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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
  • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
  • A61P25/16—Anti-Parkinson drugs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/06—Free radical scavengers or antioxidants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

• the present invention relates generally to the fields of cell biology and medicine. More particularly, it concerns methods of, treatments using, and devices for restoring the regenerative capability for mesenchymal stem cells and isolating and expanding a subpopulation of less defective mesenchymal stem cells from the bone marrow stromal cells of people with decreased quantity and/or quality of bone marrow-derived mesenchymal stem, such as elderly people.
• BM-MSCs Bone marrow-derived mesenchymal stem cells
• BM-MSC based therapies have been shown great potential for treatment of many age-related degenerative diseases. Due to biosafety concerns and FDA regulations, a patient's own (autologous) stem cells are considered preferable for cell-based therapies.
• BM-MSCs BM-MSCs
• methods used to isolate and expand the BM-MSCs would likely affect treatment outcomes.
• the quantity and quality of BM-MSCs decrease with aging, limiting the therapeutic potential of a patient's own stem cells.
• stem cell banking companies request that clients donate early, because elderly stem cells lose their potency.
• a strategy must be developed for rescuing the regenerative capacity of aging stem cell populations.
• Extracellular matrix is an important component of the cellular niche in tissues, supplying critical biochemical and physical signals to initiate or sustain cellular functions (Chen, et al, 2007; Lai, et al, 2010).
• a tissue-specific ECM microenvironment may be essential in vivo and in vitro to provide chemical and physical cues to direct/govern multipotent stem cells for tissue regeneration and repair (Chen, 2010; Costa, et al, 2012).
• the various scaffold biomaterials have been developed to mimic ECMs for tissue regeneration or repair (Nagaoka, et al, 2010).
• the materials that have been used to support the proliferation and differentiation of progenitor cells include chitosan, polyglycolic acid (PGA), poly-(l)-lactic acid (PLLA), poly (lactic-co- glycolic acid) (PLAG), and poly(ethylene glycol)-terephthalate (PEFT/poly (butylene terephthalate (PBT) (Kagami, et al, 2008; Chan, et al, 2012; Chen, et al, 2005).
• these polymeric scaffolds can induce inflammation resulting from the acidity of their degradation products (Athanasiou, et al, 1996; Cancedda, et al, 2003).
• Matrigel contains basement membrane proteins secreted by EHS mouse sarcoma cells and has been used to grow primary epithelial cells in culture (Maria, et al., 2011). Although varying levels of success have been achieved with this product, it is not consistent with the long term goal to revitalize bone marrow stromal cells.
• Natural scaffold materials are desirable due to their wide ranges of elasticity (allowing tissue-specific scaffold formation), pore sizes (allowing tissue specific nutrition and oxygen access), low bacterial adherence, biodegradability, and low toxicity and immunogenicity (Leal-Egana & Scheibel, 2010).
• ECM extracellular matrix
• BM- MSCs human and mouse bone marrow-derived mesenchymal stem cells
• the inventors have developed a novel approach that allows the isolation and expansion of a clinically valuable subpopulation of mesenchymal stem cells (MSCs) from the bone marrow stromal cells of people with decreased quantity and/or quality of bone marrow-derived mesenchymal stem cells, such as elderly people.
• MSCs mesenchymal stem cells
• the inventors have disclosed cell culture systems and compositions comprising the aforementioned MSCs.
• the inventors have developed methods of obtaining these MSCs and methods of administering them to subjects in need of stem cell therapies who may have age-related degenerative diseases and conditions.
• the inventors have developed a novel approach to restore the regenerative capability of bone marrow stromal cells.
• the methods, compositions, and techniques described herein can also apply to MSCs derived from sources other than bone marrow, e.g. adipose tissue, umbilical cord blood, etc.
• BM-MSCs bone marrow derived mesenchymal stem cells
• ROS reactive oxygen species
• the inventors describe a small population of cells found in BM-MSCs isolated from older donors that are similar in size to the size of the BM-MSCs from young donors.
• the inventors disclose that the cells of small size from older donors may be more similar to young cells in several respects, including proliferation, differentiation, protein expression, and ROS profile.
• the inventors also disclose that the environmental conditions of the cells in older subjects may suppress proliferation capacities of the cells in older subjects.
• the inventors here disclose that to improve the performance of BM-MSCs isolated from an older donor, it is desirable to isolate cells that are small in size and also express SSEA-4 (SSEA+). This population more closely resembles cells from young donors in several properties that include an increased rate of proliferation, increased adenosine triphosphate (ATP) content per cell, higher telomerase levels, and a greater concentration of stem cells.
• SSEA-4 SSEA-4
• the inventors further disclose that culturing the cells on ECM, especially ECM derived from BM-MSCs, exaggerate the differences between the small cells that are SSEA-4 positive.
• the inventors also disclose that small cells that are SSEA-4 negative also appear to recover significantly when cultured on ECM, especially ECM derived from BM-MSCs. Thus, both SSEA-4 positive and SSEA-4 negative populations are potentially valuable for clinical applications.
• BM-MSCs small size bone marrow-derived mesenchymal stem cells
• step (b) sorting the BM-MSCs from step (a) by size and optionally SSEA-4 expression
• step (f) optionally storing the small size BM-MSCs from step (e), and
• step (g) administering the small size BM-MSCs from step (e) and/or (f) to the subject.
• the subject is the same as the first donor whereby the small size BM- MSCs are autologous.
• the small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension.
• the small size BM-MSCs expressed SSEA-4 (SSEA-4 +) at the time of isolation.
• the subject is 50 years of age or older, 65 years of age or older, or 70 years of age or older.
• the small BM-MSCs are cultured on TCP or on extracellular matrix (ECM) derived from a second set of BM-MSCs obtained from a second donor. In some embodiments, the second donor is 25 years of age or younger.
• the subject has decreased quantity and/or quality of BM-MSCs, and/or is in need of stem cell therapy.
• the subject has an age-related degenerative disease, and/or has a disease or condition that compromises the quantity or quality of BM-MSCs, and/or has or will receive treatments that compromise the quantity or quality of BM-MSCs.
• a cell culture system comprising culture media and isolated small size BM-MSCs isolated from a first set of BM-MSCs obtained from a first donor, wherein the isolated small size BM-MSCs expressed SSEA-4 (SSEA-4+) at the time of isolation, and wherein the first donor was 65 years of age or older at the time the first set of BM-MSCs were donated.
• the cell culture system does not comprise BM-MSCs that were not small in size at the time of isolation.
• the cell culture system does not comprise BM-MSCs that did not express SSEA-4 at the time of isolation.
• the isolated small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension.
• the cell culture system further comprises an extracellular matrix (ECM) derived from a second set of BM-MSCs.
• ECM extracellular matrix
• the second set of BM- MSCs were obtained from a second donor 25 years of age or younger.
• the first donor had decreased quantity and/or quality of BM-MSCs, and/or had an age-related degenerative disease, and/or had a disease or condition that compromises the quantity or quality of BM-MSCs at the time the first set of BM-MSCs were donated.
