METHOD AND APPARATUS FOR RECOVERY OF UMBILICAL CORD TISSUE DERIVED REGENERATIVE CELLS AND USES THEREOF

Abstract

Methods for the extraction and processing of umbilical stem cells for therapeutic and diagnostic purposes are disclosed. The methods provide high regenerative cell numbers and high cell viability. Further, the methods do not require culturing the cells in serum or growth factors. Certain aspects of the present invention concern methods of processing tissue for use in regenerative medicine. In another aspect of the invention, the tissue is processed and the resulting cell preparation administered within one and the same medical procedure.

Claims

1. A method for matched transfer of umbilical cord tissue-derived cells from a donor to a recipient, comprising the steps of: collecting a section of umbilical cord from a newborn donor; extracting nucleated tissue from the umbilical cord to derive a stem cell population therefrom; and administering stem cells from the derived population to a recipient having HLA markers matching the HLA markers of the donor for treating a disease or disorder of the recipient.

2. The method of claim 1, wherein the donor and the recipient are the same.

3. The method of claim 2, wherein the steps of the method are performed in a single medical procedure.

4. The method of claim 3, wherein the steps of the method are performed at the site of delivery of the donor as a point of care site.

5. The method of claim 1, wherein the transfer is allogeneic, and including: extracting and analyzing a nucleated cell sample of the donor to predict the HLA type of the donor cells upon their maturation, and preserving the umbilical cord tissue-derived stem cell population for subsequent administration to the recipient having HLA markers matching those of the donor.

6. The method of claim 1, wherein the transfer is allogeneic, and including: extracting and analyzing a sample of cells derived from umbilical cord blood of the donor to determine therefrom the HLA type of the donor umbilical cord tissue derived-cells upon their maturation, and preserving the umbilical cord tissue-derived stem cell population for subsequent administration to the recipient having HLA markers matching those of the donor.

7. The method of claim 6, including: cryopreserving the umbilical cord tissue-derived cell population for storage in a cell bank according to the HLA type of the stored cells to distinguish from stored cells of a different HLA type in the bank.

8. The method of claim 7, including: cataloging the stored cells of different HLA types in the bank for rapid retrieval of stored cells of a particular HLA type to be administered for allogeneic transfer to a matching recipient.

9. The method of claim 8, including: posting the catalog of stored cells by different HLA type in the bank on a remotely accessible electronic database for retrieval of matching available donor cells.

10. The method of claim 7, including: cryopreserving stem cells derived from blood of the umbilical cord of the donor for storage together with the umbilical cord tissue-derived stem cells of the same donor in the cell bank to enhance identification of the HLA type of the stored tissue-derived cells of the donor.

11-19. (canceled)

20. A system for processing umbilical cord tissue from a newborn donor at site of delivery of the donor comprising: a tissue processing unit for processing the umbilical cord tissue in close proximity to the delivery site; wherein the processing is carried out by introduction of a reagent into said unit; and a receptacle for recovery of a cell preparation from processing of the umbilical cord tissue in the presence of the reagent such that the recovered cell preparation contains regenerative cells in a sufficient number to be used for therapeutic purposes without expansion.

21. The system of claim 20, wherein the reagent comprises a mixture of one or more of a collagenase and a protease.

22. The system of claim 20, including means for retaining the recovered cell preparation for administration to the donor.

23. The system of claim 22, wherein the retaining means retains a number of regenerative cells in the recovered cell preparation sufficient to alleviate conditions associated with pregnancy or delivery related complications suffered by the donor.

24. The system of claim 23, wherein the complications include any among cerebral hypoxia, cerebral palsy, and low APGAR scores.

25. The system of claim 20 further including means for dividing the recovered cell preparation into a plurality of aliquots, for administration of a first aliquot to the donor within the first 24 hours of delivery and subsequent administration of a second aliquot to the donor.

26. The system of claim 22 wherein the retaining means is configured for administration by one of intravenously, intra-arterially, intrathecally, infusion, direct injection, and to a mucosal membrane.

27. A system for matched transfer of umbilical cord tissue-derived cells from a donor to a recipient comprising: a collector of a sample of human leukocyte antigen expressing cells from a potential donor; and an analyzer of the sample to predict the human leukocyte antigen expression pattern of the umbilical cord tissue derived cells to be assumed after transfer to a recipient.

28. The system of claim 27 wherein the analyzer uses the results of the sample analysis to confirm that the potential donor is a match to the recipient's HLA.

29-32. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG. 1 is a schematic enlarged perspective representation of a section of an umbilical cord, illustrating the umbilical cord tissue (UCT) including vessels, surrounded by the tissue structure known as Wharton's jelly and stroma, wherein the Wharton's jelly encompasses the vascular and subamnionic regions and is walled by the amnion epithelium.

[0030] FIG. 2 is a photographic sequence of a dissection of an umbilical cord sample, illustrating in 2A, the umbilical cord anatomy; in 2B, removal of the umbilical cord amnion epithelium with a hemostat; in 2C, 2D, expansion of the umbilical cord vessels using medium size scissors to facilitate the separation of the vessel from the Wharton's jelly intervascular tissue; in 2E-G, stepwise dissection of the Wharton's jelly tissue from other structures such as the vessel walls; in 2H, umbilical cord tissue including vessel, stroma and Wharton's jelly tissue after dissection; and in 2I, the resulting umbilical cord tissue.

[0031] FIG. 3 is a multi-step diagram illustrating steps of the processing of umbilical cord tissue including Wharton's jelly tissue using a semi-automated system, wherein in step 1, dissection of the umbilical cord tissue (UCT) and subsequent mincing of the UCT is performed; in step 2, processing buffer and MATRASE™ Reagent are added to the minced tissue and incubated for 1 to 4 hours in an InGeneron™ ARC processing unit at 37 to 40° C. under automated constant agitation; in step 3, supernatant of the processed tissue is passed through a 100 μm filter to remove debris and unprocessed tissue; in step 4, after filtration the resulting cell suspension is spun down and the cell pellet is washed two to three times to remove any remaining enzyme and tissue debris following the decomposition of the umbilical cord tissue with, for example, the MATRASE™ Reagent; and in a final step 5, the final cell pellet can be re-suspended in desired carrier solution and administered or cryopreserved.

[0032] FIG. 4A is a stepwise appearance of umbilical cord tissue and cells during processing and culture, showing 1, umbilical cord tissue including Wharton's jelly, vessels, and stroma after one hour of processing with MATRASE™ Reagent with automated mechanical agitation; 2, cell pellet appearance after the first filtration and concentration step; and 3, cell pellet appearance after washes, exhibiting umbilical cord regenerative cells (UC-RC). FIG. 4B is a table indicating total nucleated cell counts, percent viability, and percent CFU-F (i.e., colony forming unit microblast) of freshly isolated cells from umbilical cord tissue. FIG. 4C illustrates light microscopy of passage 0 (i.e., first culture following the isolation of cells from tissue; also called the primary culture) adherent cell fraction (umbilical cord content of perinatal, plastic adherent mesenchymal stem cells, or UC-MSC) from umbilical cord tissue after 4 days in culture.

