another consistent guidance from the prior art is that initiating differentiation in three-dimensional cultures in suspension is required for proper maturation of the pancreatic cells. See Pagliuca F W, et al.Cell,154(2):428-439 (2014), Rezania et al.Nature Biotechnology, 32(11):1121-33 (2014). As discussed in more detail below, following such guidance may actually inhibit differentiation of insulin-secreting cells from various stem cell lines.

Thus, there remain needs for improved and predictable methods of generating therapeutic, insulin-producing cells for the treatment of diabetes. The present disclosure fulfills those needs.

SUMMARY

Described herein are cells and cellular compositions that produce insulin and may be used to treat diabetes, as well as methods of making and identifying the same.

In one aspect, the present disclosure provides methods of producing mammalian insulin-secreting cells, comprising: culturing mammalian stem cells in adhesion, thereby allowing the mammalian stem cells to spontaneously form three-dimensional structures; and culturing of the three-dimensional structures in suspension; wherein the culturing steps comprise at least a 20-day exposure to retinoic acid and cyclopamine, and do not comprise exposing the stem cells of three-dimensional structures to Wnt3A.

In another aspect, the present disclosure provides methods of producing insulin-secreting cells, comprising: culturing mammalian stem cells on an adhesive substrate in a first medium comprising Activin-A and Wortmannin, wherein the mammalian stem cells are not exposed to Wnt3a; further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine; and transferring the cells to a suspension culture when the cells form three-dimensional cell structures; wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

In some embodiments of the foregoing aspects, the mammalian stem cells may be human stem cells, while in some embodiments the mammalian stem cells may be non-human primate stem cells. In some embodiments of the foregoing aspects, the mammalian stem cells were derived from a cell line.

In one aspect, the present disclosure provides methods of producing mammalian insulin-secreting cells, comprising culturing mammalian stem cells in a first medium comprising an endoderm-inducing factor, thereby differentiating the mammalian stem cells into endoderm cells; and culturing the endoderm cells in a second medium comprising an endocrine-inducing factor, thereby differentiating the endoderm cells into endocrine cells; wherein the mammalian stem cells were not exposed to keratinocyte growth factors (KGF) prior to differentiation into endoderm cells.

In some embodiments, the mammalian stem cells may be human stem cells, non-human primate stem cells, or stem cells derived from another mammal, including but not limited to a pig, cow, sheep, horse, dog, or cat.

In some embodiments, the endoderm-inducing factor comprises Activin-A, retinoic acid, and/or cyclopamine. In some embodiments, the first medium may further comprise Wortmannin. In some embodiments, the first medium does not comprise Wnt3A. In some embodiments, the cells are cultured in the first medium for 1-3 days.

In some embodiments, the second medium may comprise noggin and/or KGF. In some embodiments, the second medium may comprise retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the second medium for 1-4 days.

Some embodiments of this aspect may comprise further culturing the endocrine cells in a third medium comprising KGF, thereby differentiating the endocrine cells into pancreatic progenitor cells. In some embodiments, the third medium comprises noggin and/or epidermal growth factor (EGF). In some embodiments, the third medium comprises retinoic acid and cyclopamine. In some embodiments the cells are cultured in the third medium for 1-4 days.

Some embodiments of this aspect may comprise further culturing the pancreatic progenitor cells in a fourth medium comprising noggin, EGF, γ-secretase inhibitor XXI, and/or Alk5i II. In some embodiments, the fourth medium may comprise T3. In some embodiments, the fourth medium may comprise retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the fourth medium for 1-4 days.

Some embodiments of this aspect may comprise further culturing the pancreatic progenitors in a fifth medium comprising Alk5i II and/or retinoic acid. In some embodiments, the fifth medium may comprise T3. In some embodiments, the fifth medium may comprise retinoic acid and cyclopamine. In some embodiments, the cells are cultured in the fifth medium for 1-5 days.

Some embodiments of this aspect may comprise further culturing the pancreatic progenitors in a sixth medium comprising Alk5i II, nicotinamide, and/or insulin-like growth factor (IGF)-I. In some embodiments, the sixth medium may comprise T3 and/or BMP4. In some embodiments, the sixth medium may comprise retinoic acid and cyclopamine. In some embodiments, the sixth medium may comprise glucagon. In some embodiments, the cells are cultured in the sixth medium for 1-9 days.

In another aspect, the present disclosure provides methods of producing insulin-secreting pancreatic cells, comprising culturing human stem cells in a first medium comprising Activin-A and Wortmannin, thereby differentiating the human stem cells into endoderm cells, wherein the human stem cells were not exposed to keratinocyte growth factors (KGF) prior to differentiation into endoderm cells; and culturing the endoderm cells in a second medium comprising retinoic acid and cyclopamine, thereby differentiating the endoderm cells into endocrine cells; culturing the endocrine cells in a third medium comprising KGF, noggin, and EGF, thereby differentiating the endocrine cells into pancreatic progenitor cells; and culturing the pancreatic progenitor cells in a fourth medium comprising noggin, EGF, γ-secretase inhibitor XXI, and Alk5i II, thereby differentiating the pancreatic progenitor cells into insulin-producing pancreatic cells.