• compositions comprising isolated small size BM-MSCs isolated from a first set of BM-MSCs obtained from a first donor, wherein the isolated small size BM-MSCs expressed SSEA-4 (SSEA-4+) at the time of isolation, and wherein the first donor was 65 years of age or older at the time the first set of BM-MSCs were donated.
• the composition does not comprise BM- MSCs that were not small in size at the time of isolation.
• the composition does not comprise BM-MSCs that did not express SSEA-4 at the time of isolation.
• the isolated small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension.
• the composition further comprises a carrier.
• the isolated small size BM-MSCs were cultured on an ECM derived from a second set of BM-MSCs.
• the second set of BM-MSCs were obtained from a second donor 25 years of age or younger.
• the first donor had decreased quantity and/or quality of BM-MSCs, and/or had an age-related degenerative disease, and/or had a disease or condition that compromises the quantity or quality of BM-MSCs at the time the first set of BM-MSCs were donated.
• BM-MSCs suitable for administration to a subject, the method comprising:
• step (b) sorting the BM-MSCs from step (a) by size and optionally SSEA-4 expression
• step (f) optionally storing the small size BM-MSCs from step (e).
• the small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension. In some embodiments, steps (b) and (c) are conducted using a flow cytometer. In other embodiments, the small size BM-MSCs expressed SSEA-4 (SSEA-4 +) at the time of isolation.
• the first donor is 50 years of age or older, or the first donor is 65 years of age or older, or the first donor is 70 years of age or older.
• the small BM-MSCs are cultured on TCP or on extracellular matrix (ECM) derived from a second set of BM-MSCs obtained from a second donor. In some embodiments, the second donor is 25 years of age or younger.
• the first donor has decreased quantity and/or quality of BM-MSCs, and/or is in need of stem cell therapy.
• the first donor has an age-related degenerative disease, and/or has a disease or condition that compromises the quantity or quality of BM-MSCs, and/or has or will receive treatments that compromise the quantity or quality of BM-MSCs.
• Tissues produced in vitro yet retaining physiological features of in vivo tissues provide a particularly useful tool for monitoring the effects of proposed therapies or molecules on the physiological functions of the tissues. Accordingly, there is disclosed a method of testing the biological activity of a substance, the method comprising:
• the substance is a candidate therapeutic to treat a disease and/or condition.
• the disease and/or condition is caused by or compromises the quantity or quality of BM-MSCs.
• the disease and/or condition is an age related disease or the aging process itself.
• the condition is reduced regenerative capacity of aging stem cell populations, or a side effect of a medication or radiotherapy.
• the substance is a cellular growth factor or cellular differentiation factor. Adding the substance to the cell culture system can comprise adding the substance to the culture medium.
• the culture medium can be exchanged for a culture medium comprising a particular substance or combination of substances to monitor the effects of the culture medium change on the physiological functions of the cells.
• Measuring a parameter of the cell culture system can include, for example, observing growth rates or morphological features of cells. Any biologically relevant parameter can be measured and monitored to determine the biological effect of exposing the cells to a substance or of changing any conditions of growth. Changes in the parameter being measured or monitored can be attributed to the presence of the substance or the change in growth conditions if a corresponding control does not show the same change.
• a number value with one or more decimal places can be rounded to the nearest whole number using standard rounding guidelines, i.e. round up if the number being rounded is 5, 6, 7, 8, or 9; and round down if the number being rounded is 0, 1, 2, 3, or 4. For example, 3.7 can be rounded to 4.
• compositions and methods for their use can "comprise,” “consist essentially of,” or “consist of any of the ingredients or steps disclosed throughout the specification.
• the MSCs and BM-MSCs include any progeny cells produced thereof.
• the term "progeny cell” is used to indicate a cell that is derived from another cell, such as a parent cell.
• the progeny cell may retain the same characteristics as the parent cell or may have different characteristics, such as a progeny cell that has differentiated.
• “decreased quantity and/or quality" of bone marrow- derived mesenchymal stem cells is used to indicate that the number of stem cells is decreased and/or stem cell function is diminished along one or more dimensions relative to those of a young, healthy subject population's.
• Non-limiting examples are shown herein of properties of sternness (i.e. SSEA-4, self-renewal, differentiation capacity) and properties of aging (senescence, reactive oxygen species, annexin-5).
• aging can cause a decreased quantity and/or quality of bone marrow-derived mesenchymal stem cells.
• the term “aging” is used to indicated the sum of processes, by which stem cell populations decrease in quantity and/or quality.
• the term “young” refers to humans (male or female) age 25 years and under, and also refers to the cells obtained from them.
• bone marrow-derived mesenchymal stem cells indicate that the cells have been removed from an organism and/or other bone marrow-derived mesenchymal stem cells with different characteristics.
• bone marrow-derived mesenchymal stem cells may be isolated from a human.
• bone marrow-derived mesenchymal stem cells that are small in size and/or express SSEA-4 may be isolated from bone marrow-derived mesenchymal stem cells that are not small in size and/or do not express SSEA-4 or express SSEA-4 to a lesser extent than the separated cells.
• small and small size when used in reference to BM-MSCs cell size means that the "small size” BM-MSCs have a smaller diameter when suspended in solution (“suspended diameter") than the average suspended diameter of the set of BM-MSCs obtained from a donor at a given time.
• the small size BM-MSCs are isolated from the set of BM-MSCs obtained from a donor.
• the terms "small” and “small size” when used in reference to BM-MSCs cell size means BM-MSCs having a median cell diameter of less than 33 microns, or less than 30 microns, or less than 25 microns, or less than 20 microns, or less than 19 microns when measured in suspension.
• the terms “large” or “large size” when used in reference to BM-MSCs cell size means “large size” BM-MSCs have a larger diameter when suspended in solution (“suspended diameter") than the average suspended diameter of the set of BM- MSCs obtained from a donor at a given time.
• the terms “large” and “large size” when used in reference to BM-MSCs cell size means BM-MSCs having a median diameter of greater than 35 microns, or 33 microns and greater when measured in suspension.
• cells that express SSEA-4 that are "SSEA-4 positive,” SSEA-4+, or are "positive for SSEA-4 expression", or any variation of these terms, indicates that the cells express SSEA-4 or express SSEA-4 to an extent that is measurable in cells when compared to a negative control.
• the SSEA-4 expressing BM-MSCs are isolated from the set of BM-MSCs obtained from a donor.
• cells that do not express SSEA-4 that are "SSEA-4 negative,” SSEA-4-, or are negative for SSEA-4 expression, or any variation of these terms, indicates that the cells do not express SSEA-4 or express SSEA-4 to an extent that is not measurable in cells when compared to a negative control.
• the SSEA-4 non-expressing BM-MSCs are isolated from the set of BM-MSCs obtained from a donor.
• Embodiment 1 is a method of administering small size bone marrow-derived mesenchymal stem cells (BM-MSCs) to a subject, the method comprising:
• step (b) sorting the BM-MSCs from step (a) by size and optionally SSEA-4 expression, (c) isolating the small size BM-MSCs,
• step (f) optionally storing the small size BM-MSCs from step (e), and
• Embodiment 2 is the method of embodiment 1, wherein the subject is the same as the first donor.