[0033] FIG. 5 represents multiple images of a differentiation potential of umbilical cord tissue stem cells in culture. UC-MSCs obtained using the semi-automated protocol were cultured for 2-3 weeks under differentiation-inducing conditions. The Figure comprises light microscopy images of UC-MSC differentiated in chondrogenic (mesoderm), osteogenic, hepatogenic (endoderm), senescence, adipogenic, and neurogenic (ectoderm) media.

[0034] FIG. 6A is a comparison of gene MSC expression profiling of UC-MSCs isolated with this presently-preferred protocol (WJSCs, i.e., Wharton's jelly stem cells) compared to adipose tissue MSCs (ADSCs) and bone marrow MSCs (BMSCs). In FIG. 6B, a scatter plot comparison of the normalized expression of genes associated with stemness and mesenchymal characteristics (MSC array, SABioscience, Qiagen, Inc., Valencia, Calif.) UC-MSC (in this figure, designated USCs) expression levels (darker circles) were plotted against adipose derived stem cells (in this figure, designated ASCs, lighter circles), and in FIG. 6C, plotted against bone marrow-derived stem cells (BMSCs, lighter circles), to quickly visualize possible differences in gene expression. The central sloped line in FIGS. 6B and 6C indicates unchanged gene expression. Genes above the central lines indicated higher expression levels; whereas, genes below central lines indicate lower expression levels for UC-MSC.

[0035] FIG. 7 consists of sets of light microscopy images showing, in the set of the upper row, that the muscle cells of a patient with Duchenne disease (Duchenne Muscular Dystrophy, or DMD) are not able to express the dystrophin gene, including images of DAPI (i.e., 4′,6-diamidino-2-phenylindole, a fluorescent stain that binds strongly to A-T rich regions in DNA), dystrophin and overlay thereof, taken at 50 microns (μm). The corresponding set of images of the lower row of FIG. 7 show that after injection into the patient, matched allogeneic donor stem cells induce the dystrophin expression in the recipient patient's muscle cells.

[0036] FIG. 8 is a photograph of injection of 1 million matched allogeneic regenerative cells into four muscle groups together with 0.5 million regenerative cells given intravenously (IV) in a laboratory test mouse.

[0037] FIG. 9 is a chart showing muscle strength versus treatment regimen for a single injection of stem cells into the muscles and cells given intravenously (IV). The chart shows a significant increase in muscular strength (measured as amount of time spent in a wire hanging test) in MDX laboratory mice (i.e., a strain of mice that has a hereditary disease of the muscles caused by a mutation on the X-chromosome used as a disease model for human muscular dystrophy) that have had such an injection, for (i) MDX untreated, (ii) MDX+fresh ADSC injection, and (iii) MDX+cultured ADSC injection; in each case, measured from a baseline, at 4 weeks, and at 2 week intervals thereafter through 10 weeks.

[0038] FIG. 10 is a light microscopy image showing that ASCs from matched allogeneic donors engraft and transdifferentiate to new skeletal muscles expressing dystrophin.

DETAILED DESCRIPTION

[0039] Introduction: Certain aspects of the disclosure are directed to a method for recovering regenerative cells from umbilical cord tissue, not from the blood of an umbilical cord (except for certain combined applications), and the use of those cells. The use and application of stem cells recovered from the tissue of umbilical cord can be found in several previous patent applications and references.sup.(9,10,11). Cells recovered from the tissue of that) umbilical cord have been described.sup.12,13,14,15 as being capable to differentiate better than cells derived from, for example, bone marrow. These cells are able typically to differentiate into endoderm, ectoderm, mesoderm in humans, and in animals such as horses and dogs.sup.16,17,18. Methods of recovery of these cells have been described.sup.19, as well as cryopreservation of these cells.sup.20. Importantly, however, certain aspects of the present invention provide an effective means of recovering a high number (population) of such cells, such as greater than typically 1 million cells per gram of umbilical cord tissue, not heretofore accomplished by the prior methods and techniques. In such aspects, about 10% or more of the cells recovered are able to form colonies, which indicates their stemness (i.e., an essential characteristic of a stem cell that distinguishes it from ordinary cells) and identify them as being primarily pluripotent stem cells.

[0040] One aspect of the present invention is to use the umbilical cord tissue-derived stem cells from a donor in a matched allogeneic transfer into a recipient in order to alleviate issues relating to the age-related limitations of the recipient's own (old and often only slowly proliferating) autologous cells for therapeutic purposes, such as repair, regeneration, or rejuvenation of damaged, diseased, or aged tissues, or in some other instances in order to correct a genetic deficiency present in the cells of the recipient, but not present in the cells of the donor, including for example schizophrenia. The characteristics of stem cells of an aged individual include: slow replication and increased doubling time, limited differentiation capacity, shortened telomeres and a high degree of senescence that potentially—especially with advanced age and concomitant diseases such as diabetes—limits the use of these aged autologous cells.

[0041] To overcome these obstacles, aspects of the disclosure pertain to the use of cells derived from umbilical cord tissue of one individual (donor) in another individual (recipient). Characteristics of these umbilical cord tissue-derived cells include, in comparison to aged cells: high genetic stability, a low degree of DNA breaks, a low degree of methylation, a fast doubling time, and a high and fast and efficient rate of differentiation into all three lineages of endoderm, ectoderm, and mesoderm. In addition, a specific genetic mutation present eventually in the cells of the recipient is not present in the cells of the donor and, therefore, these umbilical cord tissue-derived cells are, when used in allogeneic transfer, able to alleviate conditions associated with the genetic mutations in the recipient. Typically, such cells are used under the conditions that the immune surface characteristics of HLA (in humans, also called MHC, or major histocompatibility complex, constituting a set of cell surface molecules encoded by a large gene family that controls a major part of the immune system in all vertebrates), MHC1 (i.e., one of two primary classes of MHC molecules and found on nearly every nucleated cell of the body, their function being to display fragments of proteins from within the cell to T cells; healthy cells will be ignored, while cells containing foreign proteins will be attacked by the immune system) and MHC2 (i.e., MHC class II molecules constituting a family of molecules normally found only on antigen-presenting cells such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells) characteristics are matched between umbilical cord donor and recipient, in procedures where the donor is the umbilical cord donor and the recipient is other than the donor and in need of administration of stem cells.

[0042] The cells can be administered in the recipient either intrathecally, intravenously, intra-arterially, into ducts such as the ductus pancreaticus, into the spinal fluid, or directly injected into tissue such as subcutaneous structures or organs such as heart, liver, kidney, brain, joints, bone or lungs. Also, an application to the epithelial layers or mucosa of, for example, the nose, mouth, or lungs, is considered.

[0043] In certain prevalent aspects of the invention, allogeneic therapies are employed. In the case of intended or desired allogeneic use of cells from an umbilical cord donor, the immune surface characteristics of these cells can be predicted by analyzing the HLA markers/type of the umbilical cord donor. Typically, this is accomplished by drawing a tiny sample of blood, of nucleated cells, or other suitable bodily fluid from the donor, such as in an amount of 100 microliters, and in the example of blood, from the umbilical cord, a vein, an artery, or an ear lobe of the donor, to determine the typical surface antigen structure HLA, HLB (i.e., human B cell differentiation antigen), DR (HLA-DR is an MHC class II antigen that maps to chromosome 6) and that even more finely serve to characterize the immune surface cell type of the donor. This information is usable to predict the future immunotype of the umbilical cord tissue stem cells when they are injected into a recipient. Typically, stem cells do not express the HLA, MHC1, or MHC2 markers, but when they differentiate in the new microenvironment into adult tissue cells of the recipient, they commence to express these markers. The matching of the markers of the recipient with the markers of the donor is important to avoid rejection of the differentiated cells or graft vs. host disease.

