Non-hematopoietic cells from peripheral blood and uses thereof

Abstract

Provided are methods of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample containing non-hematopoielic cells, which rely on culture media supplemented with serum and in certain embodiments also lacking epidermal growth factor, methods of editing mammalian peripheral blood-derived pericytes, and cells generated these methods. Also provided are serum-free defined cell media containing PDGF-beta; VEGF-C; a Notch ligand; a fibroblast growth factor; a TGF-beta superfamily member; an insulin agonist; transferrin; a hepatocyte growth factor; and a Wnt antagonist. The serum-free defined cell media can be used to derive and / or culture pericytes.

Description

NON-HEMATOPOIETIC CELLS FROM PERIPHERAL BLOOD AND USES THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 746,665 (filed on January' 17, 2025) which is incorporated by reference in its entirety'.FIELD OF THE INVENTION

[0002] This disclosure relates to methods of isolating non-hematopoietic cells from peripheral blood and uses of such cells, in, for example, reproductive cloning.BACKGROUND OF THE INVENTION

[0003] Somatic cell nuclear transfer (SCNT) followed by Embryo Transfer (ET), generally known as reproductive cloning, typically uses a somatic cell such as a fibroblast cell as the nuclear donor. Derivation of dermal fibroblast lines from human or animal patients usually requires a painful and invasive skin biopsy requiring closure by sutures or dermal glue and exposing the patient to the risk of infection. Commercial cat, dog, and horse cloning sendees currently7rely on the use of dermal fibroblasts isolated from skin biopsies or stromal tissue from neuter or spay procedures.

[0004] Fibroblasts typically fail to expand beyond passage 15, hitting a Hayflick limit and undergoing senescence. This window limits the ability to perform multiple rounds of geneediting. In addition, single fibroblasts typically undergo senescence when plated alone into a single well.

[0005] Furthermore, using these techniques and over conventional techniques, it is difficult to obtain cells in sufficient quantities to generate extinct animals, via techniques such as gene-editing and SCNT. Blood provides an easily accessible source of cells that could be used. Groups have previously cloned mammalian embryos from mice to humans using nucleated blood cells, both terminally differentiated white cells as well as bone marrow derived hematopoietic stem cells, as well as somatic cells like fibroblasts and cumulus cells.

[0006] In blood, non-hematopoietic cells are cells that still have the potential to develop into other cell types, essentially acting as progenitor cells for tissues outside the blood system, like endothelial cells or mesenchymal stem cells. As such, these cells are a potential source for cells for reproductive cloning. However, no one has yet reported on creating cloned offspring from non-hematopoietic cells (pericytes and EPCs) circulating in peripheral blood.4930-0302-8870.1 1

[0007] Accordingly, what is needed are protocols for isolating non-hematopoietic cells (pericytes and EPCs) circulating in peripheral blood.SUMMARY OF THE INVENTION

[0008] This disclosure provides protocols for isolating pericytes and EPCs from peripheral blood and uses of these cells. Scientists at Colossal Biosciences, Inc. are the first to document that this can be done, and have produced cloned wolf fetuses, and a living cloned dog from adherent pericytes taken from peripheral blood of adult animals. In addition, the protocols of the instant disclosure can be used to isolate mammalian peripheral blood-derived pericytes and mammalian peripheral blood-derived EPCs from any species, human or animal, such as mammals, carnivores, e.g., cats (Felis catus), dogs (Cants lupus familiaris), and wolves (Cams lupus), and herbivore species, e.g., bison (Bison bison), fallow deer (Dama dama), roan antelope (Hippotragus equis), and sable antelope (Hippotragus niger).

[0009] One aspect of the disclosure is directed to a method of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a serum-supplemented cell culture medium under adherent conditions for a period of time sufficient to allow for growth of endothelial progenitor cells and pericytes, thereby deriving the cell population. In some embodiments, the serum-supplemented cell culture medium lacks or is not supplemented with epidermal growth factor. In certain embodiments, the method is used to isolate mammalian peripheral blood-derived pericytes (“pericytes”). In some embodiments, when used to isolate pericytes, the method may include culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a serum-supplemented cell culture medium under adherent conditions for a period of time sufficient to allow for growth of pericytes. In other embodiments, the method is used to isolate mammalian peripheral blood-derived endothelial progenitor cells (“endothelial progenitor cells”). In certain embodiments, when used to isolate endothelial progenitor cells, the method may include culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a serum-supplemented cell culture medium under adherent conditions for a period of time sufficient to allow for grow th of endothelial progenitor cells.

[0010] In one embodiment, the cell culture medium is EGM. In another embodiment, the cell culture medium is a conditioned medium generated by culturing pericytes. The culture medium can be further supplemented. In certain embodiments, the cell culture medium is4930-0302-8870.1 2supplemented with pituitary extract, such as e.g., bovine pituitary extract, and / or fetal calf serum.

[0011] In certain embodiments, the adherent conditions include culturing the cells on collagen, fibronectin, or cell-culture treated plastic. In other embodiments, the method further includes obtaining the mammalian peripheral blood sample from the mammal. In some embodiment, the mammal has been treated with an agent having alpha-adrenergic binding affinity prior to obtaining the peripheral blood sample. In other embodiments, the mammal has been treated with an alpha2-adrenergic agonist, dexmedetomidine, medetomidine, xylazine, opioids, and / or Butorphanol.

[0012] In certain embodiments, the mammalian peripheral blood-derived endothelial progenitor cells express at least three markers selected from CD34. platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), E-Selectin, Vascular Endothelial-Cadherin (VE-cadherin or Cadherin 5), and SCARF-1.

[0013] In other embodiments, the mammalian peripheral blood-derived pericytes express at least three cell surface membrane proteins selected from c-Met. chondroitin sulfate proteoglycan 4 (CSPG4), platelet derived growth factor receptor beta (PGDFRb), platelet derived growth factor receptor alpha (PGDFRa), CD82, melanoma cell adhesion molecule (CD 146), endosialin (CD248), kinase insert domain receptor (KDR), ty rosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), fms-related receptor tyrosine kinase 4 (FLT4), C-X3-C motif chemokine receptor 1 (CXC3CR1), Notch 2 receptor. Notch 3 receptor, insulin growth factor receptor 2 (IGRF2), fibroblast growth factor receptor 1 (FGFR1), and bone morphogenetic protein receptor 2 (BMPR2). In other embodiments, the pericytes express one or more intracellular structural proteins chosen from actin alpha 2 (ACTA2), desmin, nestin, glial fibrillary acid protein (GFAP), Myl9, myosin heavy chain 11 (Myhl 1), transgelin (TAGLN), and tropomysin 2 (TPM2).

[0014] The method can also include isolating the mammalian peripheral blood-derived pericytes. In certain embodiments, the pericytes lack expression of platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4).

[0015] In some embodiments of the methods, the isolating the pericytes includes screening for cells that are negative for platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4).

[0016] In certain embodiments, the method further includes isolating the mammalian peripheral blood-derived endothelial progenitor cells. The isolating may be achieved by a4930-0302-8870.1 3variety of different techniques including comprises fluorescence activated cell sorting (FACS), magnetic cell separation (MACs), or differential adherent culture.

[0017] The disclosure also includes isolated mammalian peripheral blood-derived pericytes derived by these methods. In addition, the disclosure includes isolated mammalian peripheral blood-derived endothelial progenitor cell derived by these methods.

[0018] Another aspect of the disclosure is a method of gene editing a mammalian peripheral blood-derived pericyte which includes editing a gene of interest in an isolated mammalian peripheral blood-derived pericyte obtained by culturing a peripheral blood sample in a cell culture medium under adherent conditions for a period sufficient to allow for growth of pericytes, whereby the cell culture medium is supplemented with serum. An aspect of the disclosure is a method of gene editing a mammalian peripheral blood-derived endothelial progenitor cells which includes editing a gene of interest in an isolated mammalian peripheral blood-derived endothelial progenitor cell obtained by culturing a peripheral blood sample in a cell culture medium under adherent conditions for a period sufficient to allow for growth of endothelial progenitor cells, whereby the cell culture medium is supplemented with serum. In certain embodiments, the culture medium lacks or is not supplemented with epidermal growth factor. In certain embodiments, the culture medium lacks or is not supplemented with epidermal growth factor.Yet another aspect of the disclosure is a method of gene editing mammalian peripheral blood-derived pericytes including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period sufficient to allow for grow th of pericytes thereby deriving the mammalian peripheral blood-derived pericytes, wherein the cell culture medium is supplemented with serum; and editing a gene in the mammalian peripheral blood-derived pericytes to produce a mammalian peripheral blood-derived pericyte having an edited gene. A further aspect is a method of gene editing mammalian peripheral blood-derived cells including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium supplemented with serum under adherent conditions for a period sufficient to allow for growth of mammalian peripheral blood-derived cells thereby deriving the mammalian peripheral blood-derived cells; and editing a gene in the mammalian peripheral blood-derived pericytes to produce a mammalian peripheral blood-derived pericyte comprising an edited gene, wherein the mammalian peripheral blood-derived cells are mammalian peripheral blood-derived pericytes and / or mammalian peripheral blood-derived cells. In certain embodiments, the culture medium lacks or is not supplemented with epidermal growth factor.4930-0302-8870.1 4

[0019] In one embodiment, the cell culture medium is EGM. In another embodiment, the cell culture medium is a conditioned medium generated by culturing pericytes. In a further embodiment, the cell culture medium is supplemented with pituitary extract, such as a bovine pituitary extract. In certain embodiments, the adherent conditions include culturing the cells on collagen, fibronectin, or cell-culture treated plastic.

[0020] In certain embodiments, the method further includes culturing the mammalian peripheral blood-derived pericyte having an edited gene to produce a clonal line. In further embodiments, the method further includes culturing the mammalian peripheral blood-derived endothelial progenitor cell having an edited gene to produce a clonal line.

[0021] In certain embodiments, the method includes FACS sorting, limiting dilution and / or plating on a Cell Raft system. In certain embodiments, the method further includes isolating a mammalian peripheral blood-derived pericyte having an edited gene and / or isolating a mammalian peripheral blood-derived endothelial progenitor cell having an edited gene.

[0022] Another embodiment is an isolated mammalian peripheral blood-derived pericyte having an edited gene produced by the method. Yet another embodiment is a method of producing a transgenic animal which includes using the isolated edited mammalian peripheral blood-derived pericyte produced by the methods above.

[0023] An alternate embodiment is an isolated mammalian peripheral blood-derived endothelial progenitor cell having an edited gene produced by the method. Yet another embodiment is a method of producing a transgenic animal which includes using the isolated edited mammalian peripheral blood-derived endothelia progenitor cell produced by the methods above.

[0024] Yet another aspect of the disclosure is directed to culture media. One embodiment is a serum-free defined cell culture medium containing: PDGF-beta; VEGF-C; a Notch ligand; a fibroblast growth factor; a TGF-beta superfamily member (such as a bone morphogenic protein); an insulin agonist; transferrin; a hepatocyte growth factor; and a Wnt antagonist. In certain embodiment, the medium lacks or is not supplemented with epidermal growth factor.

[0025] In one embodiment, the Notch ligand is Jagged 1, Jagged 2, or a mixture thereof. In another embodiment, the fibroblast growth factor is bFGF. In yet another embodiment, the TGF-beta superfamily member is a bone morphogenic protein. In a further embodiment, the TGF-beta superfamily member is BMP10. In one embodiment, the insulin agonist is natural insulin, recombinant insulin, and / or IGF2. In another embodiment, the transferrin is apo4930-0302-8870.1 5and / or holo-transferrin. In an alternate embodiment, the Wnt antagonist is Dickkopf-1, Dickkopf-2, Dickkopf-3. or a WTN antagonist antibody.

[0026] Yet another embodiment is directed to a conditioned cell culture medium generated by culturing the isolated pericyte obtained via the methods described above.

[0027] Another embodiment is directed to a method culturing pericytes in a cell culture medium described above including the serum-free defined cell culture medium or the conditioned cell culture medium.

[0028] Yet another aspect of the disclosure is directed to a method of generating a population of peripheral blood-derived pericytes which includes the following steps: culturing a mammalian peripheral blood sample in a serum-free defined cell culture medium or the conditioned cell culture medium as described above in an adherent cell culture for a period sufficient to allow for grow th of peripheral blood-derived pericytes; and isolating peripheral blood-derived pericytes from the sample after culturing. In certain embodiments, the blood sample includes non-hematopoietic cells. In other embodiments, the culturing includes culturing the mammalian peripheral blood sample on collagen, fibronectin, or cellculture treated plastic.

[0029] In certain embodiments, the isolating includes FACS, MACs, or differential adherent culture. In some embodiments, the isolating includes screening for cells expressing at least three markers chosen from CSPG4, PGDFRb, PGDFRa, CD82, CD146, CD248, KDR, TIE1, FLT4, CXC4CR1, Notch 2 receptor. Notch 3 receptor. IGRF2, FGFR1. and BMPR2.

[0030] Yet another aspect of the disclosure is directed to a method of isolating endothelial progenitor cells (EPCs) and pericytes from a peripheral blood sample which includes the following steps: isolating the peripheral blood sample from a mammal; and culturing the peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period sufficient to allow for growth of endothelial progenitor cells and pericytes, thereby deriving the EPCs and / or pericytes, wherein the cell culture medium is supplemented with serum. In certain embodiments, the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0031] Yet another aspect of the disclosure is a method of producing an induced pluripotent stem cell from an isolated mammalian peripheral blood-derived pericyte using the methods described above, which includes transfecting the isolated pericyte with one or more of OCT4, KLF4, SOX2, and optionally c-MYC. Yet an alternate aspect of the disclosure is a method of producing an induced pluripotent stem cell from an isolated mammalian peripheral blood-4930-0302-8870.1 6derived endothelial progenitor cell using the methods described above, which includes transfecting the isolated pericyte with one or more of OCT4, KLF4, SOX2. and optionally c-MYC.

[0032] In any of the above-mentioned aspects, the mammal is a mouse, a rat, a marsupial, a bovine, an ovine, a caprine, a pig, an elephant, an equid, a cat, a dog, a wolf, or a deer. In one embodiment, the bovine is a cow, a bison, or an antelope. In another embodiment, the equid is a horse, a zebra, a donkey, a tapir, or a rhinoceros. In a further embodiment, the mammal is Felis cams Canis lupus familiaris. Cants lupus. Bison bison, Dama dama, Hippotragus equinus, or Hippotragus niger.

[0033] Other features and advantages of the invention will be apparent from the detailed description and examples that follow.BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended figures. For the purpose of illustrating the invention, the figures demonstrate embodiments of the present invention. It should be understood, however, that the invention is not limited to the precise arrangements, examples, and instrumentalities shown.

[0035] FIG. 1 shows images of adherent endothelial progenitor cells (EPCs) (left) and pericytes (right) derived from peripheral blood of adult gray wolves (Canis lupus) using the protocols of the disclosure.

[0036] FIG. 2 shows an image of cleavage of genetically engineered gray wolf embryos produced by interspecies somatic cell nuclear transfer (iSCNT) with EPCs.

[0037] FIG. 3 shows an image of an ultrasound providing confirmation of a successful pregnancy with a transgenic wolf fetus.

[0038] FIG. 4A and 4B show a primary cluster of adherent primary pericytes 4 days (FIG.4A) and 8 days (FIG. 4B) after replating the mononuclear fraction of peripheral blood cells from dog CLF-003.

[0039] FIG. 5 shows an image of expanding pericytes from blood from dog CLF-003 that form stellate clusters of cells after passaging, which are morphologically distinct from endothelial EPC cells.

[0040] FIG. 6 shows that adherent pericytes derived from peripheral blood of adult domestic dog, Canis lupus familiaris , (CLF-003) expanded for 4 passages maintain a normal diploid female dog karyotype (2n=78) using the protocols of the disclosure.4930-0302-8870.1 7

[0041] FIG. 7 shows an image of obvious cleavage within 4 of 10 SCNT dog embryos reconstructed with 23CLF-003 after overnight culture prior to embryo transfer.

[0042] FIG. 8 shows a healthy puppy at 3 weeks of age cloned from CLF-003 pericytes. The sex, coat color and markings and body shape matched the original female donor. (At the time of filing of the provisional application, the female dog cloned from 23CLF-003 pericytes was still alive and healthy at 13 months of age).

[0043] FIG. 9 shows the results of DNA testing performed by a third-party, genetic diagnostic testing lab based on canine Short-Tandem Repeat (STR) markers. The profiles of 23 STR’s from the donor pericytes, CLF-003 (Sample VGK2762) were identical to the oral swab of the puppy (Sample VGK2742) cloned from pericytes CLF-003.