• Embodiment 3 is the method of embodiments 1 or 2, wherein the small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension.
• Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the small size BM- MSCs expressed SSEA-4 (SSEA-4 +) at the time of isolation.
• Embodiment 5 is the method of any one of embodiments 1 to 4, wherein the subject is 50 years of age or older.
• Embodiment 6 is the method of any one of embodiments 1 to 4, wherein the subject is 65 years of age or older.
• Embodiment 7 is the method of any one of embodiments 1 to 4, wherein the subject is 70 years of age or older.
• Embodiment 8 is the method of any one of embodiments 1 to 7, wherein the small BM-MSCs are cultured on TCP or on extracellular matrix (ECM) derived from a second set of BM-MSCs obtained from a second donor.
• ECM extracellular matrix
• Embodiment 9 is the method of embodiment 8, wherein the second donor is 25 years of age or younger.
• Embodiment 10 is the method of any one of embodiments 1 to 8, wherein the subject has decreased quantity and/or quality of BM-MSCs, and/or is in need of stem cell therapy.
• Embodiment 11 is the method of any of embodiments 1 to 10, wherein the subject has an age- related degenerative disease, and/or has a disease or condition that compromises the quantity or quality of BM-MSCs, and/or has or will receive treatments that compromise the quantity or quality of BM-MSCs.
• Embodiment 12 is a cell culture system comprising culture media and isolated small size
• BM-MSCs isolated from a first set of BM-MSCs obtained from a first donor, wherein the isolated small size BM-MSCs expressed SSEA-4 (SSEA-4+) at the time of isolation, wherein the cell culture system does not comprise BM-MSCs that were not small in size at the time of isolation or BM-MSCs that did not express SSEA-4 at the time of isolation, and wherein the first donor was 65 years of age or older at the time the first set of BM-MSCs were donated.
• Embodiment 13 is the cell culture system of embodiment 12, wherein the isolated small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension.
• Embodiment 14 is the cell culture system of embodiment 12 or 13, wherein the cell culture system further comprises an extracellular matrix (ECM) derived from a second set of BM-MSCs.
• ECM extracellular matrix
• Embodiment 15 is the cell culture system of embodiment 14, wherein the second set of BM- MSCs were obtained from a second donor 25 years of age or younger.
• Embodiment 16 is the cell culture system of any one of embodiments 12 to 15, wherein the first donor had decreased quantity and/or quality of BM-MSCs, and/or had an age- related degenerative disease, and/or had a disease or condition that compromises the quantity or quality of BM-MSCs at the time the first set of BM-MSCs were donated.
• Embodiment 17 is a composition comprising isolated small size BM-MSCs isolated from a first set of BM-MSCs obtained from a first donor, wherein the isolated small size BM-MSCs expressed SSEA-4 (SSEA-4+) at the time of isolation, wherein the composition does not comprise BM-MSCs that were not small in size at the time of isolation or BM-MSCs that did not express SSEA-4 at the time of isolation, and wherein the first donor was 65 years of age or older at the time the first set of BM- MSCs were donated.
• SSEA-4+ SSEA-4
• Embodiment 18 is the composition of embodiment 17, wherein the isolated small size BM- MSCs have a median diameter of less than 30 microns when measured in suspension.
• Embodiment 19 is the composition of embodiment 17 or 18, wherein the composition further comprises a carrier.
• Embodiment 20 is the composition of any of embodiments 17 to 19, wherein the isolated small size BM-MSCs were cultured on a ECM derived from a second set of BM- MSCs.
• Embodiment 21 is the composition of embodiment 20, wherein the second set of BM-MSCs were obtained from a second donor 25 years of age or younger.
• Embodiment 22 is the composition of any one of embodiments 17 to 21, wherein the first donor had decreased quantity and/or quality of BM-MSCs, and/or had an age-related degenerative disease, and/or had a disease or condition that compromises the quantity or quality of BM-MSCs at the time the first set of BM-MSCs were donated.
• Embodiment 23 is a method of obtaining small size BM-MSCs suitable for administration to a subject, the method comprising:
• step (b) sorting the BM-MSCs from step (a) by size and optionally SSEA-4 expression
• step (f) optionally storing the small size BM-MSCs from step (e).
• Embodiment 24 is the method of embodiment 23, wherein the small size BM-MSCs have a median diameter of less than 30 microns when measured in suspension.
• Embodiment 25 is the method of embodiment 23 or 24, wherein steps (b) and (c) are conducted using a flow cytometer.
• Embodiment 26 is the method of any one of embodiments 23 to 25, wherein the small size BM-MSCs expressed SSEA-4 (SSEA-4 +) at the time of isolation.
• Embodiment 27 is the method of any one of embodiments 23 to 26, wherein the first donor is 50 years of age or older.
• Embodiment 28 is the method of any one of embodiments 23 to 26, wherein the first donor is 65 years of age or older.
• Embodiment 29 is the method of any one of embodiments 23 to 26, wherein the first donor is 70 years of age or older.
• Embodiment 30 is the method of any one of embodiments 23 to 29, wherein the small BM- MSCs are cultured on TCP or on extracellular matrix (ECM) derived from a second set of BM-MSCs obtained from a second donor.
• ECM extracellular matrix
• Embodiment 31 is the method of embodiment 30, wherein the second donor is 25 years of age or younger.
• Embodiment 32 is the method of any one of embodiments 23 to 30, wherein the first donor has decreased quantity and/or quality of BM-MSCs, and/or is in need of stem cell therapy.
• Embodiment 33 is the method of any one of embodiments 23 to 32, wherein the first donor has an age-related degenerative disease, and/or has a disease or condition that compromises the quantity or quality of BM-MSCs, and/or has or will receive treatments that compromise the quantity or quality of BM-MSCs.
• Embodiment 34 is a method of testing the biological activity of a substance, the method comprising: (a) obtaining the cell culture system of any one of embodiments 12 to 16,
• Embodiment 35 is the method of embodiment 34, wherein the substance is a candidate
• Embodiment 36 is the method of embodiment 35, wherein the disease and/or condition is caused by or compromises the quantity or quality of BM-MSCs.
• Embodiment 37 is the method of any of embodiments 34 to 36, wherein the substance is a cellular growth factor or cellular differentiation factor.
• FIG. 1 - MSCs from old and young donors express unique phenotypes. Differences in the percentage of cells positive for stage-specific embryonic antigen 4 (SSEA-4), human major histocompatibility complex (MHC) class II HLAdr, and reactive oxygen species (ROS) is shown between BM-MSCs from old donors (left columns) and young donors (right columns).
• SSEA-4 stage-specific embryonic antigen 4
• MHC human major histocompatibility complex
• ROS reactive oxygen species
• FIG. 2 Large MSCs are more likely to express aging markers. Scatter plot of BM- MSC cells sorted by size (Large and Small) and presence of ROS (Positive) or absence of ROS (Negative). Large MSCs are significantly more likely to express aging markers.
• UL- Quad indicates the upper left quadrant (Ql)
• UR-Quad indicates the upper right quadrant (Q2-2)
• LL-Quad indicates the lower left quadrant
• LR-Quad indicates the lower right quadrant.