[0044] In instances where autologous therapy is to be performed, the umbilical cord donor is to be the recipient of the cells derived from the umbilical cord. In such a situation, the donor is known or found at birth to have certain abnormal conditions such as may have resulted from the pregnancy, birth or delivery-related complications. These may include but are not limited to cerebral hypoxia, cerebral palsy, or low APGAR (Activity, Pulse, Grimace, Appearance and Respiration) score attributable to ischemic complications during a delivery. In any such event, issues of immune matching or need therefor are moot because donor and recipient are one and the same. The same holds true if the umbilical cord tissue-derived cells are initially frozen and later thawed to be administered to the donor of the cord at some time, possibly years after birth, to treat conditions such as autism, allergies, muscle, joint or soft tissues diseases.

[0045] Typically, umbilical cord is a discarded material and, in principle, is available from every newborn delivery. The collection of umbilical cords in larger numbers allows for the establishment of a bank that incorporates cells that have been recovered from a multitude of umbilical cords and a database identifying respective immune-matching data obtained from each donor's mature cells, preferably, white blood cells or other nucleated cells, that could be used to predict the future differentiation of those umbilical cord tissue-derived cells when administered in therapeutic indications. Typically, with a standard matching of HLA markers, about 10-50 thousand donors, depending on the ethnic diversity of a population, are required to find a 100% HLA match between donor and recipient. In contrast, adaptive matching through the use of umbilical cord tissue-derived cells and umbilical cord blood-derived cells obtained from the same donor, and cryostored in a bank, can reduce this number to 3000 to 5000 donors to obtain a 100% immune-compatibility HLA matching between donor and recipient.

[0046] For storage of umbilical cord tissue-derived cells, freezing and cryopreservation and thawing of cells have been part of an established method. Even cells that have been stored at temperatures colder than −130° C. (theoretically, for 50 years) can be recovered without damage to the cells themselves, except where damage is incurred during the freezing process or the thawing process, which is readily preventable by following known proper procedures. Therefore, banked umbilical cord tissue and tissue-derived cells, as well as umbilical cord blood and blood-derived cells can be preserved for therapeutic and diagnostic purposes. HLA typing of recipients for identification and retrieval of stored cells with matching markers from the bank(s) becomes an efficient, safe and effective technique highly likely to produce positive results. For example, hematopoietic progenitor cells with CD34 marker obtained in this manner engraft successfully with bone marrow in cancer patients.

[0047] In certain aspects of the invention, both the umbilical cord tissue and the cells therefrom are separated into multiple vials, to allow thawing of the different vials of the like cells at different times. Since these cells have a very fast doubling time of 24 hours or less, it is even sufficient to freeze vials of one million cells or small samples of the umbilical cord tissue individually that, if subjected to a cell culturing in a dish or bioreactor, would allow recovery of more than 100 million cells within less than 10 days. Due to the high genetic stability of these cells, 7 or 8 doublings will typically not induce any kind of genetic aberration or genetic instability. The freezing and application of these cells at a later point in time has a further potential to regenerate not only the cells of the donor in the form of an autologous cell transfer, which has a complete immunological match, but also an enhanced probability to be available to immunologically matched family members for regenerative purposes or use in the event of genetic or acquired diseases. The chance that these cells are an immunological match is 1 out of 4 if used for siblings and very high that they could be used for other family members such as parents, grandparents and cousins.

[0048] Diseases and disorder amenable to therapy by administration of umbilical cord tissue-derived stem cells described herein include: Alzheimer's, Parkinson's, neurodegenerative diseases such as Multiple Sclerosis (MS), Duchenne Muscular Dystrophy (DMD), Multi System Atrophy (MSA), Amyotrophic Lateral Sclerosis (ALS), osteoporosis, heart failure, limb ischemia, cerebral hypoxia or perfusion defects, stroke, muscle wasting, renal insufficiency, liver diseases and insufficiency, the whole spectrum of genetic disorders including rare storage diseases, osteogenesis imperfecta, aplastic anemia, myelodystrophy, Hemophilia, Down Syndrome, Schizophrenia, Hematopoietic diseases with an underlying mutation such as for example JAK2 (Janus kinase 2 is a non-receptor tyrosine kinase implicating in signaling by members of the type II cytokine receptor family), orthopedic conditions such as diseases of joints, cartilage defects, spine disease, bone fractures, tendon diseases, skin burns, diabetes, cerebral palsy, lung fibrosis, asthma and chronic obstructive lung disease, atrophy of the nose mucosa, vascular associated diseases such as arterial peripheral vascular disease, Crohn's Disease, Colitis ulcerosa, Lupus and other autoimmune diseases, HIV including modified cells to provide a mutant CCR5 receptor (i.e., C-C chemokine receptor type 5, also known as CD195, is a protein on the surface of white blood cells that is involved in the immune system as it acts as a receptor for chemokines) or combinations thereof. Also, these cells are ideal for cancer therapy in a matched or unmatched allogeneic transfer, especially after they have been genetically modified to serve as a “Trojan Horse” and when given to a patient, unload their cancer therapeutic freight within the tumor.

[0049] Delivery of matched allogeneic cells may also be used to treat a growing fetus that has a genetic disease. As molecular diagnostics have significantly increased over the last years genetic diseases of the fetus are visible not only by amniocentesis, but also from DNA from the blood in the mother and from circulating erythrocytes—which are nucleated of the baby—in the blood of the mother, to determine specific genetic diseases in the growing fetus. At present, no therapy is available for these conditions. Matched allogeneic umbilical cord tissue cells from a donor can be injected during the pregnancy into the umbilical cord arteries of the growing fetus when it is located in the uterus of the mother. This injection contains matched allogeneic cells from a donor in which genetic determination has shown that the specific mutation of the growing fetus, is not present. Repetitive injections of those cells will help, that the transplanted stem cells will partly take over the function of the modified gene and restore diseased gene related protein expression in the baby. It is a known practice to locate the umbilical cord inside the uterus by ultrasound and with a transcutaneous injection into the circulation of the growing fetus to apply those stem cells from a donor. If this were performed in a matched allogeneic transplant way, graft versus host disease must be considered.