[0044] FIG. 10 shows a schematic of the protocols used to isolate progenitor cells in Example 4. Upon new cell line generation, blood draws show enhanced properties for biobanking, genome assembly, and potentially for cloning. Fibroblasts are slower to generate and involve marring the animal’s hide. Both collection methods produce viable options for downstream applications, but blood draws have an expedited turnaround time.

[0045] FIG. 11 A and FIG. 1 IB show the results of proteomic analyses for replicate samples of each cell type. FIG. 11A shows a heatmap depicting relative expression of canonical protein markers for cell type determination in canid and bovid cells. Protein expression was normalized, and color intensity reflects z-scores from -2.58 (lowest) to 2.58 (highest); gray indicates undetected protein. Log2(fold-change) and -Logio(p-value) for each gene are provided in Tables 4-4 and 4-5. FIG. 1 IB shows the PC A of all samples of all three taxa using 18 canonical markers present in all cell lines: VIM, COL 1 Al, ANXA5, COL3A1, CSPG4, ITGA5, ITGBL CD44, ENG, ICAM1,

[0046] FIG. 12A-FIG. 12C show the morphology, size, and doubling rates of cells isolated in Example 4. FIG. 12A shows the results for canid cells. Canid cells are compatible with commercially available antibodies and show the expected phenotypes. Pericytes have a subpopulation that is CD90+Pericytes are CD34", fibroblasts are CD90+, and Endothelial Progenitor Cells (EPCs) have populations that are CD34+and are CD90'. The EPCs have a longer longevity than the pericytes, but both have an average doubling rate to start at ~15 hours. FIG. 12B shows the results for bison cells. Bison fibroblasts tend to average between 35-40 hours doubling rate and have shown to have a long lifespan. Bison EPCs however, tend to have an average doubling rate of 15 hours but a longevity study has not yet been conducted. FIG. 12C shows the results for equid cells. Equid EPCs also have around a 15-hour doubling rate and are stable through passage 15. In each of FIG. 12A-12C, the left-hand4930-0302-8870.1 8panels show photos taken under phase imaging of the Incucyte. The right-hand panels show data collected via Incucyte Cell Imaging with the Cell-by-Cell Al Analysis software trained to analyze each cell type. Cells were imaged daily to capture live cell count, which was used to calculate average doubling rate across each passage.

[0047] FIG. 12D shows the results of FACS analyses of CD34 and CD90 expression on gray wolf cell populations. Single parameter histograms and dual-parameter dot plots detected a 12% sub-population of CD34‘, CD90+canid pericytes. While canid fibroblasts were 100% CD90+with a 3.5% sub-population co-expressing CD34 and CD90. Canid EPCs had a discrete 25% sub-population that were CD34+and all were CD90". Forward and side scatter gating was used to collect data on singlets and exclude cell clusters, and thresholds were set using cells stained with isotype controls (not shown). Quadrant percentages for dot plots are summarized in Table 4-6.

[0048] FIG. 12E and FIG. 12F show that canid cell lines are compatible with commercial antibodies for FACS analyses. A negative or antibody dilution buffer control, and corresponding isotype controls were used to ensure that no background fluorescence was observed. The CL001 fibroblasts were used as a positive control for CD90, while the CL004 EPCs were used as a positive control thanks to the known expression from the proteomics data. In addition, the antibodies were incubated alone and in conjunction to ensure no increased background.

[0049] FIG. 13A-FIG. 13C show the results of functional assays, which were performed to establish a fast and efficient method of characterization. FIG. 13A shows the results of the angiogenesis assay. Canid EPC, pericyte, and EPC + pericyte images were taken using the Zeiss Primovert with a lOx Objective. The remaining images were taken using the Incucyte at a 10X objective. The angiogenesis assay showed that EPCs formed tubule networks, while pericytes adhered to the outer layer of the EPC-tubule network. Pericytes and fibroblasts did not form tubules. FIG. 13B shows the results of the Low-Density Lipoprotein & Acylated Low Density Lipoprotein Uptake assays.below a bar indicates negative controls, while E, P, and F indicate EPCs, pericytes, and fibroblasts, respectively. Cell fluorescence was measured using Incucyte Cell-by-Cell Al Analysis software trained on these cell types. FIG.13C shows fluorescent microscopy images of cells taken by the Incucyte at a lOx objective with the green and orange fluorescence channels to generate positive cell fluorescence data. Fibroblasts have a lower level of BodipyFL-tagged LDL uptake than those of blood derived4930-0302-8870.1 9cell lines, while pericytes have lower levels of AlexaFluor594-tagged AcLDL uptake than EPCs and fibroblasts from the same species.

[0050] FIG. 14A-FIG. 14C show Optical Genome Mapping (OGM) plots summarizing chromosome copy numbers for each cell line. FIG. 14A shows the results of Canid samples. Canid samples were aligned to NCBFs mCanLorl.2 genome assembly, revealing 38 autosomes with a copy number of 2 and single copies of each sex chromosome (chr39: X, chr40: Y). Independent manual chromosome counting via microscopy (KaryoLogics) confirmed these findings, showing concordance between both methods. FIG. 14B shows the results for Bovid samples. Bovid samples were aligned to the tamu_bb3_ucsc genome assembly, displaying 29 autosomes with a copy number of 2. The X chromosome (chr30) indicated BBis08 as female (copy number 2), while BBisl7 EPC and FB were male (copy number 1). FIG. 14C shows the results for Equid samples. Equid samples were aligned to NCBFs Equus Caballus GCA_041296265.1 genome assembly, showing 31 autosomes with a copy number of 2. The X chromosome (chr32) exhibited a copy number of 2, confirming a female sex.

[0051] FIG. 15A-E show brightfield images of pre-implantation embryos and post implantation ultrasound images. Early cleavage stage embryos are visible in overnight cultures of iSCNT gray wolf embryos reconstructed with EPCs (FIG. 15 A) and pericytes (FIG. 15B). iSCNT plains bison embryos reconstructed with EPCs with three cavitating blastocysts (FIG. 15C) and one hatching blastocyst after seven days of culture (FIG. 15D). iSCNT plains zebra embryos reconstructed with EPCs with six compacting morulae by seven days of culture (FIG. 15E). A transabdominal ultrasound image show s a sagittal image of one of six viable gray wolf fetuses at 23 days after ET (FIG. 15F).DETAILED DESCRIPTION

[0052] This disclosure is based on the discovery that non-hematopoietic cells of endothelial or pericyte origin, which are capable of single-cell, adherent clonal grow th in defined culture conditions, can be isolated from peripheral blood. Accordingly, this disclosure provides for methods of isolating these cells, compositions containing these cells, and uses of these cells.

[0053] Without being bound by theory, this disclosure enables gene-editing and Somatic Cell Nuclear Transfer (SCNT) to produce genetically identical and modified individuals from any mammalian species, where reproductive cloning is possible.

[0054] Both mammalian peripheral blood-derived endothelial progenitor cells (EPCs) and surprisingly mammalian peripheral blood-derived pericyte cultures were established in an4930-0302-8870.1 10EPC medium on collagen, fibronectin, or cell-culture treated plastic from peripheral blood and expanded in culture.

[0055] While EPCs that circulate in the blood at low frequency have been routinely isolated from peripheral blood, pericytes (PCs) are normally present on the outside of capillaries and post-capillary venules and have not been isolated from circulation before. Pericytes typically wrap around the outside (adluminal side) of endothelial cells in peripheral tissues and the blood brain barrier, and to date have been isolated by dissecting and digesting tissues. The isolation of EPCs and pericytes from peripheral blood (following the protocols of the disclosure) for subsequent stable expansion in culture provides a minimally invasive way to collect and viably bank cells from individual animals.

[0056] Isolation of mammalian peripheral blood-derived, adherent EPCs and mammalian peripheral blood-derived pericytes using the protocols of the disclosure enables easy collection and banking of cells for subsequent genomic analyses, gene editing and cloning.

[0057] Cells obtained via the protocols of the disclosure remain karyotypically normal after expansion and can be used for SCNT or gene-editing followed by SCNT to produce genetically identical or purposefully modified cloned animals.

[0058] The general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as defined in the appended claims. Other aspects of the present invention will be apparent to those skilled in the art in view of the detailed description of the invention as provided herein.

[0059] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into subsections that describe or illustrate certain features, embodiments, or applications of the present invention.A. Definitions

[0060] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods, and materials are now described.

[0061] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are4930-0302-8870.1 11cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0062] It is noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0063] Each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0064] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the invention.

[0065] As used herein, the term “about” when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ± 20% or ± 10%, more preferably ± 5%, even more preferably ± 1%, and still more preferably ± 0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0066] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains” or “containing,” or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers and are intended to be non-exclusive or open-ended. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary', “or” refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any4930-0302-8870.1 12one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0067] As used herein, the conjunctive term “and / or” between multiple recited elements is understood as encompassing both individual and combined options. For instance, where two elements are conjoined by “and / or,” a first option refers to the applicability of the first element without the second. A second option refers to the applicability of the second element without the first. A third option refers to the applicability of the first and second elements together. Any one of these options is understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or” as used herein. Concurrent applicability of more than one of the options is also understood to fall within the meaning, and therefore satisfy the requirement of the term “and / or.”

[0068] As used herein, the term “consists of,” or variations such as “consist of’ or “consisting of,” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, but that no additional integer or group of integers can be added to the specified method, structure, or composition. As used herein, the term “consists essentially of,” or vanations such as “consist essentially of’ or “consisting essentially of.” as used throughout the specification and claims, indicate the inclusion of any recited integer or group of integers, and the optional inclusion of any recited integer or group of integers that do not materially change the basic or novel properties of the specified method, structure, or composition. See M.P.E.P. § 2111.03.

[0069] The words “right,” “left,” “lower,” and “upper” designate directions in the drawings to which reference is made.

[0070] It should also be understood that the terms “about,” “approximately,” “generally,” “substantially” and like terms, used herein when referring to a dimension or characteristic of a component of the preferred invention, indicate that the described dimension / charactenstic is not a strict boundary or parameter and does not exclude minor variations therefrom that are functionally the same or similar, as would be understood by one having ordinary skill in the art. At a minimum, such references that include a numerical parameter would include variations that, using mathematical and industrial principles accepted in the art (e. , rounding, measurement or other systematic errors, manufacturing tolerances, etc.), would not vary the least significant digit.

[0071] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same,4930-0302-8870.1 13when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0072] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0073] Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat ’I. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison. WI), or by visual inspection (see generally, Current Protocols in Molecular Biology, F.M. Ausubel et al., eds.. Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

[0074] Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1997) Nucleic Acids Res.25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology7Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0075] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value;4930-0302-8870.1 14the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).

[0076] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul. Proc. Nat’l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0077] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0078] As used herein, the term “polynucleotide,’’ synonymously referred to as “nucleic acid molecule,” “nucleotides” or “nucleic acids,” refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA or modified RNA or DNA. “Polynucleotides” include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that can be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “polynucleotide” refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with4930-0302-8870.1 15backbones modified for stability or for other reasons. “Modified"’ bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short nucleic acid chains, often referred to as oligonucleotides.

[0079] As used herein, the term “vector” is a replicon in which another nucleic acid segment can be operably inserted so as to bring about the replication or expression of the segment.

[0080] As used herein, the term “host cell” refers to a cell comprising a nucleic acid molecule of the present disclosure, such as, for example an isolated vector comprising an isolated nucleic acid of the invention. The “host cell” can be any type of cell, e.g., a primary cell, a cell in culture, or a cell from a cell line. In one embodiment, a “host cell” is a cell transfected with a nucleic acid molecule of the invention, e.g., a recombinant host cell. In another embodiment, a “host cell” is a progeny or potential progeny of such a transfected cell. A progeny of a cell may or may not be identical to the parent cell, e.g, due to mutations or environmental influences that can occur in succeeding generations or integration of the nucleic acid molecule into the host cell genome. A host cell can be, for example, any type of prokaryotic, eukaryotic, or archaeal cell. In some instances, the host cell is a bacterial cell. In some instances, the host cell is a mammalian cell. In some embodiments, the mammalian host cell is a canine cell.

[0081] The term “expression” as used herein, refers to the biosynthesis of a gene product. The term encompasses the transcription of a gene into RNA. The term also encompasses translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications.

[0082] As used herein, the terms “peptide,” “polypeptide,” or “protein” can refer to a molecule comprised of amino acids and can be recognized as a protein by those of skill in the art. The conventional one-letter or three-letter code for amino acid residues is used herein. The terms “peptide,” “polypeptide,” and “protein” can be used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, it can comprise modified amino acids, and it can be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.4930-0302-8870.1 16Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0083] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of the amino acids are known, it is the L-form of the amino acid that is represented unless otherwise expressly indicated.

[0084] The term “heterologous nucleic acid" or “heterologous polypeptide” refers to a nucleic acid or a polypeptide whose sequence is not identical to that of another nucleic acid or polypeptide naturally found in the same host cell or the same host. As used herein, the “heterologous nucleic acid” or “heterologous polypeptide” can be heterologous to the bacterial cell and / or the mammalian host.

[0085] As used herein, the term “transform” or “transformation” refers to the transfer of a nucleic acid fragment into a host cell, such as a host bacterial cell, resulting in genetically stable inheritance. Host cells comprising the transformed nucleic acid fragment are referred to as "recombinant” or “transgenic” or “transformed” organisms.

[0086] As used herein, the term “isolated” means a biological component (such as a nucleic acid, peptide, or protein) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, z.e., other chromosomal and extrachromosomal DNA and RNA, and proteins.Nucleic acids, peptides and proteins that have been “isolated” thus include nucleic acids and proteins purified by standard purification methods. “Isolated” nucleic acids, peptides and proteins can be part of a composition and still be isolated if the composition is not part of the native environment of the nucleic acid, peptide, or protein. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0087] As used herein, “gene” refers to a nucleic acid comprising an open reading frame encoding a polypeptide, including both exon and (optionally) intron sequences.

[0088] As used herein, a “promoter” is an example of a transcriptional regulatory sequence and is specifically a nucleic acid sequence generally described as the proximal region of a gene located 5' to the start codon. The transcription of an adjacent nucleic acid segment is initiated at the promoter region. A repressible promoter’s rate of transcription decreases in response to a repressing agent. An inducible promoter’s rate of transcription increases in response to an inducing agent. A constitutive promoter’s rate of transcription is not4930-0302-8870.1 17specifically regulated, though it can vary' under the influence of general metabolic conditions.

[0089] The term '‘gene product,” as used herein, refers to any product encoded by a nucleic acid sequence. Accordingly, a gene product may, for example, be a primary transcript, a mature transcript, a processed transcript, or a protein or peptide encoded by a transcript. Examples for gene products, accordingly, include mRNAs, rRNAs. hairpin RNAs (e.g., microRNAs, shRNAs, siRNAs, tRNAs), and peptides and proteins, for example, reporter proteins or therapeutic proteins.

[0090] Throughout this specification, unless the context requires otherwise, the word “comprise” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0091] As used herein, the term “consisting of’ excludes any element, step, or ingredient not specified in the claim element.

[0092] Before certain embodiments are described in greater detail, it is to be understood that this invention is not limited to certain embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing certain embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0093] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0094] A “mammal” as used therein, may be a human or non-human mammal. Non-human mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline, marsupial, and caprine; an elephant, an equid, a cat, a dog, a wolf, or a deer; and murine mammals, e.g. , a mouse or a rat. A bovine can be a cow, a bison, or an antelope. An equid can be a horse, a zebra, a donkey, a tapir, or a rhinoceros. In some embodiments, the mammal may be Felis catus. Cams lupus familiaris. Cams lupus. Bison bison, Dama dama, Hippotragus equinus, ox Hippotragus Niger.4930-0302-8870.1 18

[0095] Ranges: throughout this disclosure, various aspects of the invention can 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 invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.B. Methods of Deriving Pericytes and Endothelial Progenitor cells

[0096] One aspect of the disclosure is directed to methods of deriving pericytes and / or endothelial progenitor cells from a mammalian blood sample. In one embodiment, the methods can be used to derive both pericytes and endothelial progenitor cells. In other embodiments, the methods can be used to derive pericytes. In yet another embodiment, the methods can be used to derive endothelial progenitor cells. The mammalian blood sample contains non-hematopoietic cells.