• FIG. 3 Cell spread area was measured for cells in culture on TCP. The spread areas of young and elderly BM-MSCs are reported here as a relative frequency of BM-MSCs at different spread areas. Median spread area is substantially larger for elderly MSCs relative to young.
• FIG. 4A - 4D Young ECM benefits MSCs from young, but not elderly, donors.
• B Cells were detached and counted after 7 days culture in order to determine cell density.
• FIG. 5A - 5D - Young and elderly MSCs may be distinguished across a variety of parameters.
• FIG. 6A - 6C - Elderly MSCs are capable of inhibiting function of healthy cells through expression of the senescence associated secretory phenotype.
• A Elderly MSC populations have a substantially higher frequency of senescent cells relative to young MSC populations.
• B Conditioned media collected from elderly BM-MSCs contains factors that slow proliferation of young MSCs relative to controls.
• C Elderly MSCs secrete a cytokine profile indicative of the senescence associated secretory phenotype. The chart summarizes the cytokine expression of 44 SASP associated cytokines measured in elderly BM-MSCs relative to young BM-MSCs. *P ⁇ 0.05, vs. young BM-MSCs.
• FIG. 7A - 7B Fluorescence activated cell sorting to isolate a youthful sub- population.
• A Flow cytometry reveals that while young BM-MSCs reliably express a small size, SSEA-4 (+) phenotype, elderly BM-MSCs are much more heterogenous along these dimensions.
• B Using FACS analysis enables one to obtain a "youthful" phenotype subpopulation from elderly donors. After doublet discrimination, cells are separated based on size (small vs. large) and SSEA-4 expression (positive vs. negative). Roughly 5-10% of elderly MSCs express the "youthful" (small (+)) phenotype.
• FIG. 8 - Cell proliferation was determined for BM-MSCs from young donors grown in growth media (Regular GM as Control), growth media conditioned with secretions from young donor BM-MSCs (Young CM), and growth media conditioned with secretions from elderly donor BM-MSCs (Old CM).
• Elderly conditioned media inhibits proliferation of young MSCs.
• the proliferation rate of BM-MSCs from young donors was inhibited as determined by cell count after a set period of time when cultured in Old CM, while culturing in Young CM showed little to no difference.
• FIG. 9 Self-renewal capacity of small cells from older donors. Cell proliferation was determined for unsorted, small size, and large size BM-MSCs from old donors. Small cells grew 65% more cells than large cells in the same period of time. Small cells also grew faster than cells that were unsorted by size.
• FIG. 10A - 10E Small(+) MSCs exhibit characteristics of more youthful MSCs.
• A Photographs of culture plates of CFU-F, CFU-AD, and CFU-OB assays of young and elderly BM-MSCs cultured on TCP.
• CFU assays were quantified and reported as frequency/ 100 cells. There are no statistically significant differences between small(+) and young BM- MSCs in any of the CFU assays stained for markers of differentiation into fibroblast (CFU- F), osteoblast (CFU-OB), and adipocytes (CFU-AD).
• FIG. 12 Expression and ROS phenotype of small size, large size, and unsorted cells from older donors. The percentage of cells expressing several proteins associated with youth and aging and percentage of cells with detectable ROS levels were determined in cells from old donors with small size (Small), large size (Large), and unsorted size (Unsorted). Cells with small size expressed higher SSEA-4 and platelet-derived growth factor PDGF than large cells and lower HLAdr and intracellular ROS.
• FIG. 13 Cells with higher ATP concentrations within cell subpopulations before culture.
• FIG. 14 ATP concentration of cell subpopulations before culture.
• FIG. 15 Graph of Telomerase levels measured for Young and Elderly BM-MSCs; and Large -, Large +, Small -, and Small + populations of BM-MSCs from elderly donors. Telomerase levels are slightly higher for small cells that express SSEA-4.
• FIG. 16 Large cell size conditioned media inhibits proliferation of young MSCs.
• Cell proliferation was determined for MSCs from young donors grown in growth media conditioned with secretions from small size cells (Small), secretions from large size cells (Large), conditioned with secretions from cells unsorted by size (Unsorted), and growth media conditioned with secretions from young donor MSCs (young CM).
• the proliferation rate of MSCs from young donors was inhibited as determined by cell count after a set period of time when cultured in Large and Unsorted conditioned media, while culturing in Small and Young conditioned media showed little to no difference.
• FIG. 17 Rescuing stem cells in BM-MSCs from young donors and small size, large size, and unsorted size BM-MSCs from old donors.
• the number of stem cells in BM-MSCs from young donors and small size, large size, and unsorted size BM-MSCs from old donors was determined by counting the number of colony forming units before and after culturing on tissue culture plastic (TCP) and bone marrow extracellular matrix (BM-ECM). Culturing small cells from an older donor showed an increase in stem cells rescued in comparison to large and unsorted cells from an old donor. Further, culturing in BM-ECM showed an increase in stem cells in comparison to culturing on TCP.
• TCP tissue culture plastic
• BM-ECM bone marrow extracellular matrix
• FIG. 18 - CFUs from unsorted BM-MSCs from young donors and old donors, large size cells from old donors, and small size cells from old donors were grown on ECM and TCP and stained for markers of differentiation into fibroblast (CFU-F), osteoblast (CFU-OB), and adipocytes (CFU-AD).
• CFU-F fibroblast
• CFU-OB osteoblast
• CFU-AD adipocytes
• FIG. 19A - 19F - ECM expansion enhances replication of small MSCs.
• A Small MSCs proliferate rapidly on ECM and become highly confluent after 6 days of culture.
• B Graph of cell density of unfractionated (young; elderly) and fractionated elderly BM-MSCs following 7 Days in culture on TCP vs. ECM derived from BM-MSCs from young donors (young ECM). Total cell proliferation for each population is shown as cell density after 7 days in culture and equal seeding density (3,000 cells/cm 2 ). Cell proliferation was determined quantitatively by counting the number of cells in the culture dishes after culturing the BM- MSC subpopulations for 7 days on TCP or young ECM.
• Small+ cells showed increased cell proliferation on both TCP and young ECM and showed a 4 fold increase of number of cells when compared to non-separated cells grown on TCP. The results suggest that young ECM increases proliferation rate of all of the subpopulations. Further small cell size also seems to increase proliferation rates.
• C Following expansion on TCP or ECM cells were seeded at clonal density for CFU assays.
• FIG. 20 - Mesenchymal stem cells can be harvested from subjects with lower quality and quantity of MSCs.
• the cells are sorted by flow cytometry based on size and stage- specific embryonic antigen 4 (SSEA-4) expression.
• Small cells that are SSEA-4 positive (Small+) are plated on ECM made by marrow stromal cells from young donors (young ECM).
• the cells are then expanded using young ECM, divided, and stored.
• large numbers of "youthful" MSCs can be obtained from donors with lower quality and quantity of MSCs, such as elderly donors, for autologous MSC-based therapies and autologous MSC banking.