[0050] Another aspect of the disclosure is to further select, recover and distribute modified umbilical cord tissue-derived cells with a specific genetic mutation with or without culturing or freezing and thawing. A sample of more than 100 million cells of umbilical cord tissue-derived regenerative cells can serve a large population of diseased or aged persons. For example, cells that have a mutation in CCR5 can be used to treat patients with HIV. Cells that, for example, lack the same genetic mutation as it is present in the future recipient, are valuable to be used to correct inborn genetic diseases. In this case, the cells are used in a matched allogeneic manner. These cases are, for example, the transfer of umbilical cord tissue-derived cells from one donor sibling without a genetic disease to a recipient immune-matched sibling with an existing genetic disease such as DMD or Hemophilia with, for example, Factor 8 deficiency. Injection of umbilical cord tissue-derived cells from a donor having no genetic disease into an allogeneic-matched recipient having a genetic disease such as Hemophilia results in the production of Factor 8 by newly formed endothelial cells derived from the transplanted umbilical cord tissue-derived cells in the recipient with the genetic defect.

[0051] As another example, in certain instances therapy for genetic diseases is achieved through a method based on the absence of a genetic mutation in the donor umbilical cord tissue-derived cells to treat a matched recipient with a genetic disease. For example, one method herein disclosed is the identification of a genetic deficiency such as a mutation in the dystrophin gene that causes DMD or Becker muscular dystrophy; isolation, storage and characterization of umbilical cord tissue-derived cells from a donor that lack the identified genetic mutation; matching of the HLA, MHC1 and/or MHC2 surface immune-markers of a potential recipient with the umbilical cord tissue-derived cells of the donor; and application to the recipient of those umbilical cord tissue-derived cells that are genetically normal compared to the cells of the recipient for therapeutic and diagnostic purposes. In case of DMD, for example, injected donor umbilical cord tissue-derived cells need to have at least one normal X chromosome that does not carry the genetic mutation.

[0052] Injection of matched allogeneic cells from a donor in a recipient with DMD is highlighted in FIG. 7. The muscle cells of a recipient with DMD disease are typically not able to express dystrophin. As can be seen, only the nuclei of the muscle are stained, and staining for dystrophin shows no presence of the respective dystrophin protein, which is typical of this disease (FIG. 7, upper row). Matched allogeneic stem cells from a donor without the genetic disease are able to induce the dystrophin expression in the recipient's muscle cells, as shown in the overlay on the right side of FIG. 7, lower row. Here, multi-nucleated cells form between the recipient's nuclei and the nuclei from the matched allogeneic cells that do not carry the mutation, thereby inducing a protein expression of the missing dystrophin.

[0053] After treatment of the recipient's diseased muscle cells that have the DMD genetic mutation with donor umbilical cord tissue-derived cells that have a normal genetic profile and do not have the DMD mutation, the recipient's muscle cells are able to show an expression of the missing dystrophin protein and are able to convert this into muscular strength, as is evidenced by the following experiment. In a MDX mouse that resembles the human genetic deficiency of dystrophin, one million regenerative stem cells have been injected into the four major muscle groups of fore and hind limbs (FIG. 8). In addition, half a million of these normal cells without the genetic muation are given intravenously to the respective mouse. Before the injection, the mice have been subjected to a wire hanging test in order to evaluate their musclar strength. Normal mice (wild type) are able to hold for 300 seconds when hanging on a thin wire until they fall down. The mice with the genetic disease (MDX untreated) are able to hold up for 20 seconds at baseline and over time the musclar strength is reduced to about 14 seconds after 10 weeks (FIG. 9).

[0054] Injection of fresh, cultured, or frozen and thawed matched allogeneic cells into the muscles and given intravenously (IV) effects a significant increase in muscular strength enabling those genetically compromised mice to hold themselves on the wire about 100 seconds until they release and fall. The gradual increase of muscular strength over time indicates that the cell transfer is therapeutically beneficial and converted into building new muscles respectively inducing a dystrophin expression in already existing muscles (as FIG. 10 shows). Central nuclei in the histology section indicate the formation of new muscles. The transplanted allogeneic matched donor cells are able to differentiate and able to express dystrophin, which can be shown 10 weeks after the injections.

[0055] The application of allogeneic umbilical cord tissue-derived cells in patients in need of the cells is typically done repeatedly. In this way, deficient cells of a recipient are replaced more and more and over time and with normal stem and progenitor cells of a donor. In order to prevent a rejection or GVH (graft versus host) phenomenon, the cells are applied in a matched allogeneic transplant mode. Therefore, common standard and known methods such as determination of the markers of HLA-A, HLA-B, HLA-C (this belongs to MHC class I heavy chain receptors, the C receptor being a heterodimer consisting of a HLA-C mature gene product and β2-microglobulin), HLA-D or B1, HLA-DQB1 (this is a major histocompatibility complex, class II, in which DQ beta 1 is a human gene and also denotes the genetic locus that contains this gene.sup.1; the protein encoded by this gene is one of two proteins that are required to form the DQ heterodimer, a cell surface receptor essential to the function of the immune system) and further molecular biological typing is performed in order to find the right high probability of an allogeneic matched donation, and the typing including the determination of antibodies is performed. Aside from the standard typing, there is also an enhanced epitope typing applicable to reduce the need for higher numbers of donors to find a perfect or acceptable match of HLA immune markers for banking cells of typically about 30 thousand individuals to potentially down to only three thousand samples if the ethnic group included in the matching is restricted to certain geographic areas of ancestral lineage.

[0056] Use of both cord tissue-derived cells and cord blood-derived cells from the same donor applied together to a recipient provides additional beneficial effects. While cord tissue-derived cells are truly pluripotent and are able to differentiate into any of the three germ layers with guidance from the microenvironment, cord blood-derived cells very rarely have this pluripotency. The yield of nucleated cells from a donor's specimen of cord blood is very high (500 to 750 million nucleated cells), but only every second donor's cord blood contains pluripotent cells, and if they are found at all, then only in very limited numbers; so typically, culturing is required before usage if it is the aim to transfer pluripotent stem cells derived from cord blood. However, cord blood cells contain hematopoetic progenitor cells that are capable of differentiation into bone marrow cells. This makes them available in case of an ablation of the bone marrow for cure of hematopoetic malignancies, such as a bone marrow transplantation.

[0057] The two cell types are complementary, since it has been shown that the reconstitution of an ablated bone marrow is easier, when not only hematopoetic progenitors are transplanted, but when they are transplanted together with pluripotent stem cells. In accordance with this invention, this is achieved by administering umbilical cord tissue-derived cells in combination with umbilical cord blood-derived cells from the same donor.

[0058] The particulars disclosed herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. This includes methods and apparatus of the applications and other publications incorporated by reference in their entireties herein.

[0059] Umbilical cord represents and contains an abundant source of perinatal, plastic adherent mesenchymal stem cells (UC-MSCs). UC-MSCs typically exhibit robust stemness and strong immunosuppressive and regenerative effects in vivo. Processing of umbilical cord tissue by the method of the invention efficiently isolates large numbers of fresh nucleated umbilical cord regenerative cells (UC-RCs) that exhibit similar characteristics of UC-MSCs. This can alleviate the need for culture expansion to obtain large numbers of cells required for clinical application especially if the cells are used in the peri-natal setting immediately after delivery in order to be used for therapy of pregnancy, birth or delivery-related complications, such as those resulting from cerebral hypoxia, cerebral palsy or low APGAR conditions.

