5 This patent application claims the benefit of priority from U . S. Provisional Application Serial No . 63/295, 114, filed December 30, 2021, teachings of which are herein incorporated by reference in their entirety.

10 FIELD

The present disclosure relates to methods for the isolation, culture expansion and differentiation of nonhuman mammalian umbilical cord and placenta derived stem cells, compositions of these cells, and methods for use of these

15 cells in production of cultured meat .

BACKGROUND OF INVENTION

As it has been shown for human placentas and the attached umbilical cord, these tissues are known to contain

20 several types of stems cells (Weiss and Troyer, 2006) . These cells are characterized by their ability to differentiate into other cell types such as adipocytes ( fat cells) , chondrocytes (cartilage cells) , myoblasts (muscle cells) , etc . A pluripotent stem cell is able to differentiate into

25 all 3 primary germ layers including ectoderm, mesoderm and endoderm. These stem cells are also characterized by certain cell surface markers including CD10, CD29, CD44 , CD105 and others (Weiss and Troyers, 2006) . Like humans, mammals such as cows and pigs also deliver a placenta and umbilical cord

30 along with the calf and the piglet . As such, similar umbilical cord stem cells have also been identified in bovine placentas (Raoufi et al, 2010) . Xiong et al (2014 ) have Isolated bovine umbilical cord mesenchymal stem cells (UCMSCs) expressing genes for CD29, CD44 , CD73, CD90, and

CD166. In a separate study, Cardoso et al (2012) isolated stem cells with cell surface markers including CD105+, CD29+, CD73+ and CD90+ .

5

SUMMARY

This disclosure focuses on unique methods for isolation of nonhuman mammalian multi-potent cells, culture expansion methods to maintain cell surface phenotype and

10 differentiation methods to adipocytes (fat cells) , muscle cells, cartilage and endothelial vascular cells . Further, this disclosure includes combining or co-culturing the culture expanded and differentiated cells with a nonhuman mammalian derived placental extracellular matrix or ECM to

15 generate a combination product that is fit for human consumption .

An aspect of this disclosure relates to methods of isolation of stem cells from nonhuman mammalian umbilical cord, placenta and vasculature and compositions of those

20 cell types as defined by cell surface markers .

Another aspect of this disclosure relates to methods for producing the compositions of the nonhuman mammalian placental and umbilical cord cells after cell culture expansion as defined by their phenotype (surface markers) .

25 Another aspect of this disclosure relates to methods for differentiation of the expanded cells into adipocytes (fat cells) , myocytes (muscle cells) , chondrocytes (cartilage) and endothelial cells (blood vessel) and other cell types critical to formation of cultured meat .

30 Yet another aspect of this disclosure relates to use of the differentiated cells and culturing the cells on decellularized nonhuman mammalian placenta scaffolds thereof in production of nonhuman mammalian cultured meat for consumption.

BRIEF DESCRIPTION OF THE FIGURES

5 FIGs . 1A and IB are phase contrast images of bovine umbilical cord stromal cells (BUSC) isolated from bovine umbilical cord tissues (P0) . Representative images for the cell fraction BUSC (C) (FIG. 1A) and tissue fraction BUSC (T)

(FIG. IB) are shown . The scale bar is equal to 100 pm.

10 FIGs. 2A, 2B, 2C and 2D are phase contrast images of BUSC cells at different passages . Representative images of BUSC at different passages (P0 (FIG. 2A) , Pl ( FIG. 2B) , P2 (FIG. 2C) and P4 (FIG. 2D) ) are shown . The scale bar is equal to 100 μm.

15 FIG. 3 provides representative graphs of flow cytometry analyses with antibodies against human antigens, which showed cross reactivity to bovine cells .

FIG. 4 shows representative images from oil red O staining of human mesenchymal stem cells (hMSCs) and BUSC

20 cells after 12 days of adipogenic differentiation, 2xl0« of cells were seeded to each well of 24-well plate and induced to differentiate for 12 days . Representative images are shown. The scale bar is equal to 50 pm.

25 DETAILED DESCRIPTION

Various embodiments and aspects of the inventions will be described with reference to details discussed below and will illustrate the various embodiments . The following description of the invention is not to be construed as

30 limiting the invention. Numerous specific details are described to provide a thorough understanding of various embodiments of the present invention. However, in certain instances, well-known or conventional details are not described in order to provide a concise discussion of embodiments of the present inventions . Reference in the specification to "one embodiment" or "an embodiment" or "another embodiment" means that a particular feature,

5 structure, or characteristic described in conjunction with the embodiment can be included in at least one embodiment of the invention. The appearances of the phrase "in one embodiment" in various places in the specification do not necessarily all refer to the same embodiment .