• FIG. 21A - 21C - Example gating strategy to obtain small cells that are SSEA-4 positive
• FIG. 22 Light microscopy micrographs of BM-MSCs subpopulations from a 71- year old male donor cultured on TCP and ECM derived from BM-MSCs from young donors (young ECM) for 7 days. Small cells positive for SSEA-4 showed an increase in cell proliferation under both conditions. Further, cells cultured on young ECM showed improvements in cell proliferation in comparison to cells cultured on TCP.
• FIG. 23 - Cell proliferation was determined quantitativly by counting the number of cells in the culture dishes after culturing the BM-MSC subpopulations for 7 days on TCP or young ECM.
• the populations tested included non-separated BM-MSC (Unf), large cells positive for the presence of SSEA-4 (L+) or negative for SSEA-4 (L-) and small cells positive for the presence of SSEA-4 (S+) or negative for SSEA-4 (S-).
• S+ cells showed increased cell proliferation on both TCP and young ECM and showed an 8-fold increase of number of cells when compared to non-separated cells grown on TCP.
• FIG. 24 Cell proliferation was determined quantitativly by counting the number of cells in the culture dishes after culturing young and old BM-MSCs (Young and Old, respectively) and old subpopulations of BM-MSCs (S+, L+, S-, and L-) on TCP (2D) (left column) or ECM (right column). S+ cells showed increased cell proliferation on both TCP and young ECM and S- cells also showed improved cell proliferation. [0065] FIG.
• 25 - Percentage of cells expressing SSEA-4 was determined following culture of young and old BM-MSCs (Young and Unsorted, respectively) and old subpopulations of BM-MSCs (Small(+), Small(-), Large(+), and Large(-)) on TCP (left column) or ECM (right column). Small(+) cells showed high expression of SSEA-4, and in most groups culture on ECM increased SSEA-4 expression.
• FIG. 26 - ROS expression was determined following culture of young and old BM- MSCs (Young and Unsorted, respectively) and old subpopulations of BM-MSCs (Small(+), Small(-), Large(+), and Large(-)) on TCP (left column) or ECM (right column). Small(+) cells showed lower intracellular ROS than other groups, and culture on ECM further reduces mean intracellular ROS for most groups.
• FIG. 27 - ATP expression was determined following culture of young and old BM- MSCs (Y and U, respectively) and old subpopulations of BM-MSCs, Small(+), Small(-), Large(+), and Large(-) (S+, S-, L+, and L-, respectively), on TCP (left column) or ECM (right column).
• S+ cells cultured on tissue culture plastic (TCP) have significantly higher ATP concentration.
• ECM increased ATP content in most groups.
• FIG. 28 - Telomerase activity was determined following culture of young and old BM-MSCs (Y and U, respectively) and old subpopulations of BM-MSCs, Small(+), Small(-), Large(+), and Large(-) (S+, S-, L+, and L-, respectively), on TCP (left column) or ECM (right column). S+ cells cultured on tissue culture plastic (TCP) have significantly higher telomerase activity.
• TCP tissue culture plastic
• FIG. 29 The number of SSEA-4 positive cells after 5 passages of BM-MSCs from young donors and small size, large size, and unsorted size BM-MSCs from old donors was determined after culturing on ECM and Tissue Culture Plastic (2D). Culturing small cells from an older donor showed an increase in SSEA-4 positive cells after 5 passages in comparison to large and unsorted cells from an old donor. Further, culturing in ECM showed an increase in stem cells in comparison to culturing on 2D.
• FIG. 30A - 30C - Small (+) MSCs expanded on ECM maintain a youthful phenotype.
• A Graph of ⁇ -Galactosidase expression of unfractionated (Young; Elderly) and fractionated elderly BM-MSCs. ⁇ -galactosidase expression was measured in Young, Elderly, Small +, and Small - populations following culture on TCP or ECM derived from BM-MSCs from young donors (young ECM). Small size sub-populations maintain low levels of senescence during culture. Small cells isolated from elderly donors maintain low expression during culture.
• (B) Graph of ATP levels of unfractionated (Young, Elderly) and fractionated elderly BM-MSCs following culture on TCP and ECM derived from BM-MSCs from young donors (young ECM). ATP expression was determined following culture of young and elderly BM-MSCs and elderly subpopulations of BM-MSCs (S+ S-) on TCP or young ECM. S+ cells cultured on tissue culture plastic (TCP) have significantly higher ATP concentration. Young ECM increased ATP content in for most groups.
• FIG. 31 Histograms of Flow Cytometry Analysis of SSEA-4, ROS, and Annexin-5 in unfractionated (Young; Elderly) and elderly fractionated BM-MSCs.
• Expression was determined by flow cytometry following culture of young and elderly BM-MSCs and elderly subpopulations of BM-MSCs (Small +, Small -) on TCP or young ECM.
• Small + MSCs cultured on young ECM display increased SSEA-4, reduced ROS, and reduced annexin-5 expression relative to TCP. Expression was quantified by comparing fluorescent intensity of samples (black line), to negative controls (shaded region).
• FIG. 32 Graph of total fold change of unfractionated (Young; Elderly) and fractionated Elderly BM-MSCs after 4 passages.
• the cells were passaged every 7 days in culture on TCP vs.
• ECM derived from BM-MSCs from young donors (young ECM) using a seeding density of 1500 cells/cm 2 .
• the number of SSEA-4 positive cells after 4 passages of BM-MSCs from young and elderly BM-MSCs and elderly subpopulations of BM-MSCs (Small +, Small -) on TCP or young ECM increased dramatically.
• Serial passage on young ECM resulted in a fold change of roughly 6,000 when small+ cells were expanded on young ECM.
• the inventors of the present application have developed novel approaches that allow the isolation and expansion of a subpopulation of less defective mesenchymal stem cells (MSCs) from the bone marrow stromal cells of people with decreased quantity and/or quality of bone marrow-derived mesenchymal stem cells (BM-MSCs), such as elderly people, and thus have discovered a strategy for rescuing the regenerative capacity of aging stem cell populations. Further, the inventors have disclosed cell culture systems, techniques and compositions of the aforementioned MSCs, which can be very effective to rescue human stem cells from a donor with decrease quantity and/or quality of human stem cells.
• MSCs mesenchymal stem cells
• the inventors have developed methods of obtaining these MSCs and methods of administering them to subjects in need of stem cell therapies who may have age-related degenerative diseases and conditions. Still further, the inventors have developed a novel approach to restore the regenerative capability of bone marrow stromal cells.
• the phenotype of MSC cells from old and young donors are different, but there is overlap. See FIG. 1 and FIG. 4.
• the present inventors disclose herein that small BM-MSCs are more likely to express markers of early stage stem cells and large cells are more likely than small cells to express aging markers. See FIG. 2 for cells sorted based on size and the levels of ROS.
• the inventors describe a small population of cells found in BM-MSCs isolated from older donors that are similar in size to the size of the BM-MSCs from young donors. See FIG. 3.
• the inventors disclose that the MSCs of small size from older donors may be more similar to young cells in several respects, including proliferation, differentiation, protein expression, and ROS profile.