[0060] Dissociation is achieved with a blend of collagenase and neutral protease with agitation at 37° C. to 40° C. in a semi-automatic system. The average yield is about or more than one million nucleated cells/gram tissue with more than 80% viability. Viability greater than 85 to 95% has been achieved. The procedure to recover cells from umbilical cord tissue utilizing the method of the invention is often less than 30 minutes for umbilical cord segmentation and less than 180 minutes for processing and recovering a cell preparation. Indeed, the time for recovering a cell preparation can be less than 120 minutes, and even less than 60 minutes if the tissue is finely minced and prepared before processing thereof. Quickly obtaining a large number of regenerative cells that have pluripotent differentiation capacity without the complexity and risks of culture expansion simplifies and expands the use of regenerative cells in clinical therapeutic as well as research applications.

[0061] This is especially true for so-called immediate “point of care” processing and applications, i.e., the recovery of regenerative cells immediately after delivery, processing and reapplication to the baby in need. Diseases or disorders of cerebral hypoxia, low APGAR values and cerebral palsy following complicated deliveries are exemplary of those calling for processing the umbilical cord tissue in close proximity to the delivery site. This allows for administration of umbilical cord tissue-derived cells to the umbilical cord donor within 24 hours of the donor's delivery. The umbilical cord tissue-derived cells can be aliquoted, to allow a first aliquot to be administered within 24 hours of the delivery of the donor, and additional aliquots to be administered subsequently. The subsequent cell applications are part of a therapeutic regimen for the disorders mentioned immediately above. Also, diagnosed inborn genetic diseases might benefit from an early stem cells transfer, in an autologous or matched allogeneic way. The cells can be administered intravenously, intra-arterially, intrathecally, into the mucosa or surface of the airway system, including, for example, mouth, nose or lungs, or into a duct, spinal fluid, tissue or organ.

[0062] Mesenchymal stem cells (MSCs) typically define a population of cells with the potential to differentiate into all three germ layers in vitro. In vivo, MSCs promote tissue regeneration and healing, and elicit potent immune-modulatory effects. These characteristics of MSCs have generated significant interest in their use for cell-based regenerative therapies. MSCs reside in all tissues in the body and can be obtained from primary cell isolates of different sources in adult tissues and fetal tissues since they adhere to plastic in cell culture. While isolation methods for adult tissues have been mainly established for adipose tissue and bone marrow, the main source for fetal tissue is the umbilical cord (UC). Due to their origin from fetal tissue, the perinatal MSCs in the umbilical cord exhibit higher stemness and immune-modulatory properties compared to MSCs originating from adult tissues without the limitations and negative aspects of embryonic stem cells.

[0063] The umbilical cord of most mammals consists of two arteries and one or two veins (matrix) that are embedded in a jelly-like ground substance of hyaluronic acid and chondroitin sulfate (FIG. 1), named Wharton's jelly after Thomas Wharton, who first described it in 1656. Recent research has shown that the Wharton's jelly contains plastic adherent MSCs (UCT-MSCs) that exhibit properties close to but different from embryonic stem cells. In addition to their potential for differentiation to cells of all three germ layers. UCT-MSCs have a higher rate of proliferation and may have more prolonged potential for self-renewal compared to adult MSCs. It has been shown that the presence of longer telomeres in UCT-MSCs is responsible for this increased capacity for self-renewal prior to senescence. Due to their close developmental relationship to embryonic tissue (without the teratoma risk of embryonic cells), UCT-MSCs possess broad plasticity and immune-modulatory characteristics in vivo. However, in contrast to embryonic stem cells, UCT-MSCs have important and critical advantages, including absence of ethical concerns, abundant availability since the umbilical cord is part of every newborn delivery and is typically discarded material, and produce neither tumor formation nor carcinogenicity. Moreover, the low immunogenicity, ability to home in on sites with ongoing tissue inflammation, and ability to promote neovascularization and tissue regeneration of UCT-MSCs provide, in appropriate instances, a compelling rationale for allogeneic transplantation. Since Wharton's jelly and the matrix provide a rich perinatal source of UCT-MSCs, a method for efficiently isolating large numbers of cells quickly serves to advance the use of UCT-MSCs in research and clinical applications.

[0064] Isolation of MSC from the Wharton's jelly was first described by McElreavey et al. His group cultured minced Wharton's jelly without prior enzymatic processing up to two weeks in order for the UCT-MSCs to migrate out of the tissue and adhere to the culture dish. This method yielded a relatively low initial number of cells and required more extensive culture expansion after the initial two-week period. Currently, MSCs are isolated from the umbilical cord by the use of collagenase followed by a selection for plastic adherent subpopulation in cell culture. As shown in Table 1 all published protocols for UCT-MSCs isolation commonly result in a relatively low cell yield and require some kind of cell culture expansion to obtain the high cell numbers that are recommended and required for preclinical studies and, especially, clinical usage.

TABLE-US-00001 TABLE 1 Date Author Journal Material Duration No. Cells/cord Viability January H. Wang et al. Stem Cells WJ ≈18 hrs 30 — — 2004 November M. Bailey et al. Tissue WJ ≈1 week 4 — — 2007 Engineering March D. Campard et al. Gastroenterology WJ ≈1 day 15 1.22M ± 1.09M — 2008 cells December N. Tsagias et al. Transfusion cUC ≈4.5 hrs 12 0.96M 81% 2010 Medicine cells March I. Christodoulouet Stem Cells WJ ≈2.5 hrs 5 2.28M ± 1.55M 94.3% ± 2.2% 2013 et al. International cells October J. Hua et al. Cell Biology WJ ≈1-4 hrs — Explant: — 2013 International 0.042-0.0201M cells Enzyme: 0.003-0.027M cells

[0065] Given that these protocols entail several days, weeks to months of culture expansion, differences in cell characteristics including regenerative potential, induction of chromosomal changes and changes in cell surface antigens, the cells are rendered somewhat less effective and useful when used in clinical setting. In addition, expansion in culture has potential for exposure to xenogenic proteins and contamination as well as additional complexity and cost associated with the previous described methods. Also, immediate cell therapy in the peri-delivery timeframe of 12 to 48 hours is not feasible.

[0066] Ex vivo expansion and differentiation of stem cell populations is considered to be substantial manipulation by the United States Food and Drug Administration and the European Medicines Agency. For adipose tissue, fresh preparation of nucleated regenerative cells using a novel semi-automated system (generally referred to as the stromal vascular fraction or SVF), enables high cell yield and therefore obviates the need for cell culture expansion to obtain a high number of pluripotent cells to be used immediately after delivery or later on after freezing, storage and thawing in an autologous or matched allogeneic matter. The protocol of the present invention aims to facilitate the ability to efficiently and more rapidly isolate fresh regenerative cells from umbilical cord (UC-RCs or umbilical cord regenerative cells) with high cell yield to enable potential therapeutic application without the need for culture expansion.

[0067] This protocol of the invention has been developed based on a protocol for isolating the SVF from adipose tissue without ex vivo cell culture expansion. The SVF resulting from the aforementioned isolation process has been designated as a non-ATMP (i.e., not an advanced therapy medicinal product) by the European Medicines Agency when used for regeneration, repair, or replacement of weakened or injured subcutaneous tissue. (EMA/129056/2013 is the scientific recommendation on classification of advanced therapy medicinal products—Summary for Public Release for “Adult Autologous Regenerative Cells for Subcutaneous Administration”). This designation includes a determination that the SVF preparation was not subjected to a substantial manipulation. Additionally, this protocol has been successfully used for the isolation of SVF from debrided skin and adipose tissue from burn victims as well as from equine lipoaspirate samples.