[0097] Any mammalian blood sample can be used in the methods. In one embodiment, the mammal is a mouse, a rat, a marsupial, a bovine, an ovine, a caprine, a pig, an elephant, an equid, a cat, a dog, a w olf, or a deer. In a specific embodiment, the mammal is bovine selected from a cow . a bison, or an antelope. In another embodiment, the mammal is an equid selected from a horse, a zebra, a donkey, a tapir, or a rhinoceros. In a specific embodiment, mammal is Felis catus, Cams lupus familiar is, Canis lupus, Bison bison. Dama dama. Hippotragus equinus, or Hippotragus niger.

[0098] In certain embodiments, a minimum of 15 rnL of blood is necessary. In other embodiments, a minimum of 25 mL of blood is necessary. In alternate embodiments, at least 15 mL to at least 25 mL of blood are used. In some embodiments, the methods are used to derive cells from Canid species. In these embodiments, a minimum of 15 rnL of blood is used. In other embodiments, the methods are used to isolate cells from bovine (bison) or equid species. In these embodiments, a minimum of 25 mL of blood is used.

[0099] One embodiment is a method of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under4930-0302-8870.1 19adherent conditions for a period of time sufficient to allow for grow th of endothelial progenitor cells and pericytes, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum and lacks or is not supplemented with epidermal growth factor. In certain embodiments of the method, the cell culture medium lacks or is not supplemented with epidermal growth factor and is supplemented with serum and a pituitary7extract. Another embodiment is a method of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow7for growth of endothelial progenitor cells and pericytes, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum and lacks or is not supplemented with epidermal growth factor. In certain embodiments of the method, the cell culture medium lacks or is not supplemented with epidermal growth factor and is supplemented with serum and a pituitary extract. In certain embodiments, the cell population contains pericy tes and endothelial progenitor cells.

[0100] Another embodiment is a method of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for growth of pericytes, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum. An alternative embodiment is a method of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for growth of pericytes, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum and lacks or is not supplemented with epidermal growth factor.

[0101] In certain embodiments, the methods can be used to derive mammalian peripheral blood-derived pericytes. In those embodiments, the methods include culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow7for growth of pericytes. In certain embodiments, the methods can be used to derive mammalian peripheral blood-derived endothelial progenitor cells. In those embodiments, the methods include culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture4930-0302-8870.1 20medium under adherent conditions for a period of time sufficient to allow for growth of endothelial progenitor cells.

[0102] Yet another embodiment is a method of deriving a cell population containing endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for growth of endothelial progenitor cells, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum. An alternate embodiment is a method of deriving a cell population containing endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for growth of endothelial progenitor cells, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum and lacks or is not supplemented with epidermal growth factor.

[0103] Yet another alternate embodiment is a method of deriving a cell population containing endothelial progenitor cells from a peripheral blood sample including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for growth of endothelial progenitor cells, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum and a pituitary extract, and lacks or is not supplemented with epidermal grow th factor.

[0104] A variety of different culture media can be used in the methods. In certain embodiments, the culture medium lacks epidermal growth factor, i. e. , the culture medium does not contain any epidermal growth factor. In other embodiments, the culture medium is not supplemented with epidermal growth factor, i.e., no epidermal growth factor is added to the culture medium beyond what may be present as part of the medium formulation. In one embodiment, the culture medium is endothelial growth medium (EGM), such as e.g. Lonza EGM®. In another embodiment, culture medium is endothelial growth medium (EGM) that is not supplemented with EGF. In another embodiment, particularly suitable for deriving mammalian peripheral blood-derived pericytes, the cell culture medium described in Section C. below7can be used. In certain embodiments, the endothelial growth medium contains FGF, ascorbic acid, hydrocortisone, glutamine, IGF-1, heparin, and FBS. In other embodiments, the cell culture medium is a conditioned medium generated by culturing pericytes. In one embodiment, the cell culture medium is a conditioned medium generated by4930-0302-8870.1 21culturing pericytes and it is used in methods to isolate cell compositions containing pericytes, such as a population cell population containing pericytes and endothelial progenitor cells. In further embodiments, the cell culture medium is a conditioned medium generated by culturing endothelial progenitor cells.

[0105] The cell culture media (EGM or conditioned media) can further be supplemented with a pituitary extract. In one embodiment, the cell culture media are supplemented with bovine pituitary extract. In another embodiment, the cell culture media are supplemented with fetal calf serum. In yet another embodiment, the cell culture media are supplemented with both fetal calf serum and bovine pituitary' extract.

[0106] A variety of different adherent conditions can be used for methods. In certain embodiments, the adherent conditions include culturing the cells on collagen, fibronectin, or cell-culture treated plastic.

[0107] In certain embodiments, the methods further include obtaining the mammalian peripheral blood sample from the mammal. The blood sample can be obtained using conventional techniques, such as e.g, venipuncture. In some embodiments, the mammal is sedated before the peripheral blood sample is taken from the mammal. Thus, in certain embodiments, the methods include sedating animal prior to obtaining a blood sample.

[0108] The mammal can also be treated prior to obtaining the peripheral blood sample. In certain embodiments, the mammal has been treated with an agent having alpha-adrenergic binding affinity prior to obtaining the peripheral blood sample. Thus, in some embodiments, the mammal has been treated with an alpha2-adrenergic agonist, dexmedetomidine, medetomidine, xylazine, opioids, and / or Butorphanol.

[0109] In certain embodiments, the primary pericytes may be identified and isolated from peripheral blood by Fluorescence-activated Cell Sorting (FACS), Magnetic-activated cell sorting (MACs), or differential adherent culture by enrichment for the expression of a profile of key proteins present on the cell surface membranes. This profile of pericyte markers is relatively w ell conserved. Pericyte markers include:• Chondroitin sulfate proteoglycan 4 (CSPG4, also known as NG2);• Platelet Derived Growth Factor Receptors beta (PGDFRb) and alpha (PGDFRa);• KAI (CD82);• Melanoma cell adhesion molecule, MCAM (CD 146);• Endosialin (CD248);• C-MET receptor;4930-0302-8870.1 22• VEGF Receptors: KDR, TIE1, FLT4;• CX3CR1;• Notch 2 and 3 receptors;• Insulin Grow th Factor Receptor 2 (IGFR2);• Fibroblast Growth Factor Receptor 1 (FGFR1); and• Bone Morphogen Protein Receptor 2 (BMPR2).In certain embodiments, one or more, tw o or more, three or more, four or more, five or more, six or more of these pericyte markers are used to identify and isolate primary pericytes.

[0110] By way of example, but by no means limiting, the identity of pericytes may be confirmed by the expression of intracellular structural proteins, such as:• Actin alpha 2, smooth muscle (ACTA2);• Desmin;• Nestin;• Glial Fibrillary7Acid Protein (GFAP);• Myl9, which regulates muscle contraction by modulating the ATPase activity of myosin heads, are all identified as potential pericyte markers in both the lung and kidney (37- 39);• Myosin heavy chain 11 (Myhl 1);• Transgelin (TAGLN); and• Tropomyosin 2 (TPM2).In some embodiment, the identity of pericytes can be confirmed by expression of one or more, two or more, three or more, four or more, five or more, or six or more of these intracellular structural proteins.[OHl] Pericytes are negative for the following markers that may be used to isolate and identify primary endothelial progenitor cells from peripheral blood by FACS, MACs, or differential adherent culture by enrichment for the expression of a few key proteins present on the cell surface membranes:• PEC AM- 1 (CD31);• CD34;• VC AM (CD 102);• E-Selectin; and• IB4 (Isolectin).4930-0302-8870.1 23In some embodiments, one or more, two or more, or three or more of these markers can be used to distinguish between mammalian peripheral blood-derived pericytes and mammalian peripheral blood-derived endothelial progenitor cells.

[0112] In certain embodiments of the methods, the endothelial progenitor cells express at least three markers, at least four markers, at least five markers selected from CD34, platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), E-Selectin, Vascular Endothelial-Cadhenn (VE-cadherin or Cadherin 5), and SCARF- 1.

[0113] In other embodiments of the methods, the pericytes express at least three, alternatively at least four, alternatively at least five, alternatively at least six cell surface membrane proteins selected from c-Met, chondroitin sulfate proteoglycan 4 (CSPG4), platelet derived growth factor receptor beta (PGDFRb), platelet derived growth factor receptor alpha (PGDFRa), CD82, melanoma cell adhesion molecule (CD 146), endosialin (CD248), kinase insert domain receptor (KDR), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), fms-related receptor tyrosine kinase 4 (FLT4), C-X3-C motif chemokine receptor 1 (CXC3CR1), Notch 2 receptor, Notch 3 receptor, insulin growth factor receptor 2 (IGRF2), fibroblast growth factor receptor 1 (FGFR1), and bone morphogenetic protein receptor 2 (BMPR2).

[0114] In further embodiments, the pericytes express one or more intracellular structural proteins chosen from actin alpha 2 (ACTA2), desmin, nestin, glial fibrillary acid protein (GFAP), Myl9, myosin heavy chain 11 (Myhl 1), transgelin (TAGLN). and tropomysin 2 (TPM2).

[0115] In certain embodiments, the pericytes lack expression of platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM), E-Selectin. and isolectin B4 (IB4).

[0116] In certain embodiments, the methods of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample also include isolating the pericytes. The pericytes can be isolated using the any of the markers (cell surface proteins and / or cell surface proteins) mentioned above. In certain embodiments, the pericytes lack expression of platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4) and the isolating the population containing pericytes comprises screening for cells that are negative for platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM). E-Selectin. and isolectin B4 (IB4).4930-0302-8870.1 24

[0117] In further embodiments, the presence of pericytes is based on testing for three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more of the following: ITGB5, VWF, VIM, FN1, COL1A1, ANX5, COL3A1, ITGB1, ITGA5, CSPG4, TNC, CD44, ENG, FAP, MMP14, PDGFRA, DCN, THY1, COL4A2, PDGFRB, TGFBI, and IGFBP3. In other embodiments, the presence of pericytes is based on testing for three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more of the following: VIM, COL1A1, ANXA5, COL3A1, CSPG4, ITGA5, ITGB1, CD44, ENG, ICAM1, PDGFRA, MMP14, THY1, COL4A2, PDGFRB, DCN, TGFBI, and ITGB5

[0118] In certain embodiments, the methods of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample and the methods of deriving a cell population containing endothelial progenitor cells also include isolating the endothelial progenitor cells.

[0119] A variety of different techniques can be used to isolate the pericytes and / or endothelial progenitor cells. In certain embodiments, the isolating includes fluorescence activated cell sorting (FACS), magnetic cell separation (MACs), or differential adherent culture.

[0120] Yet another embodiment of the disclosure is a method of isolating endothelial progenitor cells (EPCs) and pericytes from a peripheral blood sample which includes: isolating the peripheral blood sample from a mammal; and culturing the peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period sufficient to allow for grow th of endothelial progenitor cells and pericytes, thereby deriving the EPCs and / or pericytes, wherein the cell culture medium is supplemented with serum. In certain embodiments of the method, the cell culture medium lacks or is not supplemented with epidermal growth factor. In certain embodiments, a defined cell culture medium as disclosed below can be used.

[0121] The disclosure includes isolated pericytes derived by the methods of the disclosure. The disclosure also includes isolated endothelial progenitor cells derived by the methods of the disclosure.C. Cell Culture Media

[0122] Another aspect of the disclosure is directed to cell culture media. In certain embodiments, the culture media are used to derive or culture pericytes, such as mammalian peripheral blood-derived pericytes. In other embodiments, the culture media are used to4930-0302-8870.1 25derive or culture endothelial progenitor cells, such as mammalian peripheral blood-derived endothelial progenitor cells.

[0123] One aspect of the disclosure is directed to serum-free defined cell culture media containing: PDGF-beta; VEGF-C; a Notch ligand; a fibroblast growth factor; a TGF-beta superfamily member; an insulin agonist; transferrin; a hepatocyte growth factor; and a Wnt antagonist. In one embodiment, the serum-free defined cell culture media lack epidermal growth factor.

[0124] In one embodiment, the Notch ligand is Jagged 1, Jagged 2, or a mixture thereof. In another embodiment, the fibroblast grow th factor is bFGF. In yet another embodiment, the TGF-beta superfamily member is a bone morphogenic protein. In a further embodiment, the TGF-beta superfamily member is BMP10. In yet another embodiment, the insulin agonist is natural insulin, recombinant insulin, and / or IGF2. In an additional embodiment, the transferrin is apo and / or holo-transferrin. In yet another embodiment, the Wnt antagonist is Dickkopf-1, Dickkopf-2, Dickkopf-3, or a WTN antagonist antibody.

[0125] Yet another embodiment is a serum-free defined cell culture media containing PDGF-beta; VEGF-C; hepatocyte growth factor (HGF), and a choice of a Notch ligand, a fibroblast growth factor, a TGF-beta superfamily member, an insulin agonist, transferrin, a hepatocyte grow th factor, and a Wnt antagonist shown in Table A below7. A further embodiment is a serum-free, EGF-free (z.e. lacking EGF) defined cell culture media containing PDGF-beta; VEGF-C; hepatocyte growth factor (HGF) and a choice of a Notch ligand, a fibroblast growth factor, a TGF-beta superfamily member, an insulin agonist, transferrin, a hepatocyte growth factor, and a Wnt antagonist shown in Table A below .4930-0302-8870.1 26Each of the choices of a Notch ligand, a fibroblast grow th factor, a TGF-beta superfamily member, an insulin agonist, transferrin, a hepatocyte growth factor, and a Wnt antagonist in Table A can be used together. Thus, for example, in one embodiment, Jagged 1, BFGF, BMP 10, natural insulin, apo, and Dickkopf-1 are used. In another embodiment, Jagged 2, bFGF, a TGF-beta superfamily member, IGF2, halo-transferrin, and Dickkopf-3 are used.

[0126] Another embodiment is a conditioned cell culture medium generated by culture pericytes obtained using the methods described herein. In one embodiment, the conditioned cell culture medium is further supplemented with for example a pituitary extract such as e.g.. bovine pituitary extract.

[0127] The cell culture media can be used in the methods of deriving pericytes and / or endothelial progenitor cells described above.

[0128] In another aspect, the cell culture media are used to culture pericytes. One embodiment is a method of culturing pericytes in defined culture medium of the disclosure. Another embodiment is a method of culturing pericytes in a conditioned cell culture medium of the disclosure. In another aspect, the cell culture media are used to culture endothelial progenitor cells. One embodiment is a method of culturing endothelial progenitor cells in defined culture medium of the disclosure. Another embodiment is a method of culturing endothelial progenitor cells in a conditioned cell culture medium of the disclosure.

[0129] Another embodiment is directed to methods of generating a population of peripheral blood-derived pericytes or endothelial progenitor cells which include the steps of: culturing a mammalian peripheral blood sample in a defined cell culture medium or condition medium as4930-0302-8870.1 27described above in an adherent cell culture for a period sufficient to allow for growth of peripheral blood-derived pericytes or peripheral blood-derived endothelial progenitor cells; and isolating peripheral blood-derived pericytes or peripheral blood-derived endothelial progenitor cells from the sample after culturing.

[0130] In certain embodiments, the blood sample includes non-hematopoietic cells. In one embodiment, the culturing includes culturing the mammalian peripheral blood sample on collagen, fibronectin, or cell-culture treated plastic. In another embodiment, the isolating includes FACS, MACs, or differential adherent culture.

[0131] In other embodiments, the isolating includes screening for cells expressing at least three markers chosen from CSPG4, PGDFRb, PGDFRa, CD82. CD146, CD248, KDR, TIE1, FLT4, CXC4CR1, Notch 2 receptor. Notch 3 receptor, IGRF2, FGFR1, and BMPR2.D. Methods of sene editins and producing transgenic animals

[0132] The disclosure also includes gene editing the isolated pericytes derived and / or endothelial progenitor cells. In addition, the disclosure includes methods of producing transgenic animals from these cells. The disclosure also includes generating induced pluripotent stem cells from the isolated pericytes and or endothelial progenitor cells.

[0133] These uses of the cells are based on the discovery that it is possible for first isolate pericytes using the protocols described in Section A. above and then isolate and expand clonogenic lines of pericytes after gene-editing, meaning a cell can give rise to millions of cells. This is a critical requirement to be able to genotype and cryopreserve single-cell derived lines after gene-editing. In contrast, fibroblasts typically do not expand beyond passage 15, hitting a Hayflick limit and undergoing senescence. This window limits the ability to perform multiple rounds of gene-editing. In addition, single fibroblasts typically undergo senescence when plated alone into a single well. It has been demonstrated that it is possible to expand pericytes obtained using the methods of the disclosure, as single cells into clonal lines after gene editing by three methods: FACS sorting, limiting dilution, and plating on a Cell Raft system.