10 This invention relates to compositions comprising stem cells and use of such stem cells isolated from nonhuman mammalian umbilical cords and related tissues such as, but not limited to, Wharton' s Jelly, placenta decidua and placental vasculature, for the generation of cultivated meat

15 fit for human consumption.

In one nonlimiting embodiment, the cells are isolated from the umbilical cord, the placenta and/or its vasculature of a nonhuman mammal such as, but not limited to, bovine (cow or buffalo) , porcine (pig) , goats, and sheep.

20 In one nonlimiting embodiment, multi-potent stem cells are isolated from the umbilical cord of the nonhuman mammal .

In another nonlimiting embodiment, the cells are taken from the umbilical cord and Wharton' s Jelly. Wharton' s Jelly is contained within the umbilical cord.

25 In another nonlimiting embodiment, the cells are taken from the placental decidua and the placental vasculature .

The multi-potent stem cells are culture expanded while maintaining their cell surface markers (phenotype) .

In one nonlimiting embodiment, the cells show a cell

30 surface phenotype, which includes one or more of the following markers CD10+, CD13+, CD29+, CD44+, CD73+, CD90+, CD105+ or CD200+ . Cells are culture expanded until the cell surface markers begin to alter or the cells achieve senescence or the loss of power to grow and divide, In one nonlimiting embodiment, the phenotype is maintained through 10 or more

5 doublings or expansions .

In one nonlimiting embodiment, the isolated stem cells are culture expanded through several passages to senescence.

In various embodiments, the culture-expanded cells are differentiated into smooth muscle cell precursors,

10 adipocytes, endothelial cells and chondrocytes .

In one nonlimiting embodiment, the cell culture expanded cells are differentiated into various cells representing the 3 germ layers ectoderm, endoderm and mesoderm. These cells include adipocytes, myocytes (or

15 muscle precursors) , endothelial (blood vessel) and chondrocytes (cartilage) .

These cells are cultured to sufficient densities to create cell banks for later seeding onto scaffolds as disclosed herein.

20 In one nonlimiting embodiment, the cell culture expanded stem cells are used to create master cell banks for storage at low temperature <-80 °C) .

In one nonlimiting embodiment, the differentiated cells are used to create master cell banks for storage at low

25 temperature <-80°C) .

In one nonlimiting embodiment, the cells are co- cultured with decellularized nonhuman mammalian placental extracellular matrix ECM to create a cultured meat product that is fit for human consumption. A nonlimiting example of

30 a matrix ECM which can be used is that described in

PCT/US2022/048680 filed November 2, 2022, teachings of which are herein incorporated by reference in their entirety. In one nonlimiting embodiment, the cultured meat is created by co-culturing the adipocytes, chondrocytes, muscle cells and endothelial cells at the same time .

In one nonlimiting embodiment, the cultured meat is

5 created by co-culturing the adipocytes, chondrocytes, muscle cells and endothelial cells in a specific order to generate a specific structure for the meat .

In one nonlimiting embodiment, the cultured meat is created by co-culturing the adipocytes, chondrocytes, muscle

10 cells and endothelial cells in different amounts to create cultured meats of different fat, protein content or physical structure .

The nonhuman mammalian placental stem cells referred to here is meant to encompass all stem cells derived from the

15 placenta and its components, the umbilical cord, cord blood, Wharton' s Jelly, the placenta decidua and the vasculature of the placenta.

The following nonlimiting examples are provided to further illustrate the present invention.

20 EXAMPLES

Example 1 . Isolation of bovine umbilical cord stromal cells

Bovine umbilical cord stromal cells (BUSC) were isolated from fresh or cryopreserved bovine umbilical tissues following a procedure modified and improved from

25 procedures of out-migration method and enzymatic digestion method as described by set forth by Xiong et al . (Anim Cells Syst 2014 18 ( 1) : 59-67) , Cardoso et al . (Bmc Biotechnol 2012 12 : 18 ) ; and Wang et al . Stem Cells 2004 22 (7 ) : 1330-7.