NON-LIMITING EXAMPLES

[0068] In an exemplary method, umbilical cord tissue is processed to recover regenerative cells therefrom. Tissue dissociation is achieved using a mammalian origin free, optimized blend of collagenase and neutral protease, and mechanical processing at elevated temperature in a novel semi-automatic system. This combination results in a high viability of the recovered cells and high yields of UC-RCs in shorter time with less operator involvement. The protease can be produced via a recombinant process.

[0069] In this exemplary method, umbilical cords are harvested; then washed and disinfected; and segmented to obtain umbilical cord tissue segments including matrix, stroma, and Wharton's jelly. The umbilical cord tissue segments may be minced for subsequent processing as described presently to obtain stem cells therefrom. In order to compare the regenerative cells acquired from umbilical cord tissue by methods of the present invention to previously published results of others, the plastic adherent fraction (UCT-MSCs) of the regenerative cells from umbilical cord tissue was characterized. Initial cell yields were quantified and cultures of the primary UCT-MCSs from passage 0 to passage 2 (the latter is the third culture following the isolation of cells from tissue; also called the tertiary tissue) were characterized by assessing their colony forming potential (FIG. 4). Additionally, their capacity to differentiate along all three germ layers, including cell types of the mesoderm such as osteocytes, chondrocytes, adipocytes, cell types of the endoderm such as hepatocytes, and cell types of the ectoderm such as neurons was assessed (FIG. 5). Gene expression profiling by RT-PCR array for MSC-specific genes was also performed (FIG. 6). RT-PCR, or reverse transcription polymerase chain reaction, is one of the many variants of PCR, and this technique is commonly used in molecular biology to detect RNA expression levels. RT-PCR is used to quantitatively detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA.

[0070] Umbilical cords were collected following parturition from normal full term pregnancies with unassisted or assisted delivery. In this aspect of method, umbilical tape was tied around the cord in 2 places; adjacent to where the cord breaks or is severed from the baby and of maximum useable length toward the placenta. The ligations were placed to limit contamination into the lumen of the cord. The isolated portion of the cord between the ligations was placed on a clean surface and any visible gross contamination physically removed with sterile surgical instruments and gauze sponges. In this aspect of this disclosure, the cord was rinsed by shaking in a 1.5 L bottle with sterile 0.9% saline solution three times and then washed with fresh solution containing disinfectant, antibiotic, and antifungal substances, and then placed in cold (4° C.) saline solution until processed. The cord then can be soaked in a solution containing substances to begin dissociation of the umbilical cord before the cord is processed within 24 hours after collection.

[0071] After collection, umbilical cords, in this aspect of the method, are prepared by washing the tissue in a wash solution of Penicillin and Streptomycin (10 IU and 10 μg/mL), gentamycin (2.5 μg/mL) and amphotericin B (250 ng/mL) to Phosphate Buffered Saline (PBS). The samples in this aspect of this disclosure were washed twice with 1-3% hydrogen peroxide in sterile water and three times with the prepared wash solution and finally rinsed with saline solution.

[0072] The umbilical cord contains two arteries (firm, thick walled) and one vein (pliable, thin walled) surrounded by the Wharton's jelly, which insulates and protects the umbilical cord vessels (see FIG. 1, FIG. 2A). The umbilical cord in this aspect of the method is segmented. The tissue samples are placed on large sterile petri dishes for dissection, and the thin squamous epithelium is removed and discarded (see FIG. 2B).

[0073] The remaining tissue is composed of the vessels, stroma, and Wharton's jelly. The fascial plane around the vessels is then dissected to separate the surrounding Wharton's jelly from the vessels. The vessels can be then discarded to isolate only the Wharton's jelly. This is preferred in cases where a low processing time is required or desired since processing of the vessel to release the cells typically takes longer than the processing of the Wharton's jelly for cell recovery. The Wharton's jelly is placed onto a separate dissecting plate and minced prior to further processing of the tissue. A higher degree of mincing beneficially affects the subsequent processing time.

[0074] Umbilical cord regenerative cells are isolated with a pre-warmed tissue processing unit, such as an ARC™ tissue processing unit from InGeneron Incorporated, of Houston, Tex. The ARC™ tissue processing unit is disclosed in U.S. patent application Ser. No. 13/385,599 (published as US 2013/0115697) and U.S. patent application Ser. No. 13/329,142 (published as US 2012/0195863), each of which is incorporated by reference in its entirety herein. In brief, the ARC™ tissue processing unit is a specially designed unit for tissue processing and centrifugation, with the capability to lock tube holders in an inverted upright position. A lactated ringer's back was pre-heated to 37° C. (FIG. 3). Approximately 10 g-25 g each of prepared minced umbilical cord tissue respectively is placed into one or more sterile processing tubes. 25 ml of the aforementioned lactated ringers is added to each tube to reach a final volume of 35 ml-50 ml per tube.

[0075] One to five units of MATRASE™ Reagent (available from InGeneron Incorporated, of Houston, Tex.), a proprietary collagenase and neutral protease enzyme blend, is added to each gram of tissue at a concentration of 10 units/ml of solution. The sample tubes are inverted to mix the enzyme with the tissue and then placed in the processing unit described above and processed for 1-4 hours under the increased temperature environment of the inner portion of the processing unit.

[0076] Following processing, the samples are placed on a rack to allow sedimentation for 2-3 minutes in this method (FIG. 3). The supernatants are collected and transferred to sterile processing tubes. The tissue slurry is filtered through a 100 micron filter. The cells are concentrated by centrifugation of the filtrate at 600×g for 3 to 5 minutes (FIG. 4). The pellets are re-suspended thereby making a cell suspension in saline solution to assess cell viability and cell counts. The cell preparation can either be used immediately or cryopreserved and stored (banked) for future applications. In one embodiment, ultrasound guided injection of the umbilical cord derived cells are delivered in a matched allogeneic way. The volume of these injections should be in a range of a 100 microliter to 1 ml. Representative examples for in vitro characterization of cultured UCT-MSCs demonstrating potential for differentiation into cell types of all three germ layers, CFU-F assay, and gene expression profile are indicated in FIGS. 4-6.

Cell Viability and Nucleated Count Assessment

[0077] As described above, the cell preparation is re-suspended in saline solution; additionally a green fluorescent nucleic stain was administered to the suspension, and the stained cells viewed under fluorescent microscopy. Trypan blue staining was used with light microscopy to visualize and count dead cells.

Cell Cryopreservation

[0078] In order to preserve cells, the pelleted cells, in this aspect of the disclosure, are suspended by centrifugation at 400×g for 3 to 5 minutes. The supernatant gets removed, and the cells are re-suspended in cryopreservation media. The cells are then transferred to a cryovial, and the cryovial is placed on a 100% isopropanol containing cryopreservation chamber. The chamber is then placed in a −80° C. freezer overnight. For long-term storage, the sample can be, either frozen directly in or transferred to the gas phase of liquid nitrogen. In one aspect of this disclosure, the cell preparation is aliquoted and each aliquot is cryopreserved for later use.