[0134] One aspect of the disclosure is directed to methods of gene editing a mammalian peripheral blood-derived pericyte. In one embodiment, the methods of gene editing a mammalian peripheral blood-derived pericyte include editing a gene of interest in an isolated mammalian peripheral blood-derived pericyte obtained by culturing a peripheral blood sample in a cell culture medium under adherent conditions for a period sufficient to allow for growth of pericytes. In another embodiment, the method of gene editing mammalian4930-0302-8870.1 28peripheral blood-derived pericytes includes: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period sufficient to allow for grow th of pericytes thereby deriving the mammalian peripheral blood-derived pericytes, wherein the cell culture medium is supplemented with serum; and editing a gene in the mammalian peripheral blood-derived pericytes to produce a mammalian peripheral blood-derived pericyte having an edited gene. In certain embodiments, the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0135] One aspect of the disclosure is directed to methods of gene editing a mammalian endothelial progenitor cell. In one embodiment, the methods of gene editing a mammalian endothelial progenitor cell include editing a gene of interest in an isolated mammalian endothelial progenitor cell obtained by culturing a peripheral blood sample in a cell culture medium under adherent conditions for a period sufficient to allow for growth of endothelial progenitor cells. In another embodiment, the method of gene editing mammalian endothelial progenitor cells includes: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period sufficient to allow for growth of pericytes thereby deriving the mammalian endothelial progenitor cells, w herein the cell culture medium is supplemented with serum; and editing a gene in the mammalian endothelial progenitor cells to produce a mammalian endothelial progenitor cell having an edited gene. In certain embodiments, the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0136] Yet another aspect includes a method of gene editing mammalian peripheral blood-derived cells including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium supplemented with serum under adherent conditions for a period sufficient to allow for growth of mammalian peripheral blood-derived cells thereby deriving the mammalian peripheral blood-derived cells; and editing a gene in the mammalian peripheral blood-derived cells to produce a mammalian peripheral blood-derived cells comprising an edited gene, wherein the mammalian peripheral blood-derived cells are mammalian peripheral blood-derived pericytes and / or mammalian peripheral blood-derived cells. In certain embodiments, the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0137] In some embodiments, the cell culture medium is a defined cell culture medium as described in Section C above. In other embodiments, the cell culture medium is EGM. In further embodiments, cell culture medium is conditioned medium generated by culturing4930-0302-8870.1 29pericytes. In certain embodiments, the cell culture medium is supplemented with pituitary extract, such as bovine pituitary extract.

[0138] In certain embodiments of the methods, the adherent conditions include culturing the cells on collagen, fibronectin, or cell-culture treated plastic. In other embodiments, the methods include culturing the mammalian peripheral blood-derived pericyte having an edited gene to produce a clonal line. The method can also include FACS sorting, limiting dilution, and / or plating on a Cell Raft system. In further embodiments, the methods include isolating a mammalian peripheral blood-derived pericyte having an edited gene.

[0139] In some embodiments of these methods, the mammal is a mouse, a rat, a marsupial, a bovine, an ovine, a caprine, a pig, an elephant, an equid, a cat, a dog, a wolf, or a deer. In another embodiment of the methods, the bovine is a cow, a bison, or an antelope. In a further embodiment of these methods, the equid is a horse, a zebra, a donkey, a tapir, or a rhinoceros. In additional embodiments, the mammal is Felis catus, Canis lupus familiaris, Canis lupus, Bison bison, Dama dama, Hippotragus equinus, or Hippotragus niger.

[0140] The disclosure also includes isolated mammalian peripheral blood-derived pericytes having an edited gene produced by the methods above. The disclosure also includes isolated mammalian peripheral blood-derived endothelial progenitor cells having an edited gene produced by the methods above.

[0141] In certain embodiments, the isolated mammalian peripheral blood-derived pericytes having an edited gene can be used to produce a transgenic animal. In other embodiments, the isolated mammalian peripheral blood-derived endothelial progenitor cells having an edited gene can be used to produce a transgenic animal. The methods involve gene-editing followed by SCNT to produce genetically identical or purposefully modified cloned animals. In certain embodiments, the method involves SCNT as outlined in Examples 3 and 4 below.

[0142] Yet another embodiment of the disclosure is directed to methods of producing an induced pluripotent stem cell from an isolated mammalian peripheral blood-derived pericyte obtained via the methods in Section B above, which include transfecting the isolated pericyte with one or more of OCT4, KLF4. SOX2, and optionally c-MYC. Yet a further embodiment of the disclosure is directed to methods of producing an induced pluripotent stem cell from an isolated mammalian peripheral blood-derived endothelial progenitor cell obtained via the methods in Section B above, which include transfecting the isolated mammalian endothelial progenitor cell with one or more of OCT4, KLF4, SOX2, and optionally c-MYC.

[0143] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the4930-0302-8870.1 30present invention and practice the claimed methods. The following working examples, therefore, specifically point out the preferred embodiments of the present invention and are not to be construed as limiting in any way the remainder of the disclosure.ExamplesExample 1 - Isolation of Pericytes and EPC

[0144] Somatic cell nuclear transfer (SCNT) followed by Embryo Transfer (ET), generally known as reproductive cloning, typically uses a somatic cell such as a fibroblast cell as the nuclear donor. Derivation of dermal fibroblast lines from human or animal patients usually requires a painful and invasive, skin biopsy requiring closure by sutures or dermal glue and exposing the patient to the risk of infection. Commercial cat, dog and horse cloning services currently rely on the use of dermal fibroblasts isolated from skin biopsies or stromal tissue from neuter or spay procedures.

[0145] While nucleated hematopoietic cells (White cells: T-cells, B-cells, Macrophage, Monocytes, Granulocytes, NK cells) can all be isolated from peripheral blood, they cannot be expanded in culture without transformation such as using Epstein Barr Virus (EBV) or reprogramming into induced pluripotent stem cells (iPSCS) using Sendai or Lentiviral Vectors. These resultant cell lines are unsuitable for subsequent SCNT to produce viable offspring, as the EBV expression can interfere with embryonic development. In addition, any offspring produced by SCNT from a primary' (non-transformed, non-expanded) peripheral B or T-cell that has undergone Variable, Diversity and Joining (VDJ) of antigen receptor (AR) genes would have a fixed haplotype and be immunologically deficient and susceptible to profound systemic infections.

[0146] In an effort to enable less invasive collection of expandable primary somatic cells for SCNT, adherent cells from peripheral blood samples obtained from a range of domestic and exotic animal species using a commercially available medium (EGM from Lonza) were collected, isolated, and cultured.

[0147] For these isolations, the following protocol was used. Pericytes were isolated from peripheral blood samples of the animal using a method to traditionally isolate and culture Endothelial Progenitor Cells (EPCs). These cultured cell lines were subsequently used as nuclear donors for SCNT. Pericytes typically sit on the outside of the endothelial layer of small blood vessels such as capillaries. While rare EPCs are known to be present in systemic circulation, pericytes are not. Isolation pericytes from peripheral blood samples by the same method was also possible. The ease of collection by standard venipuncture techniques in a4930-0302-8870.1 31clinical or field seting enables the isolation of expandable adherent non-hematopoietic cell populations for subsequent gene-editing and SCNT. Cells obtained using the protocols of this disclosure produce viable offspring.

[0148] This protocol was tested with various carnivores and herbivores and proven to successfully isolate pericytes and EPCs. Specifically, this protocol was successfully used to isolated pericytes and EPC from wolves (Canis lupus'), cats (Fells catus). dogs (Cams lupus familiaris), bison (Bison bison), fallow deer (Dama dama), roan antelope (Hippotragus equis), and sable antelope (Hippotragus niger).A. Isolation of Pericytes and EPC from CarnivoresI. Isolation of pericytes and EPC from wolves (Canis lupus)

[0149] Pericytes were isolated from blood samples collected from two male gray wolves (Cl-001 and Cl-004). Pericytes were isolated from peripheral blood of the two grey wolves using a method to traditionally isolate and culture Endothelial Progenitor Cells (EPCs). These cultured cell lines were subsequently used as nuclear donors for SCNT. Pericytes typically sit on the outside of the endothelial layer of small blood vessels such as capillaries. While rare EPCs are known to be present in systemic circulation, pericytes are not. Isolation pericytes from peripheral blood samples by the same method was also possible. The ease of collection by standard venipuncture techniques in a clinical or field seting enables the isolation of expandable adherent non-hematopoietic cell populations for subsequent geneediting and SCNT.

[0150] The pericytes collected from these gray wolves were used for an SCNT experiment using dog oocytes. Transfer of thirty-one cloned embry os into one surrogate dog resulted in the implantation of eight viable fetuses, which were isolated at mid-gestation (4 weeks). These fetuses were individually genotyped and shown to be from both of the two wolf donor pericyte lines.

[0151] Using this protocol, it has been possible to grow, deliver a range of gene editing tools (by transfection, electroporation, and viral transduction) into wolf pericytes and isolate and expand hundreds of gene-modified lines.II. Isolation of pericytes and EPC from dogs (Canis lupus familiaris)

[0152] A blood sample was obtained from a domestic dog (CLF-003) and adherent cell lines were derived. These cells were used for cloning and embryo transfer (ET). A surrogate dog became pregnant after ET of cloned-SCNT embry os using pericy tes and gave birth to a live cloned female pup. Pictures of this female pup match the coat color and patern of the female donor dog that is now deceased. DNA profiling (STR analyses) have been performed4930-0302-8870.1 32on an oral swab of the pup and the adult dog donor cells by a third-party' diagnostic lab (UC Davis). The results of this testing are shown in FIG. 4A to FIG. 9.Example 2 - Isolation of Pericytes and EPC using conditioned media

[0153] As pericytes produce autocrine growth factors, it was observed that the use of conditioned medium improved the efficiency of derivation and doubling-time or mitogenic rate of expansion of pericyte cultures. These protocols are similar to the protocols used in Example 1 above except that a conditioned medium is used.

[0154] Proteomic and gene-expression analyses have also identified the expression of key growth factors and signaling pathways that can enable more efficient derivation and sustained expansion.Example 3 - Creation of Transgenic Animals with direwolf gene variants

[0155] The animal work described herein has been approved and conducted under the oversight of an Institutional Animal Care and Use Committee (IACUC). Colossal uses the “Guide for the Care and Use of Laboratory Animals” when establishing animal research standards.Materials and Methods:Cell Engineering:

[0156] Blood-derived adherent endothelial progenitor cells (EPCs) and pericytes from wild and domestic canids represent a readily accessible, relatively non-invasive way to biobank living cells from animals (FIG. 1). Peripheral blood was collected by venipuncture from adult gray wolves, and the mononuclear fraction of cells separated from Red Blood Cells by centrifugation using a HISTOPAQUE® density gradient. The entire mononuclear fraction was plated in defined or commercial EPC medium (EGM™ or EGM™2 medium from Lonza; Basel, Switzerland) on plastic or collagen-coated cell culture plates or flasks and cultured overnight. The medium was changed the following day to remove all unattached cells. Within 5-10 days of culture, clusters of peripheral blood-derived, adherent cells were observed. These putative EPCs were reliably expanded, cryopreserved, and reconstituted.Genome Engineering:

[0157] As a proof of concept, five loci or genes of interest associated with core direwolf phenotypes were targeted. The genes are: LCORL, FGF5, MC1R, MFSD12. and MSTN. LCORL is associated with size in domestic dogs, FGF5 is associated with coat composition across mammalian species, MC1R and MFSD12 are associated with coat composition and4930-0302-8870.1 33coloring across mammalian species, and MSTN is associated with muscle composition across mammalian species. CRISPR guides were designed for full gene knock-out (KO) in all five loci in gray wolf endothelial progenitor cell lines. Tested guides for each locus were assembled into a single 5X array and delivered to the gray wolf EPCs in a single transfection. Edited EPCs were sorted based on a GFP-reporter system. Monoclonal EPCs were established from the edited population and screened for editing efficiencies on the gene KOs of interest. Genotyping verified successful editing of intended targets with a combination of homozygous and heterozygous KOs. Edited EPCs were expanded and prepped for SCNT.Somatic Cell Nuclear Transfer:

[0158] Blood-derived EPCs and pericytes from an adult male, gray wolf, Cams lupus (22CL04) were used as nuclear donors in enucleated domestic dog oocytes and activated by standard methods used in domestic dog cloning (Lee et al., '‘Dogs cloned from adult somatic cells,” Nature 436:641 (2005); Olsson et al. , “Insights from one thousand cloned dogs,” Sci. Rep. 12: 11209 (2022)). While canine SCNT is routinely performed with fibroblasts, the successful cloning from adherent, blood-derived, non-hematopoietic cells has not been reported to date. After overnight in vitro culture, cleavage to the 2-cell stage is evident in some of the iSCNT gray wolf embryos-indicative of effective activation (FIG. 2).Analysis.

[0159] Both 1-cell and 2-cell iSCNT embryos reconstructed with genetically engineered gray wolf EPCs were surgically transferred into the oviducts of domestic dog recipients in natural estrus. Uterine implantation was reliably detected by trans-abdominal ultrasound (US) as early as day 20 in embry o transfer (ET) recipients, with cardiac development and active beating observed by Day 23 using trans-abdominal ultrasound (US) (FIG. 3).

[0160] Transabdominal radiography and micro-Computed Tomography (microCT) were used to detect normal bone formation between Day 42-47 of post-implantation development of iSCNT genetically engineered gray wolf fetuses.

[0161] Cells lines and tissue were isolated from iSCNT genetically engineered wolf fetuses. Post-implantation iSCNT wildtype and gene-edited gray wolf fetuses were isolated after ovariohysterectomy between 23 to 45 days after oviductal embryo transfer. In one experiment, ET of 34 iSCNT genetically engineered gray wolf embry os resulted in the implantation of six fetuses. A timed ovariohysterectomy (OHE) was performed at Day 33 after embryo transfer, and fetuses were isolated from the uterus under sterile conditions. Tissues were collected from these genetically engineered fetuses for downstream transcriptomic analysis and comparisons in the genes of interest.4930-0302-8870.1 34

[0162] Transfer of 339 embryos split between eight recipient dogs resulted in four pregnancies of gene-edited iSCNT gray wolf cloned embryos with two singleton pregnancies going to term. Two living gene-edited, male wolf pups were delivered by c-section at day 62 and 63 post embryo transfer. Genotyping of the umbilical cords from each pup demonstrated that both were derived from the same clonal gene-edited EPC line. Both pups demonstrated phenotypic changes from the wildtype Agouti coat color in the starting adult donor wolf to pure white by the disruption of both the MC1R and MFSD12 loci preventing the production of eumelanin and pheomelanin. Additional phenoty pic and transcriptomic analysis, such as cranio-facial and whole body measurements, among others, will continue as the pups grow.Example 4 - Primary Cell Line Derivation for Conservation: A Comparative Analysis of Blood-Derived Cells and Dermal Fibroblasts

[0163] Establishing stable and proliferative primary' cell lines is critical for advancing experimental models and conservation applications. Traditionally, fibroblasts isolated from skin biopsies have been a common source for cell line derivation. This Example demonstrates that endothelial progenitor cells (EPCs), and pericytes represent a superior alternative with comparable genomic stability, enhanced cloning potential, increased culture duration, and significantly faster doubling rates. EPCs and pericytes proliferation reduces turnaround time from derivation to experimental readiness, improving efficiency in downstream applications such as cloning and cell assays. Additionally, unlike fibroblasts, which require skin biopsies, EPCs and pericytes can be readily harvested from blood samples, facilitating a potentially less invasive collection process which can be coupled with standard veterinary care and yielding a higher success rate for cell line establishment. Thus, EPCs may provide a more viable and scalable source for cell-based research, particularly in studies requiring rapid expansion and genomic integrity.

[0164] Primary' cell derivation offers significant potential for species conservation by proactively safeguarding genetic diversity, a crucial resource for species facing extinction. Major biobanks, such as the Frozen Zoo® at the San Diego Zoo Wildlife Alliance, have amassed substantial collections; its repository holds over 10,000 individual cell lines representing more than 1,100 species and subspecies, including approximately 5% of threatened mammals, birds, amphibians, and reptiles listed on the IUCN Red List (San Diego Zoo Global, 2020).