• the inventors also disclose that the environmental conditions of the cells in older subjects may suppress proliferation capacities of the cells in older subjects. See FIGS. 6B, 8 and 16.
• the inventors here disclose that to improve the performance of MSC cells isolated from a donor, it is desirable to isolate cells that are small in size and also express SSEA-4. See FIG. 21 for an embodiment of a gating strategy to obtain small cells that are SSEA-4 positive. This population more closely resembles cells from young donors in several properties that include an increased number of cells with higher ATP content, increased ATP content per cell, higher telomerase levels, lower ⁇ -Galactosidase expression, and a greater concentration of stem cells. See FIGs. 10, 11, 13 through 15.
• the inventors further disclose that culturing the cells on ECM derived from BM- MSCs from young donors (young ECM) exaggerate the differences between the small cells that are SSEA-4 positive. See FIGs. 17 through 19 and 22 through 32. [0077] The inventors also disclose that small cells that are SSEA-4 negative also appear to recover significantly when cultured on young ECM. This population is also valuable for clinical applications. [0078] In one aspect, the disclosures herein can be used in adult autologous stem cell banking. With the techniques disclosed herein one may be able to find and expand healthy cells from anyone, regardless of age, for stem cell banking and future therapeutic applications.
• Stem cell banks, clinics, and adult patients with conditions that could potentially be treated with stem cells are non-limiting examples of groups that may find the invention useful.
• using the discoveries disclosed herein one may select only the best cells from clients for stem cell banking that will enable the banks to offer higher quality services, and/or offer services to individuals they would have previously assumed were too old.
• novel approaches that allow one to isolate and expand a small subpopulation of less defective MSCs from MSCs of low quality and quantity, such as the cells of elderly people.
• the less defective MSCs express stage-specific embryonic antigen 4 (SSEA-4) and/or are small in size.
• SSEA-4 stage-specific embryonic antigen 4
• novel approaches to restore the regenerative capability of MSCs This process provides a new paradigm for treating age-related disease.
• serial administration of rejuvenated autologous BM-MSCs may not only replace old BM-MSCs, but also gradually reverse the aged microenvironment. The ultimate goal is to slow the progression and/or reverse the degenerative effects of age-related degenerative diseases, disease and/or conditions or treatments that compromise the quantity or quality of MSCs or progeny cells thereof, or the aging process itself.
• Non-limiting examples of age-related degenerative disease and diseases and/or conditions that compromise the quantity or quality of MSCs include, but are not limited to osteoarthritis and osteoporosis, sarcopenia, atherosclerosis, Parkinson's, Alzheimer's, xerostomia, macular degeneration, nonunion fractures, marrow ablation by chemotherapy or radiotherapy, and/or pneumonia.
• the method comprises isolating healthy cells from a compromised population, such as a population from an elderly subject.
• MSCs may be considered small if the cells have a smaller diameter when suspended in solution ("suspended diameter") than the average suspended diameter of the set of MSCs obtained from a donor at a given time.
• BM-MSCs bone marrow-derived mesenchymal stem cells
• the methods comprising: harvesting a first set of BM-MSCs from a first donor; sorting the BM-MSCs by size and optionally SSEA-4 expression, isolating the small size BM-MSCs, plating the small size BM-MSCs for culture, expanding the small size BM-MSCs in culture, and administering the small size BM-MSCs to the subject.
• the BM-MSCs are sorted using a flow cytometer.
• the expanded cells can be divided and/or stored in cell banks for future use.
• the subject is the same as the first donor, and in these cases, the small size BM-MSCs would be autologous.
• the small size BM-MSCs have a median diameter of less than 33 microns, or less than 30 microns, or less than 25 microns, or less than 20 microns, or less than 19 microns when measured in suspension.
• the small size BM-MSCs expressed SSEA-4 (SSEA-4 +) at the time of isolation.
• the subject is 50 years of age or older, 65 years of age or older, or 70 years of age or older.
• the cells are cultured on TCP or on extracellular matrix (ECM) derived from a second set of BM-MSCs obtained from a second donor.
• the second donor is 25 years of age or younger (providing "young" ECM).
• the subject has decreased quantity and/or quality of BM-MSCs, and/or is in need of stem cell therapy.
• the subject has an age-related degenerative disease, and/or has a disease or condition that compromises the quantity or quality of BM-MSCs, and/or has or will receive treatments that compromise the quantity or quality of BM-MSCs.
• compositions and/or cell culture systems comprising isolated small size BM-MSCs isolated from a first set of BM-MSCs obtained from a first donor, wherein the first donor was 50, 65, or 70 years of age or older at the time the first set of BM-MSCs were donated.
• the small size MSCs expressed SSEA-4 (SSEA-4+) at the time of isolation.
• the cell culture system does not comprise MSCs that were not small in size at the time of isolation.
• the cell culture system does not comprise MSCs that did not express SSEA-4 at the time of isolation.
• the isolated small size MSCs have a median diameter of less than 33 microns, or less than 30 microns, or less than 25 microns, or less than 20 microns, or less than 19 microns when measured in suspension.
• the isolated small size MSCs were cultured on a ECM derived from a second set of BM-MSCs.
• the second set of BM-MSCs were obtained from a second donor 25 years of age or younger (young ECM).
• the first donor had decreased quantity and/or quality of MSCs, and/or had an age-related degenerative disease, and/or had a disease or condition that compromises the quantity or quality of MSCs at the time the first set of MSCs were donated.
• the culture systems comprise a culture media. Suitable culture media are known by one skilled in the art.
• the compositions may further comprise a carrier. Carriers can be aqueous based.
• the compositions and cell culture systems can comprise adjuvants, e.g., to preserve the composition or maintain the viability of the cells.
• Compositions and cell culture systems can be stored at cryogenic and/or hypothermic conditions, e.g., in cell banks, and can contain ingredients to aid in preservation at these conditions, e.g., cryoprotectants.
• the extracellular matrix (ECM) derived from BM-MSCs described herein can be produced by the methods disclosed in US patents US 8,084,023, US 8,388,947, US 8,961,955, and international patent application WO 2016/070057, all of which are herein incorporated by reference.
• the methods include producing a 3D ECM by culturing bone marrow stromal cells, which can include BM-MSCs, on TCP or carriers such as microcarriers, to produce the ECM, followed by decellularizing (lysing and/or washing/removing) the cells from the ECM.
• the ECM comprises type I collagen, type III collagen, fibronectin, decorin, biglycan, perlecan, and laminin.
• the ECM comprises type I collagen, type III collagen, fibronectin, decorin, biglycan, perlecan, and laminin, and further comprises at least one of syndecan-1, collagen type V, or collagen type VI.
• the ECM comprises collagen alpha-l(XII), collagen alpha-3(VI), EMILIN-1, serpin HI, thrombospondin-1, tenascin precursor (TN) (Human), transforming growth factor-beta-induced protein, and vimentin.
• the ECM comprises collagen alpha-l(XII), collagen alpha-3(VI), EMILIN-1, serpin HI, thrombospondin-1, tenascin precursor (TN) (Human), transforming growth factor-beta-induced protein, vimentin, type I collagen, type III collagen, fibronectin, decorin, biglycan, perlecan, and laminin.