In some embodiments of this aspect, the second culture medium further comprises KGF. In some embodiments, the human stem cells are derived from pancreatic primary tissue. In some embodiments, the fourth medium may comprise a thyroid hormone, such as T3.

In some embodiments of this aspect, both the third and fourth mediums may comprise retinoic acid and cyclopamine.

Some embodiments of this aspect may comprise further culturing the cells in a fifth and/or sixth medium, wherein the fifth medium comprising Alk5I II and retinoic acid, and optionally cyclopamine and wherein the sixth medium comprising Alk5i II, nicotinamine, IGF-I, and optionally retinoic acid and cyclopamine. In some embodiments, the sixth medium may comprise glucagon.

In some embodiments of the foregoing aspects, the cells may be cultured for 30 days or less.

In another aspect, the present disclosure provides cell-based compositions for treating diabetes, comprising a population of surrogate pancreatic cells and a suitable carrier for implantation into a human subject in need thereof, wherein at least 66% of the surrogate pancreatic cells are insulin-producing pancreatic cells.

In some embodiments, at least 66% of the surrogate pancreatic cells express NeuroD1, while in some embodiments, at least 68% of the surrogate pancreatic cells express Nkx6.1 .

In some embodiments, the insulin-producing pancreatic cells were derived according to a method comprising culturing a population of human stem cells on an adhesive substrate in a first medium comprising an endoderm-inducing factor, wherein the mammalian stem cells are not exposed to Wnt3a; further culturing the cells in at least one additional medium comprising retinoic acid and cyclopamine; and transferring the cells to a suspension culture when the cells form three-dimensional cell structures; wherein the cells are exposed to retinoic acid and cyclopamine for at least 20 days.

In some embodiments, the endoderm-inducing factor comprises Activin-A and/or Wortmannin. In some embodiments, the at least one additional medium may comprise KGF, noggin, EGF, and/or a thyroid hormone, such as T3.

In some embodiments, the surrogate pancreatic cells may be encapsulated in a macro-capsule. For example, the cells may be encapsulated in a macro-capsule comprising alginate, cellulose sulfate, glucomannan, or a combination thereof.

In some embodiments of the foregoing aspects, the human stem cells are derived from pancreatic primary tissue, are human embryonic stem cells, are induced pluripotent stem cell, or are reprogrammed cells that are not pluripotent. In some embodiments, the reprogrammed cells are derived from pancreatic primary tissue, for example, by expressing reprogramming genes without incorporating the reprogramming genes into the genome of the cell. In some embodiments, the reprogramming genes may be encoded on at least one episomal expression plasmid, which does not incorporate into the genome. In some embodiments, the reprogramming genes comprise Oct4, Sox2, Klf4, and L-Myc.

In another aspect, the present disclosure provides methods of identifying undifferentiated cells that preferentially differentiate into an endodermal lineage, comprising assessing expression of BHMT2 and NAP1L1 in an undifferentiated cell and identifying the cell as having a preference for differentiating into an endodermal lineage if BHMT2 expression is down-regulated relative to a control cell and NAP1L1 expression is up-regulated relative to a control cell.

Some embodiments of this aspect further comprise assessing the expression of Cox7A1 and HSPB2 in the undifferentiated cell and identifying the cell as having a preference for differentiating into an endodermal lineage if both Cox7A1 and HSPB2 expression are down-regulated relative to a control cell. In some embodiments, the control cell may be a pluripotent cell that does not exhibit preferential differentiation to the endodermal lineage or a substantial inability to differentiate to the mesodermal lineage.

In some embodiments, BHMT2 expression is down-regulated at least 2 logs relative to the control cell and NAP1L1 expression is up-regulated at least 2 logs relative to the control cell when the assessed undifferentiated cell preferentially differentiates into an endodermal lineage. In some embodiments, both Cox7A1 and HSPB2 expression are down-regulated at least 2 logs relative to the control cell when the assessed undifferentiated cell preferentially differentiates into an endodermal lineage.

Some embodiments of this aspect further comprise assessing expression of GLIS2, CCDC58, MTX3 and C7orf29. For example, in some embodiments, GLIS2, CCDC58, and MTX3 expression may be up-regulated relative to the control cell and C7orf29 expression may be down-regulated relative to the control cell when the assessed undifferentiated cell preferentially differentiates into an endodermal lineage.

In some embodiments, the level of expression is assessed by Q-PCR and/or by microarray analysis.

The following detailed description is exemplary and explanatory, and is intended to provide further explanation of the invention.