[0165] Minimally invasive techniques like skin punch biopsies commonly yield fibroblasts (Plikus et al.. Cell 184, 3852-3872 (2021)), mesenchymal cells abundant in connective tissues4930-0302-8870.1 35such as muscle, skin, and cartilage. Fibroblast derivation success can vary dramatically across species. While some, like the Chilean shrew, have reported high success rates (e.g..90% with dermatome punches; Tovar et al., In vitro Cellular & Developmental Biology - Animal 44, 309-320 (2008)), others, such as elephants, exhibit low and inconsistent success (0%-80%) even from biopsies of the same individual (Vuuren et al., Animals (Basel) 13, 2353-2363 (2023)). In addition, skin samples are covered with commensal microbes that can contribute to the contamination of cell cultures, despite efforts to reduce this risk in the clinic, field, and lab.Materials and MethodsA. Blood Derivation

[0166] Blood samples from four species were collected: gray wolf (Canis lupus) and domestic dog (C. I. familiaris), bison (Bison bison), horse (Equus caballus), and all major zebra species (Equus quagga, E. zebra, and E. grevyi). Hereafter, these are referred to as canids, bovids, and equids.

[0167] All samples were collected in ACD blood tubes and transported overnight at room temperature. Samples required for blood derivation differed by genus, but all needed to be collected in ACD blood tubes and transported overnight at room temperature for optimal success. For Canid species, a minimum of 15 mL of blood was needed, while Bison and Equid species required at least 25 mL blood for successful derivation.

[0168] Upon receipt, the blood volume was recorded and then diluted with lx dPBS (Cat#: 14190-144; Fisher Scientific) and 3% HiFBS (Cat#: 16140071; Gibco) at a 1:3 ratio. The diluted blood was then gently and equally layered on top of 15 mL of Ficoll Paque Plus (Cat#: 95021-205; VWR) in 50 mL conical tubes. For example, 60 mL of diluted blood would be split into two 50 mL conicals with 15 mL Ficoll Paque Plus per conical. The dilute blood:Ficoll Paque Plus was then centrifuged at 800g for 25 minutes at room temperature with the break set to 0. The plasma layer was aspirated. The PBMC layer was then transferred to another 50 mL conical tube and brought up to 50 mL with dPBS. The conical tube was then centrifuged at 800g for 7 minutes with the brake set to 9. The supernatant was aspirated and resuspended in 30 mL of dPBS and centrifuged at 400g for 5 minutes. The supernatant was then aspirated and resuspended in 1 mL of EGM (20% HiFBS, + EGM (Cat#: CC-3124; Lonza)). The cell suspension was counted using AOPI Cell Staining Solution (Cat#: CS2-0106-25 mL; Fisher Scientific), resuspended in 25 mL of EGM, transferred to a Biocoat Collagen T175 Flask (Cat#: 356487; Coming), and then incubated at 37°C. The next day, the media was changed, and media changes continued every other day4930-0302-8870.1 36until colonies formed. Once colonies appeared daily half media changes were performed until the colonies were confluent or were greater than 20 total colonies per flask.

[0169] The plate was lifted using 50:50 ratio of 0.05% Trypsin (Cat#: 25300-054; Fisher Scientific) and Accutase (Cat#: SF006; Millipore) for 3-5 minutes at 37°C. Once cells were lifted, the solution was quenched with EGM and centrifuged at 400g for 5 minutes. The media was then aspirated and resuspended in 1 mL of EGM. The cells were then counted using AOPI and plated onto Biocoat Collagen T175 Flasks and seeded at l-2xl06per flask. When the cells were confluent, the same procedure was repeated and instead of the cells being replated, they were centrifuged at 400g for 5 minutes, and then resuspended at a concentration of lxlO6 / mL in EGM freezing media (30% EGM, 60% HiFBS, and 10% DMSO (Cat#: D2650-5x5 mL)).B. Fibroblast Derivation and Culture

[0170] Dermal tissue was obtained from either ear notches, or punch or dart biopsies. To account for contamination from the field, the samples were divided in half to generate an explant-derived cell line or digested-derived fibroblast cell line. Digested-derived fibroblasts were generated by mincing in collagenase IV (Cat#: C4-28-100MG; Sigma) and then incubating at 37°C for 1 hour. The cells were then washed and plated onto a tissue culture treated T75 flask (Cat#: 229340; CellTreat) in cDMEM (DMEM high glucose, + sodium pyruvate. - glutamine (Cat#: 10313-021; Fisher Scientific)), 10% FBS, IX Glutamax (Cat#: AI2860-01; Fisher Scientific). IX MEM-NEAA (Cat#: 11140-050; Fisher Scientific), IX 2-mercaptoethanol (Cat#: 21985023; Gibco)). The cells were cultured, passaged, and frozen at p2 to p3 in FB Freezing Media (30% cDMEM, 60% HiFBS, and 10% DMSO).C. Doubling Rate Calculation

[0171] The formula used to calculate the doubling rate across all species and cell types was:Doubling Rate = Hours in culture * LN(2) / LN(final cell number / initial cell number) D. Proteomics analysis

[0172] Cells were maintained under standard culture conditions until passage 5 and cultured to 80% confluency in two six-well plates for biological replicates. On the day of lysis, cells were gently washed twice with 1 mL of 1 x dPBS, and the supernatant was discarded. Lysis was performed by adding 75 pL of RIPA buffer (Cat# J63306.AP) premixed with HALT protease inhibitor (Cat# 87785). 1 pL of nuclease (Cat# 88700) was then incorporated and mixed post-lysis. Total protein concentration was quantified using Thermo4930-0302-8870.1 37Fisher’s BCA Gold assay (Pub. No. MAN0029413 Rev. B.O). Lysates were stored under -80 °C in 1.5 mL low bind Eppendorf tubes until all samples were collected for downstream mass spectroscopy library preparation.

[0173] Peptides were generated via trypsin digestion and labeled using the TMTIOplex isobaric labeling kit (Cat# 90406; Thermo Fisher). Labeled samples were pooled by species after high-pH fractionation yielded eight fractions. Each fraction was analyzed using an Orbitrap Eclipse mass spectrometer and processed with Proteome Discoverer 3.0 software. Peptides were annotated against species-specific UniProt databases. Following data acquisition, proteins were filtered based on false discovery rate (FDR), retaining only those with an FDR <1% and a normalized average intensity >2000 units across grouped samples for downstream analysis.E. Whole Genome Sequencing and Reference Genome Assemblies

[0174] For canids, the gray wolf GenBank assembly GCA_905319855.2 was used, which included autosomes, sex chromosomes, and a mitochondrial genome. For equids, the domestic horse GenBank assembly GCA_041296265.1 was used, which included autosomes, chromosome X, and a mitochondrial genome. For bovids, the autosomes, X chromosome, and mitochondrial genome from bison GenBank assembly GCA_030254855.1 were combined with the Y chromosome from Fl cattle x bison GenBank assembly GCA_018282365.1.F. FA Cs Staining and Analysis

[0175] 3x105cells were harvested per condition per line and then washed with cold blocking buffer (90% dPBS (Cat#: 14190-144; Fisher Scientific) and 10% Goat serum (Cat#: 16210-072; VWR)) for 30 minutes. The cells were pelleted and washed twice with dPBS at 400g for 5 minutes. The cells were then incubated for 1 hour at room temperature in the dark with 200uL of Flow Cytometry Antibody Dilution Buffer (Cat#: 13616; Cell Signaling Technology) plus the corresponding antibody. The Rat IgG2b Isotype Control (APC) (Cat#: 17-4031-82; eBioscience) and Mouse IgGl Isotype Control (PE) (Cat#: 12-4714-82; eBioscience) were diluted 1:200, CD90 (APC) (Cat#: 17-5900-42; eBioScience) was diluted at 1:20. CD34 (PE) (Cat#: 12-0340-42; eBioscience) was added to make 3 pg / mL, and the dual stain condition was the same with CD90 at a 1 :20 dilution and CD34 at 3 pg / mL dilution. After incubation, the cells were pelleted and washed three times with dPBS at 400g for 5 minutes and then resuspended into 200 pL of cold FACs buffer (dPBS, 2% FBS(Cat#: 16140071; Gibco). 2.5 mM EDTA pH 8.0 (Cat#: 15575020; Thermofisher), 25 mM HEPES (Cat#: 15630-080; Fisher Scientific), and 1% Pen / Strep 100X (Cat#: 15140122; Fisher4930-0302-8870.1 38Scientific) and filtered into a 5 mL FACs tube (Cat#: 196-19881-083; Spectrum Laboratory' Products). The cells were then analyzed on BD FACSDiscover™ S8 Cell Sorter, with a VBYR laser configuration, 100pm nozzle size, and standard configuration and settings while using the YG1 (575) and R1 (655) lasers.G. Angiogenesis

[0176] The Matrigel (Cat#: 354230; Coming) was thawed on ice and then 75 pL was plated per well of a 96 well plate. The plate was then incubated at 4°C for at least 1 hour. The Matrigel was then aspirated prior to plating (for blood derived cells: Cat#: 354409; Coming; for fibroblasts: Cat# 229196; Cell Treat) and then incubated at 37°C for 10 minutes. Cells were lifted by normal culture conditions and then were plated at 1x104for EPCs and Pericytes, and 2x104for fibroblasts in duplicates into both Matrigel coated wells and uncoated 96 wells with 100 pL media. The plate was then incubated at 37°C for a maximum of 24 hours and imaged at least four times over the time period. In general, the assay showed tubule formation by 12 hours.H. Low Density Lipoprotein & Acylated Low Density Lipoprotein Uptake

[0177] Fibroblasts and blood derived cells were grown under standard cell culture conditions until the cell lines had reached 65-80% confluency. On the day of passaging, l.OxlO5cells were collected from each cell line, pelleted, washed with IX DPBS, and then resuspended in serum free DMEM (DMEM, -FBS, 0.3% MACS® BSA (Cat#: 130-091-376, Miltenyi Biotec)) for fibroblasts and serum free EGM (EGM.-FBS. 0.3% MACS® BSA) for blood derived cells and transferred onto a Coming 96-well for blood derived cells and Cell Treat 96-well for fibroblasts with l.OxlO4cells per condition. The plate was then incubated at 37°C for 16 hours. Cells were then washed with LDL Assay buffer (IX DPBS, +calcium, +magnesium (Cat#: 14040133; Gibco), 0.3% MACS® BSA) and resuspended in serum free DMEM for fibroblasts and serum free EGM for blood derived cell lines. Low Density Lipoprotein from Human Plasma, BODIPY™ FL complex (Cat#: L3483, Invitrogen™) was diluted 1 : 100 for a final concentration of 10 pL / mL and added to the appropriate wells in replicates. Low Density Lipoprotein from Human Plasma, Acetylated, Alexa Fluor™ 594 Conjugate (Cat#: L35353; Invitrogen) was also diluted 1: 100 for a final concentration of 10 pL / mL and added to the appropriate wells in replicates. Cells were returned to the incubator at 37°C for 2.5 hours. After incubation, the cells were washed three times with the LDL4930-0302-8870.1 39Assay Buffer, imaged, and analyzed on the Incucyte® SX5 utilizing the Orange / Green optical modules.I. Karyotyping:

[0178] Live cell cultures were shipped to an independent cytogenetics laboratory (KaryoLogic Inc.) in two T25 flasks at 25% and 50% confluency so that they would arrive subconfluent, thus minimizing contact inhibition of the cells. Mitotic spreads were prepared, and G-banded karyotypes assembled from 20 cells per line. The average and atypical gross ploidy results were reported for each cell line with a typical karyotype provided for each.J. Optical Genome Mapping:

[0179] Optical Genome Mapping (OGM) analyses of the derived cell lines were performed using the Bionano Saphyr platform according to the manufacturer’s specifications. 500k-750k cells were used for the isolation of ultra-high molecular weight (UHMW) genomic DNA, as described in the Bionano Prep SP-G2 Fresh Cell Pellet DNA Isolation Protocol (Bionano, Doc # CG003). The UHMW gDNA was then fluorescently labeled using the Direct Label Enzyme (DLE-1) and stained for backbone visualization, following the Bionano Prep Direct Label and Stain (DLS) Protocol (Doc # 30206). The labeled and stained UHMW gDNA sample was loaded onto a Saphyr Chip and into the Saphyr System Instrument (Bionano Saphyr System User Guide, Doc # 30247). Structural variations (SV) and low variant allele fraction variants (VAF) were identified using Bionano Solve software (Bionano Solve Theory of Operation: Structural Variant Calling, Doc # CG-30110). With this method, variants were detected at a resolution of >500 bp up to the chromosome level, when confirmed by short-read WGS assemblies.K. Interspecies Somatic Cell Nuclear Transfer

[0180] Synchronization of fibroblasts. EPCs and pericyte donor cells for iSCNT was achieved by allowing cultures to grow to complete confluence to induce contact inhibition and enter cell cycle (GO) arrest. For gray wolf iSCNT, EPCs or pericytes from Canis lupus were used as nuclear donors. Oocytes from donor dogs in estrus were isolated and performed enucleation, reconstruction, electroporation, subsequent activation, and overnight in vitro culture (IVC) was performed as described previously (Kim et al. , Reproduction in Domestic Animals, 47 Supl. 6, 80-83 (2012)). For bison, cattle cumulus oocyte complexes (COCs) were isolated from abattoir ovaries for subsequent in vitro maturation (IVM), enucleation, reconstruction with female bison EPCs or fibroblasts and activation as previously published in detail for cow SCNT (Sangalli et al. , Methods in Molecular Biology. 2647, 225-2444930-0302-8870.1 40(2023)). Reconstructed Bison iSCNT embryos were then cultured in a bovine IVC medium for 7 days and cryopreserved at the blastocyst stage (Barfield, Methods in Molecular Biology, 2006, 165-177 (2019); Benham etal., Theriogenology, 160, 3-39(2021)). For equids, individual donor EPCs and fibroblasts from a male plains zebra were injected into the ooplasm of enucleated domestic horse oocytes obtained by ovum pick-up, electrofused, and activated to produce reconstructed iSCNT plains zebra embryos as performed in the successful generation of cloned Przewalski’s Horse by iSCNT (Novak et al., Animals: An Open Access Journal fromMPDI, 15(7) 2025). Reconstructed plains zebra embryos were cultured and frozen at the morulae and blastocyst stages 6-8 days post cloning (Hinrichs. Journal of Equine Veterinary Science, 2020).ResultsA. Derivation Success

[0181] Adherent cell populations were successfully derived from peripheral blood samples across three genera: Canis. including gray wolf and domestic dog, Bison, where all individuals were plains bison, and Equus, which included domestic horse and major zebra species (Table 4-1). It was observed that consistent successful derivation of canid cells required a minimum of 15mL of blood, while bovid and equid cell lines required at least 25mL of blood. Blood-derived cell cultures showed an overall derivation success rate of 78% (Table 4-1). Time to initial colony formation varied by species (FIG. 1): bison formed colonies in 4±1 days, equids in 6±1 days, and gray wolf in 7±1 days post-processing. Due to rapid proliferation, all blood-derived cultures generated 30 million low-passage (Pl-2) cells within 1.5-2 weeks of initial plating.4930-0302-8870.1 41

[0182] The relative success rates for primary cell line derivation from blood-derived cells and dermal fibroblasts are shown in Tables 4-2 and 4-3 below. Derivation of adherent blood cells in non-model organisms is a novel culture system and showed a species agnostic average success rate of 72.97%. Fibroblasts derived from ear notches have been successful on average 91.67% of the time if multiple replicates were done during the derivation process. However, for samples collected via dart or punch biopsies derivation success fell to an average of 45.45%.4930-0302-8870.1 42

[0183] In traditional fibroblast cell line derivation and generation, a large piece of tissue is processed for explant or digestion and plating methods. Both methods were used to ensure that if contamination happened in one replicate, there would be other cultures to ensure a successful cell line was generated. Following this digestion approach, the overall success rate was 91.67% (Table 4-2). This method also generates approximately 30 million low passage cells (P1-P3) in approximately 3-3.5 weeks, if no contamination occurs (FIG. 10). However, in collecting samples from non-model species, tissue is often much harder to obtain in the field. Using dart or punch biopsy protocols, typically only enough tissue was available to use the explant method of derivation, and contamination was more frequent. A 45.45% success rate via this method was observed (Table 4-3). This method has allowed for animals that are typically not accessible to be sampled, as well as not marring or scarring the animal itself. This explant approach to fibroblast generation takes about 4 weeks on average to freeze 30 million low passage cells, due to the initial sample being smaller (FIG. 10).B. Overall Proteomic Distinction of Primary Cell Populations

[0184] Relative protein expression data, quantified by mass spectrometry, was employed to characterize, and compare three primary cell populations: Endothelial Progenitor Cells (EPCs), Fibroblasts (FBs), and Pericytes (Peris). Principal Component Analysis (PCA) performed on 32 canonical cell markers revealed distinct clustering of EPC, FB, and Peri samples. Canid cell lines were used as a framework for the analyses, as canid annotations in the UniProt database both captured more genes and higher annotation scores compared to other taxa.