• the ECM comprises collagen alpha-l(XII), collagen alpha-3(VI), EMILIN-1, serpin HI, thrombospondin-1, tenascin precursor (TN) (Human), transforming growth factor-beta-induced protein, vimentin, type I collagen, type III collagen, fibronectin, decorin, biglycan, perlecan, laminin, and further comprises at least one of syndecan-1, collagen type V, or collagen type VI.
• Tissues produced in vitro yet retaining physiological features of in vivo tissues provide a particularly useful tool for monitoring the effects of proposed therapies or molecules on the physiological functions of the tissues. Accordingly, there is disclosed a method of testing the biological activity of a substance comprising obtaining any of the cell culture systems described above; adding the substance to the cell culture system; and measuring a parameter of the cell culture system or cells to determine the effect of adding the substance to the cell culture system.
• Adding the substance to the cell culture system can comprise adding the substance to the culture medium.
• the culture medium can be exchanged for a culture medium comprising a particular substance or combination of substances to monitor the effects of the culture medium change on the physiological functions of the cells.
• Measuring a parameter of the cell culture system can include, for example, observing growth rates or morphological features of cells. Any biologically relevant parameter can be measured and monitored to determine the biological effect of exposing the cells to a substance or of changing any conditions of growth. Changes in the parameter being measured or monitored can be attributed to the presence of the substance or the change in growth conditions if a corresponding control does not show the same change.
• the substance being tested is a candidate therapeutic to treat a disease and/or condition, including, for example, age-related disease or the aging process itself.
• the condition is reduced regenerative capacity of aging stem cell populations, or a side effect of a medication or radiotherapy.
• the disease and/or condition is caused by or compromises the quantity or quality of MSCs.
• the substance is a cellular growth factor or cellular differentiation factor.
• the inventors disclose that using the disclosed method allows one to isolate and expand a small subpopulation of less defective MSCs from bone marrow stromal cells of low quality and quantity, such as the cells of elderly people. Further, the inventors disclose novel methods to restore the regenerative capability of MSCs.
• BM-MSCs Bone marrow-derived mesenchymal stem cells
• the cells were sorted using flow cytometry based on size and SSEA-4 expression. Approximately 5% to 10% of the BM-MSCs harvested were small cells that are SSEA-4 positive (Small+). See FIG. 4 and FIG. 7.
• the inventors found that the Small+ enriched fraction after cell sorting was enriched in stem cells as determined by a colony forming units (CFU-F) assay compared to other subpopulations. The subpopulations were then plated on either ECM made by marrow stromal cells from young donors ( ⁇ 23 years old) (young ECM) or TCP.
• CFU-F colony forming units
• FIG. 20 illustrates an embodiment of the general approach explained above.
• FIG. 4A shows a brightfield microscopy of cells in culture at day 6.
• Young mesenchymal stem cells (MSCs) appear to have a greater number of cells and display a more spindle-like morphology relative to elderly cells. For elderly cells, there is no substantial difference in the total number of cells, nor the overall appearance for culture on TCP vs. ECM. This is substantiated by the density of cells after 7 days of culture (FIG. 4B).
• CFU-F colony forming unit -fibroblast
• AD -adipocyte
• -OB -osteoblast
• FIG. 4C colony formation and differentiation by elderly BM-MSCs is not substantially impacted by pre- expansion on ECM. Furthermore, colony formation in both groups is less than young BM- MSCs. In contrast, young BM-MSCs pre-expanded on ECM formed larger, denser, and more numerous colonies in each condition.
• Bone Marrow from Young Donors Bone Marrow from young donors was purchased from LONZA (Walkersville, MD, USA). Bone marrow samples were obtained from healthy, male donors under the age of 23. Fresh, unprocessed samples were shipped overnight on ice and upon receipt, red blood cells were lysed and mononuclear cells seeded onto TCP vessels (5xl0 5 cells/cm 2 ) in standard growth media (composition described below). When colonies began to form, media were removed, non-adherent cells washed away gently using PBS, and fresh media added. These cells were expanded for one or two passages (PI, P2) and used in the experiments.
• the cells were stored in liquid nitrogen at 2xl0 6 cells/mL in 20% serum containing media + 10% (v/v) dimethyl sulfoxide (DMSO).
• DMSO dimethyl sulfoxide
• Clinical samples were brought back to the lab within 3-4 hours of removal from the patient, cut into small pieces using sharp scissors at 4°C, and then digested for 30 minutes using collagenase type 2 (400 units/mL) dissolved in phosphate buffered saline (PBS) at 37°C with agitation. At completion, the digest was centrifuged (600 xg) for 5 minutes at 4°C and the supernatant removed. The pellet was then resuspended in isolation buffer and filtered through a 100 micron cell strainer to remove bone fragments. The bone fragments, collected on the cell strainer, were washed until bright white in color.
• collagenase type 2 400 units/mL
• PBS phosphate buffered saline
• Cell Culture - Bone-marrow MSCs were cultured in a humidified incubator at 37°C and 5% C0 2 . Unless indicated otherwise, cells were cultured in "standard growth media" containing a-Minimal Essential Medium (MEM), 15% pre-selected FBS (Atlanta Biologies, Flowery Branch, GA, USA), 2mM L-glutamine (Life Technologies, Grand Island, NY, USA), and 1% (v/v) streptomycin/penicillin (Life Technologies, Grand Island, NY, USA). One-half media changes were performed every three days during culture.
• MEM Minimum Essential Medium
• CFU-F CFU - fibroblast
• cells were seeded onto 6 well plates at 10 and 30 cells/cm 2 in standard growth media. When dense colonies formed (typically, culture day 10-14), media were removed, wells washed one time with PBS, and cells fixed at room temperature (100% methanol for 10 minutes). After fixation, cells were washed once with PBS and allowed to dry at room temperature for 10 minutes. Colonies were then stained with methyl violet (MP Biomedicals, Solon, OH, USA) for 10 minutes. Staining solution was collected for reuse and residual stain washed away with deionized water until the background appeared clear.
• methyl violet MP Biomedicals, Solon, OH, USA
• CFU - Adipocyte (AD) and Osteoblast (OB) assays cells were seeded onto 6 well plates at 20 and 60 cells/cm 2 .
• CFU-AD and CFU-OB cultures were started at the same time as those for CFU-F assays.
• CFU-AD and -OB plates were switched to their respective induction media.
• media were supplemented with 5 mM 3-isobutyl-L-methylxanthine (IBMX), 1 mM indomethacin, ⁇ dexamethasone, and 10 ⁇ g/mL insulin.
• CFU-AD and -OB plates were stained when colonies were visually observed to contain lipid or mineral, respectively, as observed by light microscopy. This was typically after 10-14 days for adipogenesis or 17-21 days for osteogenesis.
• CFU-AD cultures were stained by fixing the cells for 1 hour at room temperature with 10% formalin, followed by gentle washing with deionized water before staining with freshly prepared Oil Red O for 1 hour at room temperature. After staining, the solution was removed and the wells washed gently with deionized water until the background was clear.