[0079] The recovered preparation of regenerative cells can be used either directly after preparation or after thawing of cells that have been previously been frozen. For freezing and thawing the respective known technology is applied. The cell preparation is then prepared according to the specific indication. For IV application for example, the cells are diluted in an isotonic solution. The volume of the solution depends on the size of the patient. For a new born baby, 10-30 ml of isotonic solution will be connected to a vein, or an artery or prepared in a syringe for puncture of the cerebral fluid for intrathecal or intraductal application. For IV and intra-arterial application, about 2-20 million cells are dissolved in 10-20 ml solution. If the injection of cells occurs directly into an organ, such as a joint, a liver or into any other organ, the volume of the injection is considerably lower and ranges between ½ ml-5 ml, also dependent on the size of the patient. For adult patients with a body weight between 50-100 kilograms, typically 250 ml of saline solution and cells in a range between 20 and 100 million will be slowly infused over 30 minutes either into an intravenous application, or through a catheter into an intra-arterial location. Also if the cells are used for local treatment of an organ, the volume will be considerably lower in a range of 1-5 ml; that means the concentration of cells will be considerably higher. Also for intrathecal or intraductal application, the amount of fluids will be in a range of 1-20 ml. For intramucosal delivery a fluid volume of 1-20 ml is selected. For ultrasonic guided injection of umbilical cord derived cells in a match allogeneic way, the volume should be in a range of a 100 microliter to 1 ml.

[0080] As discussed above, cells can be either prepared within the site of the delivery where the umbilical cord initially is recovered, or at a later remote location. Usage is either in an autologous manner, meaning the cells are used only for umbilical cord donor, or in a matched allogeneic manner, meaning that the HLA markers of the umbilical cord donor will be matched with the recipient's HLA markers in order to avoid immunological reactions. In rare cases, when the cells are not expected to transdifferentiate but only have an indirect effect by release of cytokines, exosomes or application of regulatory T cells, then the cells can be used in a non-matched manner.

[0081] This holds true if a co-culturing of cells with the stem cells of a patient if intended, for example for rejuvenation purposes. In this case, the umbilical cord cells can be cultured in serum or serum free media, and cytokines and exosomes derived from those umbilical cord cells can be collected and administered to the patient directly. Or in a co-culture approach, the patient's own stem cells and progenitor cells—cultured in the presence of the cytokines and exosomes contained in the culture media in which the umbilical cord tissue were cultured—are collected and administered. It has been shown by respective studies underlying and supporting this application that the media in which a donor's umbilical cord cells have been cultured contains valuable substances usable for various diagnostic and therapeutic applications. For example, the media can be used in a direct way (injected into the donor or a recipient) or in an indirect way (namely, by culturing a recipient's own cells in the media derived from the culturing of the donor's cells).

[0082] One challenge of isolating regenerative cells from umbilical cord that can be re-administered to a (often elderly) patient as sterile cells is avoidance of contamination of samples during the process of obtaining the umbilical cord. Since newborns are usually vaginally delivered, contamination of the umbilical cord is inevitable. Tissue collection during warm weather conditions can expedite bacterial and fungal growth on tissue samples. Certain interventions can be employed to decrease the risk of contamination of cells during and downstream of the isolation process. The most important step is to minimize bacterial and fungal growth immediately after obtaining the tissue sample. If possible, a sterile field should be set up for working with the freshly obtained tissue sample. The umbilical cord should be rinsed with cold Lactated Ringer's or saline solution. Additional washes as disclosed above plus usage of a providone-iodine solution may also aid in reducing or depleting the bacterial and fungal flora on tissue samples.

[0083] To prevent pathogen growth between tissue collection and tissue processing the cord should then be kept chilled in Lactated Ringer's or saline solution containing 1% of Penicillin, 1% Streptomycin, 0.01% of Gentamycin and 0.2% of Amphotericin B. This antibiotic and antimycotic regimen corresponds with the typical combination in the regular growth media for primary MSC cultures. Identifying the bacterial and fungal flora of the tissue sample right after collection and testing for antibiotic resistance may help in adjusting the antibiotic regimen if microbial growth occurs during culture expansion. Additionally, incubating the cord in the MATRASE™ Reagent helps to release the stem cells in higher numbers from the cord in a shorter amount of time and to reduce the viability of otherwise contaminating bacteria and fungi.

[0084] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. It should further be understood that the references, patents and patent applications disclosed herein are incorporated by reference in their entirety.