[0185] While not all markers were detected across all genera (canid, bo vid, equid), necessitating species-specific PCA plots and heatmaps based on detected markers, a combined PCA plotting all genera together also supported these distinct expression profiles. This segregation validates their classification as distinct biological entities within this study and suggests that these cell types maintain unique molecular signatures bey ond traditionally4930-0302-8870.1 43used model species. The observed distinctions provide a crucial baseline for interpreting individual protein expression patterns, including those that deviate from canonical definitions. Such deviations, as discussed below, are hypothesized to reflect nuanced biological factors like cellular plasticity or context-dependent expression. Canid annotations in UniProt database were found to both capture more genes and have higher annotation scores, therefore canid cell lines are used as a framework to compare and contrast bovid samples. However, the absence of a fibroblast control for equid samples led to their exclusion from the detailed comparative analysis presented here.

[0186] The results of this testing are in FIG. 11 A, as well as Tables 4-4 (Canid) and 4-5 (Bovid) below. Tables 4-4 and 4-5 show Log2(fold-change) and -Logio(p-value) for a curated list of genes, highlighting their canonical reported associations when comparing specific cell types within the Canid and Bovid genera. Gene expression data is show n for the following comparisons: Canid Genus: Endothelial Progenitor Cells (EPC) vs. Fibroblasts (FB), Pericytes (Peri) vs. Fibroblasts (FB), and Endothelial Progenitor Cells (EPC) vs. Pericytes (Peri). Bovid Genus: Endothelial Progenitor Cells (EPC) vs. Fibroblasts (FB). A Log2(fold-change) value of 1 represents a 2-fold change in gene expression, while a value of -1 indicates a 0.5-fold change. The -Logio(p-value) is a measure of statistical significance, where a value of 3 corresponds to a p-value of 0.001. Data includes “Marker Category” and “Gene Symbol” for each gene.4930-0302-8870.1 444930-0302-8870.1 45C. Proteomic Signature of Fibroblasts (FBs)

[0187] Fibroblasts in this dataset exhibited a proteomic signature characteristic of active extracellular matrix (ECM) production and a potentially activated state (see Table 4-4 and Table 4-5). Across both bovid and canid samples, FBs demonstrated significantly higher expression of several key proteins compared to EPCs and Peris, including Fibroblast Activation Protein alpha (FAP), Insulin-like growth factor-binding protein 3 (IGFBP3), Collagen Type I Alpha 1 Chain (COL1A1), Collagen Type III Alpha 1 Chain (COL3A1), Vimentin (VIM), Platelet-Derived Grow th Factor Receptor Beta (PDGFRB), and Tenascin C (TNC). FAP, an established marker for activated fibroblasts in tissue remodeling, was highly abundant in FBs compared to EPCs in both canid and bovid genera. The Log2(Fold-Change), abbreviated as “FC’ of FAP in EPCs relative to FBs was -1.4 in canid and -8.9 in bovid, indicating a more pronounced relative FAP expression in bovid FBs. Similarly, IGFBP3, involved in modulating IGF signaling and ECM interactions, was highly expressed in FBs and showed concordance in both bovid (-0.6) and canid (-3.2) genera Log2(FC). High abundance of major structural ECM components, COL1A1 and COL3A1, aligned with the known matnx-synthesizing capacity of FBs. This was observed in both canid and bovid expression profiles, although the Log2(FC) in EPCs relative to FBs was less pronounced in bovid (COL1A1: -1.1; COL3A1: -0.4) than in canid (COL1A1: -6.2; COL3A1: -4.4). Though all these differences were noted to be above significance Logio(p- values) > 2.8; note: a Logio(p-value) of 2 corresponds to p=0.01, and 3 to p=0.001. This pattern of a less pronounced Log2(FC) differential in bovid compared to canid for several FB-associated markers (when comparing FBs to EPCs) w as a recurring observation. Other highly expressed proteins in FBs included VIM, crucial for cell structure, and PDGFRB, a key receptor in fibroblast proliferation. TNC, an ECM glycoprotein induced during tissue injury and remodeling, showed its highest abundance in FBs, despite some literature variability in its cellular categorization.D. Proteomic Signature of Pericytes (Peris)

[0188] Pericytes demonstrated a high abundance of proteins involved in cell adhesion, ECM interaction, and tissue remodeling. Notable highly expressed proteins included Integrin beta 5 (ITGB5) and Intercellular adhesion molecule 1 (ICAM1), both critical for cell-cell and cell-matrix interactions. High expression of Matrix Metalloproteinase-14 (MMP14) suggested potential for ECM degradation and turnover, key functions in vascular remodeling. Transforming growth factor beta induced (TGF(3I) and Decorin (DCN) further pointed to active ECM engagement. Melanoma Cell Adhesion Molecule (MCAM / CD146), a4930-0302-8870.1 46recognized marker for pericytes and endothelial cells that downregulates TGFB signaling to impact endothelial cells junction integrity (Chen et al., PNAS, 114(36): E7622-E7631 (2017)), was also abundant. A particularly noteworthy finding was the high expression of Integrin beta 1 (ITG01) in canid pericytes followed by moderate expression in canid EPCs and low expression in FB. This observation was also seen in bovid samples whereby EPCs expressed higher abundance compared to their FB counterparts despite canonical association as a fibroblast marker. Given that pericytes are intimately associated with the endothelial basement membrane and play vital roles in vessel stability, robust adhesion mechanisms involving ITGpl I are biologically plausible and functionally significant. Pericyte and EPC heterogeneity and their mesenchymal stem cell-like properties could also contribute to the dynamic expression of ITGpi and other adhesion molecules, underscoring that marker categorization can be context-dependent.

[0189] In addition to fibroblasts, PDGFRB is a canonical marker of human and mouse pericytes involved in their recruitment and activation (reviewed by Yao, Translational Stroke Research, 13(6): 863-865 (2022)). PDGFRB was abundant in canid pericytes and negligible in EPC.E. Proteomic Signature of Endothelial Progenitor Cells (EPCs)

[0190] The proteomic signature of EPCs confirmed their endothelial-like nature through high expression of Endoglin (ENG), a TGF-(3 co-receptor (observed in canid and bovid), and Cadherin 5 (CDEI5 / VE-Cadherin). essential for endothelial cell-cell junctions (detected in canid EPCs only). Intriguingly, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), typically categorized as a pericyte marker, showed its highest abundance in EPCs from both canid and bovid samples when compared to their fibroblast counterparts; canid pericytes exhibited the lowest PDGFRA expression. Bovid or equine pericyte data were not available to substantiate if this observation holds true for pericytes in those species. As PDGFRA is expressed by multipotent cardiovascular progenitors and other progenitor cell types, its high level in EPCs may reflect their progenitor status and capacity for differentiation.Furthermore, a panel of canonical EPC markers, including CD34. PEC AMI, SELE, NOS3, ITGA9, ESAM, EIGF, and ICAM1 (distinct from the high ICAM1 in Peris, contextually), were detected and quantified in canid EPCs but were not detected in the other canid cell types (FBs and Peris). This non-detection reflects fundamental lineage differences.4930-0302-8870.1 47F. Context-Dependent Expression and Deviations from Canonical Marker Definitions

[0191] The proteomic data revealed several instances where observed protein abundance challenged canonical marker definitions, highlighting the influence of cellular plasticity, culture context-dependent expression, and potential marker promiscuity.

[0192] Von Willebrand Factor (VWF): Traditionally an endothelial marker. VWF showed very low abundance in EPCs but surprisingly high abundance in fibroblasts (canid only; not detected in bovid or equine samples). Low VWF in EPCs could suggest incomplete maturation or context-specific suppression, while high VWF in fibroblasts might show a specific activated state.

[0193] Fibronectin 1 (FN1): Exhibited highly variable expression in canid EPCs and relatively low levels in canid fibroblasts. Conversely, in bovid samples, fibroblasts showed higher FN1 expression compared to EPCs. This aligns with FNl’s known highly context-dependent expression.

[0194] Annexin A5 (ANXA5): Categorized with pericytes, ANXA5 expression was low in EPCs and pericytes but high in both bovid and canid fibroblasts. This may reflect its broader expression in various mesenchymal cells beyond pericytes.

[0195] CD44: This adhesion molecule, variably categorized as a pericyte or fibroblast marker, was most abundant in canid EPCs. In contrast, bovid fibroblasts showed higher CD44 expression compared to bovid EPCs, again indicating species-specific or context-dependent patterns.

[0196] These results, along with the high ITG(31 in pericy tes and high PDGFRA in EPCs, underscore that protein expression is dynamic and influenced by cellular state, microenvironment, and functional demands. Such deviations from static marker definitions are likely indicative of biological nuances rather than inconsistencies in the dataset. Finally, variability observed for several key EPC markers within the EPC replicates themselves warrants further investigation to clarify the homogeneity' and stability of the EPC populations studied or to refine quantification methods for these specific proteins.G. General Principal Component Analysis

[0197] Principal Component Analysis (PCA) was performed to visualize the relationships betw een the different cell lines based on their protein expression profiles, namely equid whereby no FB control was available. Of the 32 markers analyzed, 18 were included in the PCA as they were detected and quantified in all samples across genera. The included markers were: VIM, COL1A1, ANXA5, COL3A1, CSPG4, ITGA5, ITGB1, CD44, ENG,4930-0302-8870.1 48ICAM1, PDGFRA, MMP14, THY1, COL4A2, PDGFRB, DCN, TGFBI, and ITGB5 (see FIG. 1 IB). The PC A plot aims to illustrate the dichotomy and overall similarities in expression profiles across the evaluated cell lines. The PCA revealed distinct clustering of cell types. Pericytes (shown near coordinates 4.5,3) exhibited the most distinct protein expression profile, clearly separating from other cell populations. A notable separation was observed for canid fibroblasts (near coordinates 2.5. -3). The biological replicates for this cell type are clustered tightly, with replicate 2 overlapping and obscuring replicate 1. In contrast, canid Endothelial Progenitor Cells (EPCs) (near coordinates -2.5,3) displayed a markedly different expression profile compared to both pericytes and canid fibroblasts. Bovid EPCs (near coordinates 0,0.5) also demonstrated a distinct profile when compared to their bovid fibroblast counterparts. These cells aligned more closely with canid EPCs, residing in the same quadrant of the PCA plot, though the expression differential between bovid EPCs and fibroblasts was less pronounced than that observed for canid EPCs versus their fibroblast counterparts. Finally, equid samples (near coordinates -2, -0.5) showed some similarity in their expression profile to EPC markers.

[0198] Equine blood-derived cells clustered more closely with canid and bovid EPCs than with fibroblast or pericyte populations. Some marker expression patterns deviated from canonical assignments (e g., low VWF in EPCs, high VWF in canid fibroblasts; variable FN1 and CD44 expression across species and cell types), reflecting context-dependent expression profiles.H. Growth Characteristics, Morphology, and Surface Markers

[0199] In addition to proteomics data, other cell characteristics were compared such as cellular morphology, doubling rates, and FACs characterization. Genus agnostic, fibroblasts trended together with a doubling rate over 35 hours, pericytes averaged a doubling rate of 20 hours, while EPCs had the fastest average doubling rate of 15 hours (FIGs. 12A-D). The fibroblast morphology of all genera presented as elongated spindly cells with a central nucleus. The EPCs were in general small, cobblestone-like, highly aggregated cells.Pericytes, on the other hand, were closer to fibroblast morphology’, but did not have the same elongation. FACS analysis was run with CD34 and CD90 antibodies, however, only the canid genus was run due to no compatible commercial antibodies available for the bovid or equid genera (FIG. 12D-12F). The FACS data is summarized in Table 4-6 below.

[0200] Flow cytometry' analysis of canid cells using anti-dog CD34 and CD90 antibodies showed distinct surface marker profiles (FIG. 12A-D). EPCs contained a discrete 25%4930-0302-8870.1 49population of CD34+ cells. Pericytes contained a minor -12% sub-population of CD90+ cells with negligible (< 1%) detection of CD34.

[0201] The FACs data showed that EPCs contained a discrete 25% population of CD34+cells, and less than half a percent of either CD90+alone, or CD34+and CD90+cells, while pericytes contained a minor -12% sub-population of CD90 cells with negligible (< 1%) detection of CD34. Due to the fibroblast derivation process, other cell types were present at the early passages, thus a small proportion of cells (3.51%) were CD34+, and almost 100% of the cells were CD90+.I. Functional Characterization

[0202] Additional functional assays were performed to establish a rapid charactenzation assessment, so that extensive proteomics analyses could be saved for initial genus defining studies. A standard in vitro angiogenesis assay w as performed to validate the ability7of EPCs to form tubule-like structures in the presence of an extracellular matrix, such as Matrigel (FIG. 13 A). EPCs formed tubule-like networks when plated on extracellular matrix substrate, while fibroblasts showed no morphological changes (FIG. 13 A). Pericytes adhered to the outer surface of EPC-derived tubule structures in co-culture experiments. Neither pericytes nor fibroblasts formed tubules when plated alone.

[0203] Low Density Lipoprotein (LDL) and Acetylated-LDL (AcLDL) uptake assays were also performed. This assay exhibited the difference in the internalization of LDL and AcLDL between fibroblast cell lines and blood derived cell lines (FIG. 13B and FIG. 13C). Both Pericytes and EPCs were observed to have an elevated uptake level of LDL in comparison to fibroblasts from the same species. Pericytes were found to have lower levels of AcLDL in comparison to both fibroblasts and EPCs, which once again underlines the ability7to derive multiple cell types from blood. For AcLDL uptake, EPCs demonstrated highest uptake, followed by fibroblasts, with pericytes showing the lowest uptake levels. This differential AcLDL uptake can be used to distinguish EPCs from pericytes in cultures originating from blood samples.4930-0302-8870.1 50J. Genomic Stability

[0204] The assessment of karyotypic stability is a critical step following primary cell derivation, as the process can occasionally introduce chromosomal abnormalities (Ueyama, H. et al., Journal of Cellular and Molecular Medicine 16, 72-82 (2012); Liu, A. et al. M., American Journal of Blood Research 2, 71 (2012)). In this study, optical genome mapping (OGM) was utilized to confirm the chromosome copy numbers in the canid, bovid, and equid cell lines (FIG. 14A-C). This platform was particularly advantageous for work with the aforementioned non-model species, where conventional image-based karyotyping resources and cytogenetic expertise were sparse. Furthermore, OGM provides high-resolution structural variant detection, down to 500 base pairs, which is crucial for identifying subtle genomic alterations potentially problematic for downstream research. Additionally, OGM can detect mosaic populations whereby one could quantify the percentage of the primary cell population that may carry copy number variants and structural variants. All samples successfully generated the requisite ~80x coverage for copy variant number analysis, and structural variant analysis. Confidence in the OGM methodology was significantly reinforced by the direct concordance observed in canid samples (FIG. 14A), where results matched those from independent microscopic karyotyping - a valuable comparison given the more established cytogenetic support for canids. Reassuringly, findings across all species were consistent with expected diploid karyotypes and sex chromosome compositions, confirming the successful derivation of karyotypically normal primary cells suitable for future applications. Beyond its resolution capabilities, the Bionano Saphyr OGM platform offered practical advantages, including a rapid turnaround time of 48-72 hours from cell pellet to analysis and the flexibility of using frozen cells, circumventing the live-cell preparation requirement for traditional karyotypic analyses.

[0205] OGM data from male canid samples was concordant with independent G-banded karyotype analysis (FIG. 14 A). Both male gray wolves from which cell lines were derived had 38 pairs of autosomes and one X and one Y chromosome. Bovid samples had 29 pairs of autosomes and X chromosome copy number consistent with known sex (Female 008; Male 017 EPC and fibroblasts in FIG. 14B). Equid samples had 31 pairs of autosomes and a pair of X chromosomes, consistent with female sex (FIG. 14C). No significant structural variants or aneuploidies were detected, confirming karyotypically normal primary cells suitable for downstream applications.4930-0302-8870.1 51K. Interspecies Somatic Cell Nuclear Transfer (iSCNT)

[0206] Canids. Both pericytes and EPCs successfully generated viable iSCNT gray wolf embryos. Twenty-one embryos reconstructed from pericytes and 18 from EPCs were transferred into a single recipient domestic dog in estrus (FIG. 15A, 15B). Transabdominal ultrasound at Days 21 and 23 post-embryo transfer confirmed implantation of six viable embryos with active heartbeats (FIG. 15G). Timed ovariohysterectomy at Day 33 yielded six morphologically normal fetuses. Genotyping using a custom SNP panel identified four fetuses derived from pericytes and two from EPCs, demonstrating viable implantation rates of 19% (pericytes) and 11% (EPCs).