Specifically, umbilical cord tissues were disinfected in 70%

30 ethanol for 2-3 minutes followed by washing with phosphate buffered saline (PBS) . The surrounding connective tissues were trimmed away from the disinfected tissues . Tissues were cut into 2-5 mm in length segments and digested in digestion solution (MEM-a complete medium + lx Antibiotic- Antimycotic + 1 mg/mL of collagenase type I (Worthington Biochemical Corporation Cat# LS004196, Code: CLS-1, Lot# 40N205980) ) at 5 mL/gram of tissue. The digestion was

5 incubated in the cell culture incubator with gentle rotation for 3 hours . After digestion, an equal volume of PBS was added to the digestion mixture and the mixture was passed through a 45 pm pore cell strainer. The fraction passing through the strainer is referred as "cell fraction" and the

10 undigested tissues are referred as "tissue fraction" . Both cell and tissue fractions were washed using excess volumes of PBS . The cell fraction BUSC (C) and tissue fraction BUSC (T) were then cultured in 100 mm cell culture dishes for 7-10 days (P0) (see FIG. 1A and IB, respectively) . The yield

15 of cells from umbilical cord tissues on Day 7 was about 7xl0 ⁶ cells/g of wet tissue . Cells from the cell fraction and tissue fraction were then cryopreserved as BUSC (C) P1 and BUSC (T) P1, respectively.

20 Example 2. In vitro expansion (culture) of bovine umbilical cord stromal cells

Pl cells (20x10*) were seeded onto one 100 nun cell culture dish in growth medium (MEM-a base medium + 10% fetal bovine serum (FBS) + lx Antibiotic-Antimycotic) and

25 incubated at 37°C in an incubator with 5% CO2 and 90% humidity until reaching 85% confluence . Once cells reached

85% confluence, cells were trypsinized with 1 mL trypsin (Gibco) , neutralized with 4 mL of cell growth medium and then the number of P2 cells were counted using a

30 hemocytometer. To set up the next passage, 20x10* cells of P2 cells were seeded onto one 100 mm dish and cultured until

85% confluence . This process was repeated and cell numbers at the end of each passage were recorded. The morphologies of cells at different passages are shown in FIGs . 2A through 2D.

The doubling time was calculated as follows : Doubling time (h) = Duration (h) x In (2) / In (Final cell

5 number/Initial cell number) .

The fold of amplification was calculated as follows : Final cell number/Initial cell number.

The expansion of BUSC from different batches was monitored up to passage 6 and results are shown in Table 1.

10 Table 1

Example 3. Analysis of stem cell markers on BUSC using flow cytometry

15 Cells were cultured in 100 mm cell culture dishes to 85% confluence and detached using 1 mL/dish of TrypLE (Tryple

Express Enzyme (lx) Phenol Red, FFiisshheerr SScciieennttiiffiicc,, Cat# 12-

605-010) . About 50x10* cells were resuspended in 100 μL of staining buffer (1XPBS + 5%FBS + 0. 02%NaN ₃ ) . Individual or

20 multiple antibodies (as listed in Table 2) were added to the cells at 5 μL of each antibody per IxlO ⁶ cells . The cells were incubated with antibodies in the dark for 30 minutes followed by centrifugation at 1000 rpm for 5 minutes , The supernatant was removed and the cells were washed with 1 mL

25 of PBS and then centrifuged again at 1000 rpm for 5 minutes . Following this second centrifugation step, the supernatant and the cells were resuspended cells in staining buffer at 20-

50x10* cells in 200-300 μ L of staining buffer. The cells were kept on ice and flow cytometry analysis was performed using

Gallios Flow Cytometer (Beckman Coulter) .

Since the antibodies against stem cell markers are developed against human antigens, the cross reactivities of

5 those antibodies to bovine cells must be determined. The commonly used cell surface markers for human stem cells are listed in Table 2. Among all the antibodies (conjugated with fluorophores) tested, CD105-FITC, CD44-PE/Cy5, CD146-PE and

CD-APC showed positive staining on bovine cells (see FIG. 3) .

10 Flow cytometry analyses indicated that the isolated BUSC cells contained at least a population of cells expressing mesenchymal stem cell markers .

Table 2

Example 4. Adipogenic differentiation of bovine umbilical cord stromal cells

BUSC cells were cultured to 75% confluence and

5 trypsinized and counted. 2x10 ⁴ /well of BUSC were seeded to the wells of a 24-well plate. Human bone marrow derived mesenchymal stem cells (hMSC) were used as a positive control for adipogenic differentiation. After incubation overnight, cells were induced to adipogenic differentiation using

10 adipogenic medium as described by Lee and Fried (Methods

Enzymol 2014 538 : 49-65) . Medium was changed every three days for 12 days . After differentiation, cells were stained with

Oil Red 0 in accordance with the procedure described by Ghoniem et al . Anat Cell Biol 2015 48 (2) : 85-94. Results depicted in

15 FIG. 4 are indicative of the stromal cells isolated from bovine umbilical cord tissues being stem cells which can be differentiated into adipocytes .