REFERENCES

[0085] 1. Zhang Y Y, Yue J, Che H, Sun H Y, Tse H F, and Li G R (2013) BKCa and hEag1 channels regulate cell proliferation and differentiation in human bone marrow-derived mesenchymal stem cells. J Cell Physiol doi:10.1002/jcp.24435 [0086] 2. Zhou J, Xu C, Wu G, Cao X, Zhang L, Zhai Z, Zheng Z, Chen X, Wang Y (2011) In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen-hydroxyapatite layered scaffolds. Acta Biomater 7:3999-4006 [0087] 3. Young R G, Butler D L, Weber W, Caplan A I, Gordon S L, Fink D J (1998) Use of mesenchymal stem cells in collagen matrix for Achilles tendon repair. J Orthop Res 16:406-413 [0088] 4. Hung M J, Wen M C, Huang Y T, Chen G D, Chou M M, Yang V C (2013) Fascia tissue engineering with human adipose-derived stem cells in a murine model: Implications for pelvic floor reconstruction. J Formos Med Assoc doi:pii: S0929-6646(13) 167-168. [0089] 5. Caballero M, Skancke M D, Halevi A E, Pegna G, Pappa A K, Krochmal D J, Morse J, van Aalst J A (2013). Effects of connective tissue growth factor on the regulation of elastogenesis in human umbilical cord-derived mesenchymal stem cells. Ann Plast Surg 70, 568-573 [0090] 6. Anzalone R, Lo Iacono M, Loria T, Di Stefano A, Giannuzzi P, Farina F, La Rocca G (2011) Wharton's jelly mesenchymal stem cells as candidates for beta cells regeneration: extending the differentiative and immunomodulatory benefits of adult mesenchymal stem cells for the treatment of type 1 diabetes. Stem Cell Rev 7:342-363 [0091] 7. Chen Q Q, Yan L, Wang C Z, Wang W H, Shi H, Su B B, Zeng Q H, Du H T, Wan J (2013) Mesenchymal stem cells alleviate TNBS-induced colitis by modulating inflammatory and autoimmune responses. World J Gastroenterol 19:4702-4717 [0092] 8. Wada N, Gronthos S, Bartold P M (2013) Immunomodulatory effects of stem cells. Periodontol 63:198-216 [0093] 9. Takehara N. Cell therapy for cardiovascular regeneration. (2013) Ann Vasc Dis 6:137-144 [0094] 10. Ruzzini L, Longo U G, Rizzello G, Denaro V (2012) Stem cells and tendinopathy: state of the art from the basic science to clinic application. Muscles Ligaments Tendons J 2:235-238. [0095] 11. Stewart A A, Barrett J G, Byron C R, Yates A C, Durgam S S, Evans R B, Stewart M C (2009) Comparison of equine tendon-, muscle- and bone marrow-derived cells cultured on tendon matrix. Am J Vet Res 70:750-757 [0096] 12. Izadpanah R, Trygg C, Patel B, Kriedt C, Dufour J, Gimble J M, Bunnell B A (2006) Biologic properties of mesenchymal stem cells derived from bone marrow and adipose tissue. J Cell Biochem 99:1285-1297 [0097] 13. Yoshimura H, Muneta T, Nimura A, Yokoyama A, Koga H, Sekiya I (2007) Comparison of rat mesenchymal stem cells derived from bone marrow, synovioum, periosteum, adipose tissue and muscle. Cell Tissue Res 27:449-462 [0098] 14. Harris D T (2013) Umbilical Cord Tissue Mesenchymal Stem Cells: Characterization and Clinical Applications. Curr Stem Cell Res Ther 8:394-399 [0099] 15. Ryu Y J, Seol H S, Cho T J, Kwon T J, Jang S J, Cho J (2013) Comparison of the ultrastructural and immunophenotypic characteristics of human umbilical cord-derived mesenchymal stromal cells and in situ cells in Wharton's jelly. Ultrastruct Pathol 37:196-203 [0100] 16. Kadar K, Kiraly M, Porcsalmy B, Molnar B, Racz G Z, Blazsek J, Kallo K, Szabo E L, Gera I, Gerber G, Varga G (2009) Differentiation potential of stem cells from human dental origin-promise for tissue engineering. J Physiol Pharmacol 60 Suppl:167-175. [0101] 17. Hsieh J Y, Fu Y S, Chang S J, Tsuang Y H, Wang H W (2010) Functional module analysis reveals differential osteogenic and stemness potentials in human mesenchymal stem cells from bone marrow and Wharton's jelly of umbilical cord. Stem Cells Dev 19:1895-1910. [0102] 18. Lovati A B, Corradetti B, Lange Consiglio A, Recordati C, Bonacina E, Bizzaro D, Cremonesi F (2011) Comparison of equine bone marrow-, umbilical cord matrix and amniotic fluid-derived progenitor cells. Vet Res Commun 35:103-121 [0103] 19. Liras A (2010) Future research and therapeutic applications of human stem cells: general, regulatory, and bioethical aspects. J Transl Med 8:131. [0104] 20. Warton T (1656) Adenographia: sive glandularum totius corporis descriptio. London: Wharton. pp. 243-244. [0105] 21. Weiss M L, Troyer D L (2006) Stem cells in the umbilical cord. Stem Cell Rev 2:155-162. [0106] 22. Gotherstrom C, West A, Liden J, Uzunel M, Lahesmaa R, (2005) Difference in gene expression between human fetal liver and adult bone marrow mesenchymal stem cells. Haematologica 90:1017-1026 [0107] 23. Karahuseyinoglu S, Kocaefe C, Balci D, Erdemli E, Can A (2008) Functional structure of adipocytes differentiated from human umbilical cord stroma-derived stem cells. Stem Cells 26: 682-691 [0108] 24. Sarugaser R, Lickorish D, Baksh D, Hosseini M M, Davies J E (2005) Human umbilical cord perivascular (HUCPV) cells: a source of mesenchymal progenitors. Stem Cells 23:220-229 [0109] 25. Kita K, Gauglitz G G, Phan T T, Herndon D N, Jeschke M G (2010) Isolation and characterization of mesenchymal stem cells from the sub-amniotic human umbilical cord lining membrane. Stem Cells Dev 19:491-502 [0110] 26. Cooper K, SenMajumdar A, Viswanathan C. (2010) Derivation, Expansion and Characterization of Clinical Grade Mesenchymal Stem Cells from Umbilical Cord Matrix Using Cord Blood Serum. International Journal of Stem Cells 3:119-128 [0111] 27. Prasanna S J, V S Jahnavi (2011) Wharton's Jelly Mesenchymal stem cells as off-the-shelf cellular therapeutics: A closer look into their regenerative and immunomodulatory properties The Open Tissue Engineering and Regenerative Medicine Journal 4:28-38 [0112] 28. Can A, Karahuseyinoglu S (2007) Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells. 11:2886-2895 [0113] 29. McElreavey K D, Irvine A I, Ennis K T, McLean W H (1991) Isolation, culture and characterisation of fibroblast-like cells derived from the Wharton's jelly portion of human umbilical cord. Biochem Soc Trans 19:29S [0114] 30. Baudin B, Bruneel A, Bosselut N, Vaubourdolle M (2007) A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2:481-485 [0115] 31. Iacono E, Brunori L, Pirrone A, Pagliaro P P, Ricci F, Tazzari P L, Merlo B (2012) Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse. Reproduction 143:455-468 [0116] 32. Cardoso T C, Ferrari H F, Garcia A F, Novais J B, Silva-Frade C, Ferrarezi M C, Andrade A L, Gameiro R. (2012) Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system BMC Biotechnology 12:18 [0117] 33. Weiss M L, Medicetty S, Bledsoe A R, Rachakatla R S, Choi M, Merchav S, Luo Y, Rao M S, Velagaleti G, Troyer D (2006) Human umbilical cord matrix stem cells: preliminary characterization and effect of transplantation in a rodent model of Parkinson's disease. Stem Cells 24:781-792 [0118] 34. Von Bahr L, Sundberg B, Loennies L, Sander B, Karbach H, Haegglund H, Ljungman P. GUstafsson B, Karlsson H, Le Blanc K, Ringden O. (2012) Long-term complications, immunologic effects, and role of passage for outcome in mesenchymal stromal cell therapy. Biol Blood Marrrow Transplant; 18(4):557-64 [0119] 35. European Medicines Agency, Reflection paper on stem cell-based medicinal products. 14 Jan. 2011. [0120] 36. Chan R K, Zamora D O, Wrice N L, Baer D G, Renz E M, Christy R J, Natesan S. (2012). Development of a vascularized skin construct using adipose-derived stem cells from debrided burned skin. Stem Cells Int [Epub 2012] [0121] 37. McClure, Peters R, Wolf M J, van den Broek M, Nuvolone M, Dannenmann S, Stieger B, Rapold R, Konrad D, Rubin A, Bertino J R, Aguzzi A, Heikenwalder M, Knuth A K (2010) Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture. PLoS One 5 doi: 10.1371/journal.pone.0015689. [0122] 38. Campard D, Lysy P A, Najami M, and Sokal E M. (2008) Native umbilical cord matrix stem cells express hepatic markers and differentiate into hepatocyte-like cells. Gastroenterology, 134(3):833-48. [0123] 39. Ilmer, Matthias, Jody Vykoukal, Alejandro Recio Boiles, Michael Coleman, and Eckhard Alt. (2014) “Two sides of the same coin: stem cells in cancer and regenerative medicine.” The FASEB Journal 28:2748-2761 [0124] 40. Alt E, Senst C, Murthy S N, Slakey D P, Dupin C L, Chaffin A E, Kadowitz P J, Izadpanah R. (2012) Aging alters tissue resident mesenchymal stem cell properties. Stem Cell Research; 8 (2): 215-225.