[0207] Bovids. Blood-derived EPCs were compared directly to fibroblasts from the same female bison donor for iSCNT using in vitro matured cow oocytes. Of 86 bison EPC-derived iSCNT embryos, 69 embryos cleaved (80% cleavage rate) with five developing to blastocyst stage by day 7 (7% of cleaved embryos; FIG. 15C). In comparison, 67 of 87 fibroblast-derived iSCNT embryos cleaved (77% cleavage rate) with two progressing to blastocyst stage (3% of cleaved embryos; FIG. 15D). Genotyping confirmed that all bison iSCNT blastocysts were derived from bison donor cells.

[0208] Equids. iSCNT embryos were successfully generated using male plains zebra EPCs and fibroblasts as donor cells in enucleated domestic horse oocytes. Sixteen out of 51 iSCNT zebra embryos reconstructed with EPCs (FIG. 15E) developed to the morulae and blastocyst stages (32%) within 6 to 8 days of culture and were cryopreserved. In parallel. 13 out of 31 iSCNT zebra (42%) embryos produced with fibroblasts developed to the morulae and blastocyst stages prior to cryopreservation.DiscussionBlood-derived cells offer practical advantages for conservation biobanking

[0209] The testing in this Example demonstrates that endothelial progenitor cells (EPCs) and pericytes isolated from peripheral blood are a robust alternative to dermal fibroblasts for conservation biobanking and interspecific somatic cell nuclear transfer (iSCNT). Blood-derived cells exhibit 2-3-fold faster doubling rates (15-20 hours versus >35 hours for fibroblasts) and reduce the timeline for generating banked cell lines from 3-4 weeks to 1.5-2 weeks (FIG. 10, FIG. 11A-C). Combined with genomic stability equivalent to tissue-derived cells (FIG 14A-C) and demonstrated viability for generating cloned embryos across three mammalian genera (FIG. 15), blood-derived cells offer practical advantages for accelerating genetic rescue efforts.4930-0302-8870.1 52

[0210] The collection of peripheral blood provides several operational advantages over skin biopsies. Blood sampling during routine veterinary examinations, health assessments, or translocation procedures creates opportunistic collection windows without requiring additional animal restraint or separate sampling events.

[0211] Unlike dart biopsies, which yield minimal tissue often insufficient for both explant and digestion derivation methods, blood samples provide abundant starting material. The sterile, closed-system collection of blood in anticoagulant tubes contrasts with the microbial burden inherent to skin surfaces, which contributes to the higher contamination rates observed in explant cultures compared to blood-derived cultures.

[0212] The consistency of blood-derived cell doubling rates across phylogenetically diverse species (canids, bovids, equids) suggests broad taxonomic applicability and facilitates standardized biobanking workflows across diverse conservation programs. This contrasts with reported fibroblast derivation variability, such as elephant biopsies showing 0-80% success even within the same individual (van Vuuren el al., Animals: An Open Access Journal from MDPI, 13(14) (2023)). Whole blood samples are already routinely collected with anticoagulants for genomic studies and non-viably frozen in the field without cryoprotectants for DNA extraction. With advanced planning, blood collection protocols could preserve options for both immediate genomic analysis and future cell line establishment, maximizing the value of each sampling event. This dual-purpose approach is particularly relevant for cryptic, nocturnal, or otherwise difficult-to-sample species where opportunities for biological sample collection are rare.Molecular and functional characterization confirms distinct cell populations

[0213] Comprehensive proteomic profiling established that EPCs, pericytes, and fibroblasts are biologically distinct populations with lineage-specific molecular signatures (FIG. 11 A and FIG. 11B). Fibroblasts exhibited elevated ECM production markers (COL1A1, COL3A1, FAP), pericytes showed high abundance of adhesion and vascular remodeling proteins (ITGB5, ICAM1, MMP14, MCAM), and EPCs expressed endothelial-associated markers (ENG, CDH5, PDGFRA) along with canonical EPC markers (CD34, PECAM1, SELE, NOS3) detected exclusively in EPCs (FIG. 11 A and FIG. I IB, Tables 4-4 and 4-5). The statistical significance of expression differences (Logio(p-value) > 2.8) and consistency across biological replicates confirm that blood-derived cultures yield distinct cell t pes rather than heterogeneous mixtures.

[0214] For practical biobank characterization without mass spectrometry, morphological assessment combined with doubling rate measurements provides reliable first-pass4930-0302-8870.1 53identification (FIG. 12A-C). EPCs consistently displayed cobblestone morphology and ~15-hour doubling times, while fibroblasts show spindle morphology with >35-hour doubling times. Pericytes exhibited intermediate characteristics with fibroblast-like morphology but faster doubling (~20 hours). Functional assays further distinguished cell types within 24 hours: tubule formation on extracellular matrix occurs exclusively in EPC cultures, while differential uptake of fluorescently labeled LDL versus acetylated-LDL distinguishes EPCs from pericytes (FIG. 13A-C). These species-agnostic methods should enable biobank curators to characterize cell lines from taxonomically diverse samples as collections expand to under-represented vertebrate lineages.Both EPCs and pericytes support viable iSCNT outcomes

[0215] The iSCNT experiments demonstrated that blood-derived cells generate viable embryos with efficiency comparable to or exceeding traditional fibroblast donors. In gray wolves, both pericytes (19% implantation) and EPCs (11% implantation) contributed to viable fetuses, with the combined rate (15%) exceeding prior published w olf iSCNT rates using fibroblasts (Kim et al.. CloningStem Cells, 9(1): 130-137 (2007)). The bison experiments provided direct within-individual comparison, as EPCs from the same female donor showed higher blastocyst formation (7%) than fibroblasts (3%) under identical conditions, suggesting that rapid proliferation and maintained progenitor characteristics may confer advantages for post- transfer embryonic development. These results overcome previously reported developmental blocks in bison iSCNT (Gonzalez-Grajales et al., BMC Developmental Biology, 16(1): 36 (2016)). Plains zebra iSCNT embryos generated with EPCs (32% to morula / blastocyst) and fibroblasts (42%) further demonstrate cross-species applicability. Collectively, these results indicate that cell source (blood versus tissue) and specific blood-derived cell type does not compromise cloning outcomes.Current limitations and opportunities for optimization

[0216] The stochastic emergence of either EPC-dominant or pericyte-dominant cultures from blood samples presents both a challenge and an opportunity. In this study, replating the total buffy-coat fraction resulted in primary adherent colonies of both cell types adhering to traditional cell culture substrates and media optimized for EPC derivation and expansion. Both cell types formed primary colonies in a similar time window and could be dissociated, replated, and grown rapidly. While admixture of both cell types in cultures cannot be ruled out without single-cell analyses, the formation of cultures appeared stochastic with either EPCs or pericytes forming the predominant cell type in individual cultures. Selective enrichment may be achievable through substrate modification, growth factor4930-0302-8870.1 54supplementation, or differential adhesion protocols, enabling targeted derivation based on intended downstream applications.

[0217] A decreased success of derivation from cryopreserved samples (data omitted) was observed, though successful preservation of the option for viable cell expansion at a later date remains a frequent real-world necessity7.Integration with conservation strategies and existing biobanking infrastructure

[0218] The decision between blood and tissue sampling should be guided by speciesspecific considerations including restraint safety7, veterinary access, and existing protocol success rates. For taxa where tissue biopsy success is inconsistent or anatomical considerations preclude safe sampling, blood collection offers clear advantages. The rapid expansion timeline has implications for time-sensitive interventions in species experiencing rapid population decline. Recent successes introducing cloned historically underrepresented founders into black-footed ferret and Przewalski’s horse populations (Novak el al., bioRxiv (2024); Novak et al. , Animals: An Open Access Journal from MDPI, 15(5) (2025)) illustrate how biobanked cells restore genetic variation unavailable through conventional breeding. Blood-derived cells expand the pool of individuals that can be biobanked before unique genotypes are lost.

[0219] Integration with existing repositories such as the Frozen Zoo® is straightforward. Blood-derived cells can be processed using standard cry opreservation protocols and stored under identical conditions as tissue-derived lines. Unlike the technical demands to generate and biobank stem cells (Hutchinson et al., Development (Cambridge, England), 151(20) (2024)), blood-derived cells can be processed using standard cry opreservation protocols and stored under identical conditions as tissue-derived lines. The addition of blood-derived samples would enhance representation of individuals for whom tissue biopsies were not collected, expanding the genetic diversity captured in these repositories. The demonstrated success in this Example across phylogenetically7distant mammalian orders suggests applicability7to additional vertebrate taxa, potentially including threatened avian, reptilian, and amphibian species where current biobanking and conservation breeding efforts remain limited compared to the scale and rates of extinction (Browne et al., Animals: An Open Access Journal from MDPI, 14(10) (2024)).

[0220] However, the success of any biobanking-to-cloning effort depends critically on codevelopment of Assisted Reproductive Technologies (ART) using non-threatened, related model species whenever available and appropriate. Cloning of a new species can only happen when its basic reproductive physiology is understood and requires supporting the4930-0302-8870.1 55development of foundational ART to be successful. The iSCNT results reported in this Example were possible because of established ART protocols in domestic dogs, cattle, and horses, emphasizing that cloning advances depend on robust foundational reproductive biology research (Cowl et al., Annual Review of Animal Biosciences, 12:91-112 (2024); Hildebrandt & Holtze, Reproduction in Domestic Animals, 59 Suppl. 3 (2024)). Investing in studies and methods to collect viable gametes for artificial insemination, production of embryos by IVF, and timing of donors and recipients for successful embryo transfer can have profound positive impacts on preservation of current endangered populations (Bolton et al., Reproduction and Fertility, 3(3) (2022); Hutchinson et al., Development (Cambridge, England), 151(20) (2024)).

[0221] Beyond technical advances in biobanking and ART. the selection of genetically valuable individuals for cloning should be performed in coordination with local and international community members invested in the propagation of endangered species and populations (Gray et al., 2023; Novak, et al., Animals: An Open Access Journal fromMDPI, 15(5) (2025)). Importantly, supporting both international and indigenous groups advocating for in situ and ex situ conservation efforts of transnational, endangered animal populations and their habitats is needed in parallel to biobanking and long-term cloning efforts (Bolton et al. , Reproduction and Fertility, 3(3) (2022); Hay ah et al. , Npj Biodiversity, 4(1) (2025)).

[0222] While the invention has been described and illustrated herein by references to various specific materials, procedures and examples, it is understood that the invention is not restricted to the particular combinations of material and procedures selected for that purpose. Numerous variations of such details can be implied as will be appreciated by those skilled in the art. It is intended that the specification and examples be considered as exemplary', only, with the true scope and spirit of the invention being indicated by the following claims. All references, patents, and patent apphcations referred to in this application are herein incorporated by reference in their entirety.EMBODIMENTS

[0223] The invention provides also the following non-limiting embodiments.

[0224] Embodiment 1 is a method of deriving a cell population containing pericytes and endothelial progenitor cells from a peripheral blood sample comprising: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for growth of4930-0302-8870.1 56endothelial progenitor cells and pericytes, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum.

[0225] Embodiment 2 is the method of embodiment 1 , wherein the cell population comprises pericytes and endothelial progenitor cells.

[0226] Embodiment 3 is the method of embodiments 1 or 2, wherein the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0227] Embodiment 4 is the method of embodiments 1 or 2, wherein the cell culture medium is EGM.

[0228] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the cell culture medium is a conditioned medium generated by culturing pericytes.

[0229] Embodiment 6 is the method of any one of embodiments 1 to 4. wherein the cell culture medium is supplemented with pituitary extract and / or fetal calf serum.

[0230] Embodiment 7 is the method of embodiment 6, wherein the cell culture medium is supplemented with bovine pituitary7extract.

[0231] Embodiment 8 is the method of any one of embodiments 1 to 7. wherein the adherent conditions comprise culturing the cells on collagen, fibronectin, or cell-culture treated plastic.

[0232] Embodiment 9 is the method of any one of embodiments 1 to 8, wherein the method further comprises obtaining the mammalian peripheral blood sample from the mammal.

[0233] Embodiment 10 is the method of embodiment 9, wherein the mammal has been treated with an agent having alpha-adrenergic binding affinity7prior to obtaining the peripheral blood sample.

[0234] Embodiment 11 is the method of embodiment 10, wherein the mammal has been treated with an alpha2-adrenergic agonist, dexmedetomidine, medetomidine, xylazine, opioids, and / or Butorphanol.

[0235] Embodiment 12 is the method of any one of embodiments 1 to 11, wherein the endothelial progenitor cells express at least three markers selected from CD34, platelet and endothelial cell adhesion molecule (PEC AM- 1 or CD31), E-Selectin, Vascular Endothelial-Cadherin (VE-cadherin or Cadherin 5), and SCARF-1.

[0236] Embodiment 13 is the method of any one of embodiments 1 to 11 , wherein the pericytes express at least three cell surface membrane proteins selected from c-Met, chondroitin sulfate proteoglycan 4 (CSPG4), platelet derived growth factor receptor beta (PGDFRb), platelet derived growth factor receptor alpha (PGDFRa), CD82, melanoma cell adhesion molecule (CD 146), endosialin (CD248), kinase insert domain receptor (KDR),4930-0302-8870.1 57tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), fms-related receptor tyrosine kinase 4 (FLT4), C-X3-C motif chemokine receptor 1 (CXC3CR1). Notch 2 receptor, Notch 3 receptor, insulin growth factor receptor 2 (IGRF2), fibroblast growth factor receptor 1 (FGFR1), and bone morphogenetic protein receptor 2 (BMPR2).

[0237] Embodiment 14 is the method of any one of embodiments 1 to 11 or 13, wherein the pericytes express one or more intracellular structural proteins chosen from actin alpha 2 (ACTA2), desmin, nestin, glial fibrillary acid protein (GFAP), Myl9, myosin heavy chain 11 (Myhll), transgelin (TAGLN), and tropomysin 2 (TPM2).

[0238] Embodiment 15 is the method of any one of embodiments 1 to 14, wherein the method further comprises isolating the mammalian peripheral blood-derived pericytes.

[0239] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the pericytes lack expression of platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4).

[0240] Embodiment 17 is the method of embodiments 15 or 16, wherein isolating the population containing pericytes comprises screening for cells that are negative for platelet and endothelial cell adhesion molecule (PECAM-1 or CD31), CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4).

[0241] Embodiment 18 is the method of any one of embodiments 1 to 12, wherein the method further comprises isolating the mammalian peripheral blood-derived endothelial progenitor cells.

[0242] Embodiment 19 is the method of any one of embodiments 15 to 18, wherein the isolating comprises fluorescence activated cell sorting (FACS), magnetic cell separation (MACs), or differential adherent culture.

[0243] Embodiment 20 is an isolated mammalian peripheral blood-derived pericyte derived by the method of any one of embodiments 1 to 11, 13, 16 or 17.

[0244] Embodiment 21 is a mammalian peripheral blood-derived isolated endothelial progenitor cell derived by the method of any one of embodiments 1 to 11, 13, or 15.

[0245] Embodiment 22A is a method of gene editing a mammalian peripheral blood-derived pericyte comprising editing a gene of interest in an isolated mammalian peripheral blood-derived pericyte obtained by culturing a peripheral blood sample in a cell culture medium under adherent conditions for a period sufficient to allow for growth of pericytes, wherein the cell culture medium is supplemented with serum. Embodiment 22B method of gene editing an endothelial progenitor cell comprising editing a gene of interest in an isolated mammalian peripheral blood-derived endothelial progenitor cell obtained by culturing a4930-0302-8870.1 58peripheral blood sample in a cell culture medium under adherent conditions for a period sufficient to allow for growth of endothelial progenitor cells, wherein the cell culture medium is supplemented with serum.

[0246] Embodiment 23 is a method of gene editing mammalian peripheral blood-derived pericytes comprising: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period sufficient to allow for growth of pericytes thereby deriving the mammalian peripheral blood-derived pericytes, wherein the cell culture medium is supplemented with serum; and editing a gene in the mammalian peripheral blood-derived pericytes to produce a mammalian peripheral blood-derived pericyte comprising an edited gene. Embodiment 23A is a method of gene editing mammalian peripheral blood-derived cells including: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium supplemented with serum under adherent conditions for a period sufficient to allow for growth of mammalian peripheral blood-derived cells thereby deriving the mammalian peripheral blood-derived cells; and editing a gene in the mammalian peripheral blood-derived pericytes to produce a mammalian penpheral blood-derived pencyte comprising an edited gene, wherein the mammalian peripheral blood-derived cells are mammalian peripheral blood-derived pericytes and / or mammalian peripheral blood-derived cells.

[0247] Embodiment 24 is the method of embodiments 22 or 23, wherein the cell culture medium is EGM.

[0248] Embodiment 25 is the method of embodiments 22 or 23, wherein the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0249] Embodiment 26 is the method of embodiments 22, 23 or 25, wherein the cell culture medium is a conditioned medium generated by culturing pericytes.

[0250] Embodiment 27 is the method of any one of embodiments 22 to 26, w herein the cell culture medium is supplemented with pituitary extract and / or fetal calf serum.

[0251] Embodiment 28 is the method of embodiment 27, wherein the cell culture medium is supplemented with bovine pituitary extract.

[0252] Embodiment 29 is the method of any one of embodiments 23 to 28, w herein the adherent conditions comprise culturing the cells on collagen, fibronectin, or cell-culture treated plastic.

[0253] Embodiment 30 is the method of any one of embodiments 23 to 29 further comprising culturing the mammalian peripheral blood-derived pericyte comprising an edited gene to produce a clonal line.4930-0302-8870.1 59

[0254] Embodiment 31 is the method of embodiment 30, wherein the method comprises FACS sorting, limiting dilution and / or plating on a Cell Raft system.

[0255] Embodiment 32 is the method of any one of embodiments 1 to 31 , wherein the mammal is a mouse, a rat, a marsupial, a bovine, an ovine, a caprine, a pig, an elephant, an equid, a cat, a dog, a wolf, or a deer.

[0256] Embodiment 33 is the method of embodiments 32, wherein the bovine is a cow, a bison, or an antelope.

[0257] Embodiment 34 is the method of embodiment 32, wherein the equid is a horse, a zebra, a donkey, a tapir, or a rhinoceros.

[0258] Embodiment 35 is the method of embodiment 32, wherein the mammal is Felis catus, Cams lupus familiaris, Cams lupus, Bison bison. Dama dama. Hippotragus equinus, or Hippotragus niger.

[0259] Embodiment 36 is the method of any one of embodiments 22 to 35, further comprising isolating a mammalian peripheral blood-derived pericyte comprising an edited gene a mammalian peripheral blood-derived endothelial progenitor cell comprising an edited gene.

[0260] Embodiment 37 is an isolated mammalian peripheral blood-derived pericyte comprising an edited gene produced by the method of embodiment 36. Embodiment 37A is an isolated mammalian peripheral blood-derived endothelial progenitor cells comprising an edited gene produced by the method of embodiment 36.

[0261] Embodiment 38 is a method of producing a transgenic animal comprising using the isolated mammalian peripheral blood-derived pericyte of embodiment 37. Embodiment 38A is a method of producing a transgenic animal comprising using the isolated mammalian peripheral blood-derived endothelial progenitor cell of embodiment 37A.

[0262] Embodiment 39 is a serum-free defined cell culture medium comprising: PDGF-beta; VEGF-C; a Notch ligand; a fibroblast growth factor; a TGF-beta superfamily member; an insulin agonist; transferrin; a hepatocyte grow th factor; and a Wnt antagonist.

[0263] Embodiment 40 is the cell culture medium of embodiment 39, wherein the medium lacks or is not supplemented with epidermal growth factor.

[0264] Embodiment 41 is the cell culture medium of embodiments 39 or 40, wherein the Notch ligand is Jagged 1, Jagged 2, or a mixture thereof.

[0265] Embodiment 42 is the cell culture medium of any one of embodiments 39 to 41, wherein the fibroblast growth factor is bFGF.4930-0302-8870.1 60

[0266] Embodiment 43 is the cell culture medium of any one of embodiments 39 to 42, wherein the TGF-beta superfamily member is a bone morphogenic protein.

[0267] Embodiment 44 is the cell culture medium of embodiment 31, wherein the TGF-beta superfamily member is BMP 10.

[0268] Embodiment 45 is the cell culture medium of any one of embodiments 39 to 44, wherein the insulin agonist is natural insulin, recombinant insulin, and / or IGF2.

[0269] Embodiment 46 is the cell culture medium of any one of embodiments 39 to 46 wherein the transferrin is apo and / or holo-transferrin.

[0270] Embodiment 47 is the cell culture medium of any one of embodiments 39 to 46, wherein the Wnt antagonist is Dickkopf-1, Dickkopf-2, Dickkopf-3, or a WTN antagonist antibody.

[0271] Embodiment 48 is a conditioned cell culture medium generated by culturing the isolated pericyte of embodiment 20.

[0272] Embodiment 49 is a method of culturing pericytes in a cell culture medium of any one of embodiments 39 to 48.

[0273] Embodiment 50 is a method of generating a population of peripheral blood-derived pericytes comprising: culturing a mammalian peripheral blood sample in a cell culture medium of any one of embodiments 39 to 47 in an adherent cell culture for a period sufficient to allow for growth of peripheral blood-derived pericytes; and isolating peripheral blood-derived pericytes from the sample after culturing.

[0274] Embodiment 51 is the method of embodiments 50, wherein the blood sample comprises non-hematopoietic cells.

[0275] Embodiment 52 is the method of embodiment 51 , wherein the culturing comprises culturing the mammalian peripheral blood sample on collagen, fibronectin, or cell-culture treated plastic.

[0276] Embodiment 53 is the method of any one of embodiments 50 to 52 wherein the isolating comprises FACS, MACs, or differential adherent culture.

[0277] Embodiment 54 is the method of any one of embodiments 50 to 52, wherein the isolating comprises screening for cells expressing at least three markers chosen from CSPG4, PGDFRb, PGDFRa, CD82, CD146, CD248, KDR, TIE1, FLT4, CXC4CR1, Notch 2 receptor, Notch 3 receptor, IGRF2, FGFR1, and BMPR2.

[0278] Embodiment 55 is a method of isolating endothelial progenitor cells (EPCs) and pericytes from a peripheral blood sample comprising: isolating the peripheral blood sample from a mammal; and culturing the peripheral blood sample containing non-hematopoietic4930-0302-8870.1 61cells in a serum-free defined cell culture medium under adherent conditions for a period sufficient to allow for growth of endothelial progenitor cells and pericytes, thereby deriving the EPCs and / or pericytes, wherein the cell culture medium is supplemented with serum.

[0279] Embodiment 56 is the method of embodiment 55, wherein the cell culture medium lacks or is not supplemented with epidermal growth factor.

[0280] Embodiment 57 is a method of producing an induced pluripotent stem cell from an isolated mammalian peripheral blood-derived pericyte of embodiment 20 or from an isolated endothelial mammalian peripheral blood-derived progenitor cell of embodiment 21, comprising transfecting the isolated pericyte or the isolated endothelial progenitor cell with one or more of OCT4, KLF4, SOX2, and optionally c-MYC.4930-0302-8870.1 62

Claims

1. CLAIMSWhat is claimed is:

1. A method of deriving a cell population comprising pericytes and endothelial progenitor cells from a peripheral blood sample comprising: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium under adherent conditions for a period of time sufficient to allow for grow th of endothelial progenitor cells and pericytes, thereby deriving the cell population, wherein the cell culture medium is supplemented with serum.

2. The method of claim 1, wherein the cell culture medium lacks or is not supplemented with epidermal growth factor.

3. The method of claim 1, wherein the cell culture medium is EGM.

4. The method of claim 1 , wherein the cell culture medium is a conditioned medium generated by culturing pericytes.

5. The method of claim 1, wherein the cell culture medium is supplemented with pituitary extract and / or fetal calf serum.

6. The method of claim 5, wherein the cell culture medium is supplemented with bovine pituitary extract.

7. The method of claim 1, w herein the adherent conditions comprise culturing the cells on collagen, fibronectin, or cell-culture treated plastic.

8. The method of claim 1, wherein the method further comprises obtaining the mammalian peripheral blood sample from the mammal.

9. The method of claim 8, wherein the mammal has been treated with an agent having alpha-adrenergic binding affinity prior to obtaining the peripheral blood sample.

10. The method of claim 9, wherein the mammal has been treated with an alpha2-adrenergic agonist, dexmedetomidine, medetomidine, xylazine, opioids, and / or Butorphanol.

11. The method of claim 1 , w herein the endothelial progenitor cells express at least three markers selected from CD34, platelet and endothelial cell adhesion molecule (PECAM-1 or4930-0302-8870.1 63CD31), E-Selectin. Vascular Endothelial-Cadherin (VE-cadherin or Cadherin 5), and SCARF- 1.

12. The method of claim 1, wherein the pericytes express at least three cell surface membrane proteins selected from c-Met, chondroitin sulfate proteoglycan 4 (CSPG4), platelet derived growth factor receptor beta (PGDFRb), platelet derived growth factor receptor alpha (PGDFRa), CD82, melanoma cell adhesion molecule (CD 146), endosialin (CD248), kinase insert domain receptor (KDR), tyrosine kinase with immunoglobulin-like and EGF-like domains 1 (TIE1), fms-related receptor ty rosine kinase 4 (FLT4), C-X3-C motif chemokine receptor 1 (CXC3CR1), Notch 2 receptor, Notch 3 receptor, insulin growth factor receptor 2 (IGRF2). fibroblast growth factor receptor 1 (FGFR1), and bone morphogenetic protein receptor 2 (BMPR2).

13. The method of claim 1, wherein the pericytes express one or more intracellular structural proteins chosen from actin alpha 2 (ACTA2), desmin, nestin. glial fibrillary acid protein (GFAP), Myl9, myosin heavy chain 11 (Myhl 1), transgelin (TAGLN), and tropomysin 2 (TPM2).

14. The method of claim 1, wherein the method further comprises isolating the mammalian blood-derived pericytes.

15. The method of claim 1, wherein the pericytes lack expression of platelet and endothelial cell adhesion molecule (PECAM-1 or CD31). CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4).

16. The method of claim 14, wherein isolating the pericytes comprises screening for cells that are negative for platelet and endothelial cell adhesion molecule (PECAM-1 or CD31). CD34, vascular cell adhesion molecule (VCAM), E-Selectin, and isolectin B4 (IB4).

17. The method of claim 1, wherein the method further comprises isolating the mammalian blood-derived endothelial progenitor cells.

18. The method of claim 14, wherein the isolating comprises fluorescence activated cell sorting (FACS), magnetic cell separation (MACs), or differential adherent culture.

19. An isolated mammalian blood-derived pericyte derived by the method of claim 1.4930-0302-8870.1 6420. An isolated mammalian blood-derived endothelial progenitor cell derived by the method of claim 1.

21. A method of gene editing a mammalian peripheral blood-derived pericyte comprising editing a gene of interest in an isolated mammalian peripheral blood-derived pericyte obtained by the method of claim 1.

22. A method of gene editing a mammalian peripheral blood-derived endothelial progenitor cell comprising editing a gene of interest in an isolated mammalian peripheral blood-derived endothelial progenitor cell obtained by the method of claim 1.

23. A method of gene editing mammalian peripheral blood-derived cells comprising: culturing a mammalian peripheral blood sample containing non-hematopoietic cells in a cell culture medium supplemented with serum under adherent conditions for a period sufficient to allow for growth of mammalian peripheral blood-derived cells thereby deriving the mammalian peripheral blood-derived cells; andediting a gene in the mammalian peripheral blood-derived pericytes to produce a mammalian peripheral blood-derived pericyte comprising an edited gene, wherein the mammalian peripheral blood-derived cells are mammalian peripheral blood-derived pericytes and / or mammalian peripheral blood-derived cells.

24. The method of claim 23. wherein the cell culture medium is EGM.

25. The method of claim 23, wherein the cell culture medium lacks or is not supplemented with epidermal growth factor.

26. The method of claim 23, wherein the cell culture medium is a conditioned medium generated by culturing pericytes.

27. The method of claim 23. wherein the cell culture medium is supplemented with pituitary extract and / or fetal calf serum.

28. The method of claim 27, wherein the cell culture medium is supplemented with bovine pituitary extract.

29. The method of claim 23, wherein the adherent conditions comprise culturing the cells on collagen, fibronectin, or cell-culture treated plastic.4930-0302-8870.1 6530. The method of claim 23, wherein the mammalian peripheral blood-derived cells are mammalian peripheral blood-derived pericytes.

31. The method of claim 30 further comprising culturing the mammalian peripheral blood-derived pericyte comprising an edited gene to produce a clonal line.

32. The method of claim 23, wherein the mammalian peripheral blood-derived cells are mammalian peripheral blood-derived endothelial progenitor cells.

33. The method of claim 31, wherein the method comprises FACS sorting, limiting dilution and / or plating on a Cell Raft system.

34. The method of any one of claims 1 to 33, wherein the mammal is a mouse, a rat, a marsupial, a bovine, an ovine, a caprine, a pig, an elephant, an equid, a cat, a dog, a wolf, or a deer.

35. The method of claims 34, wherein the bovine is a cow, a bison, or an antelope.

36. The method of claim 34, wherein the equid is a horse, a zebra, a donkey, a tapir, or a rhinoceros.

37. The method of claim 34, wherein the mammal is Felis cams, Canis lupus familiaris, Canis lupus. Bison bison, Dama dama, Hippotragus equinus, or Hippotragus niger.

38. The method of any one of claims 21 to 33, further comprising isolating a mammalian peripheral blood-derived pericyte comprising an edited gene or a mammalian endothelial progenitor cell comprising an edited gene.

39. An isolated mammalian peripheral blood-derived pericyte comprising an edited gene produced by the method of claim 38.

40. A method of producing a transgenic animal comprising using the isolated mammalian peripheral blood-derived pericyte of claim 39.

41. An isolated mammalian peripheral blood-derived endothelial progenitor cell comprising an edited gene produced by the method of claim 38.

42. A method of producing a transgenic animal comprising using the isolated mammalian peripheral blood-derived endothelial progenitor cell of claim 38.4930-0302-8870.1 6643. A serum-free defined cell culture medium comprising: PDGF-beta; VEGF-C; a Notch ligand; a fibroblast growth factor; a TGF-beta superfamily member; an insulin agonist; transferrin; a hepatocyte growth factor; and a Wnt antagonist.

44. The cell culture medium of claim 43, wherein the medium lacks or is not supplemented with epidermal growth factor.

45. The cell culture medium of claim 43, wherein the Notch ligand is Jagged 1, Jagged 2, or a mixture thereof.

46. The cell culture medium of claim 43, wherein the fibroblast growth factor is bFGF.

47. The cell culture medium of claim 43, wherein the TGF-beta superfamily member is a bone morphogenic protein.

48. The cell culture medium of claim 43, wherein the TGF-beta superfamily member is BMP10.

49. The cell culture medium of claim 43, wherein the insulin agonist is natural insulin, recombinant insulin, and / or IGF2.

50. The cell culture medium of claim 43 wherein the transfenin is apo and / or holo-transferrin.

51. The cell culture medium of claim 43, wherein the Wnt antagonist is Dickkopf- 1 , Dickkopf-2, Dickkopf-3. or a WTN antagonist antibody.

52. A conditioned cell culture medium generated by culturing the isolated pericyte of claim 19.

53. A method of culturing pericytes in a cell culture medium of any one of claims 43 to 51.

54. A method of generating a population of mammalian peripheral blood-derived pericytes comprising:culturing a mammalian peripheral blood sample in a serum-free defined cell culture medium of any one of claims 43 to 51 in an adherent cell culture for a period sufficient to allow for grow th of peripheral blood-derived pericytes; and4930-0302-8870.1 67isolating mammalian peripheral blood-derived pericytes from the sample after culturing.

55. The method of claims 54, wherein the blood sample comprises non-hematopoietic cells.

56. The method of claim 54, wherein the culturing comprises culturing the mammalian peripheral blood sample on collagen, fibronectin, or cell-culture treated plastic.

57. The method of claim 54 wherein the isolating comprises FACS. MACs. or differential adherent culture.

58. The method of claim 54, wherein the isolating comprises screening for cells expressing at least three markers chosen from CSPG4, PGDFRb, PGDFRa, CD82, CD146, CD248, KDR, TIE1, FLT4, CXC4CR1, Notch 2 receptor. Notch 3 receptor, IGRF2, FGFR1, and BMPR2.

59. A method of producing an induced pluripotent stem cell from an isolated pericyte of claim 19 or an isolated endothelial progenitor cell of claim 20, comprising transfecting the isolated pericyte or the isolated endothelial progenitor cell with one or more of OCT4, KLF4, SOX2, and optionally c-MYC.4930-0302-8870.1 68

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