• CFU-OB For CFU-OB, cells were fixed at room temperature for 1 hour using 10% formalin, washed with deionized water, and then stained with 1% silver nitrate (AgN0 3 ) overnight at room temperature under ultraviolet light. The next day, the AgN0 3 solution was removed and excess silver removed by treating with 5% sodium thiosulfate for 2 minutes. Wells were then washed with deionized water to prevent removal of excess stain.
• AgN0 3 silver nitrate
• Fluorescent Activated Cell Sorting - Fluorescein isothiocyanate (FITC) conjugated anti-SSEA-4 and IgG3 isotype control antibodies were purchased from BD Biosciences (San Jose, CA, USA).
• the gating strategy for selecting small and large BM-MSC populations was developed over a period of weeks using cells from a variety of young and elderly donors, with young donor cells serving as a control for small cells.
• FSC forward scatter
• Staining was performed on single cell suspensions (lOxlO 6 cells/mL) using antibody at lC ⁇ g/mL and incubation for 1 hour at 4°C. Cells were washed twice with isolation buffer and placed on ice ( ⁇ 3hrs) until subjected to sterile sorting using the FACSAria Cell Sorter from BD Biosciences (San Jose, CA, USA). The sorting was run using the FACSDiva software system.
• Flow Cytometry Mouse-anti-human non-conjugated antibodies were purchased from BD Biosciences (San Jose, CA, USA). Single cell suspensions (lxlO 5 in ⁇ .) were incubated for at least 1 hour at 4°C with primary antibody (lC ⁇ g/mL). Stained cells were washed twice with staining buffer (PBS + 5% v/v FBS + 0.01% m/v sodium azide) prior to incubation with FITC conjugated goat anti-mouse IgG for 30 minutes at 4°C. The cells were then washed twice with staining buffer and either immediately analyzed or fixed with freshly prepared 1% paraformaldehyde and analyzed within 72 hours.
• staining buffer PBS + 5% v/v FBS + 0.01% m/v sodium azide
• Adenosine Triphosphate - Adenosine triphosphate (ATP) levels were measured using the ATP Determination Kit (Molecular Probes, Eugene, OR, USA). 25,000 cells from test cultures were measured in triplicate and compared to an ATP standard curve created using an ATP standard supplied with the kit. Luminescence was due to the activity of firefly luciferase and an average ATP concentration for each sample was determined using the standard curve.
• Beta-Galactosidase - ⁇ -Galactosidase was measured using a 96-well Cellular Senescence Assay Kit (Cell Biolabs, San Diego, CA, USA).
• telomere adds telomeric repeats to the end of a biotin-labeled primer, and then the primer and elongation products are amplified by PCR.
• the PCR products are hybridized to digoxigenin-(DIG)-labeled detection probes, specific for the telomeric repeats, and then immobilized via the biotin label to a microplate. They are then detected with an antibody conjugated to horseradish peroxidase (HRP). Using absorbance values of the sample, as well as all of the experimental controls, one is able to calculate relative telomerase activities for different samples within a given experiment.
• HRP horseradish peroxidase
• Telomerase assays were performed in triplicate per independent experiment. Flow cytometry was performed once for each marker in each group in each independent experiment. All types of experiment were repeated at least 3 times using cells from different tissue donors. [00112] The replicate data for each assay were pooled (i.e. proliferation, CFU assays, Telomerase activity, ATP levels, and gene expression) and then analyzed. Mean, standard deviation, and confidence intervals were calculated and groups compared using ANOVA analysis and post-hoc tests when necessary.
• Differentiation capacity was calculated as proportions of CFU- AD and -OB to CFU-F, and confidence intervals determined using a modified Wald method.
• MSCs are known to be a heterogeneous population, and a number of recent studies in hematopoietic cells have described population level aging in cells as a shift in the clonal composition rather than aging of all cells individually. Extensive immunophenotyping was performed to understand the phenotypic composition of elderly BM-MSC populations relative to young. No differences were seen in traditional immunophenotypic markers for BM-MSCs; however, several differences in expression of markers correlated with sternness and aging via flow cytometry (FIG. 5C). Despite diminished metabolic activity, a significant number of elderly BM-MSCs have very high intracellular reactive oxygen species (ROS) content.
• ROS reactive oxygen species
• SSEA-4 has been used as a marker of potent MSCs, and may be a reliable marker of sternness (Sun, et al., 2011; Gang, et al., 2007; Kawanabe, et al., 2014; Pipino, et al., 2015). However, if this is true, one might expect that in populations of cells with a relatively small fraction of SSEA-4 (+) MSCs, the SSEA-4 phenotype would become the dominant phenotype by proliferating more quickly than other cells. In practice, this is not the case. In elderly MSC populations, SSEA-4 expression tends to decrease during serial passage, and the culture eventually fails.
• FIGs 3 and 5D The distribution of cell spread area and cell size (as measured by forward scatter in flow cytometry) of elderly and young BM-MSCs are shown in FIGs 3 and 5D, respectively. While the median size appears to be substantially larger in elderly MSCs relative to young, there is significant overlap, between the populations, and they appear to occupy a similar range.
• the BM-MSCs of small size from the elderly donors are similar in size to the size of the BM- MSCs found in young donors.
• conditioned media from elderly and young donors were analyzed using a cytokine microarray to test relative concentrations of 80 cytokines. 44 of the cytokines tested have been identified as part of the senescent associated secretory phenotype. As expected the conditioned media from elderly MSCs contained higher concentrations of most components of the senescence associated secretory phenotype. For 36 out of 44 cytokines (81.8%) were elevated in elderly BM-MSC populations. The difference in 19 out of those 36 cases was statistically significant. This data is summarized in FIG. 6C.
• Elderly BM-MSCs in contrast, are much more heterogeneous, and can easily be divided into four populations based on size and SSEA-4 expression (FIG. 7A).
• Elderly BM-MSCs were sorted into 4 populations to determine whether the small(+) sub-population of elderly BM-MSCs has youthful function and whether both these markers do correlate with cell function. See sorting strategy in FIG. 7B. After doublet discrimination, cells were separated into four populations based on expression of SSEA-4 (positive vs. negative), and cell size (small vs. large) as measured by FSC. In order to obtain populations with greater purity, cells with intermediate FSC or FITC intensity were discarded. By sorting, it was determine that only 8.24 + 3.63% of MSCs from individuals over the age of 65 possess a youthful phenotype when measured along these dimensions.
• Cell proliferation was also determined for unsorted, small size, and large size BM-MSCs from older donors by cell count. Small cells grew 65% more cells than large cells in the same period of time. Small cells also grew faster than cells that were unsorted by size (FIG. 9).
• ATP concentration, cell numbers with high levels of ATP, ATP content of cells, telomerase levels, and ⁇ -Galactosidase expression for the four sub-populations of BM- MSC cells from old donors were determined.
• the dashed line represents the mean ⁇ -Galactosidase expression of young BM- MSCs.
• Small BM-MSCs were statistically equivalent to young BM-MSCs.
• the evidence that small(+) cells have a low frequency of senescent cells fits the theory that they may represent a sub-population of elderly MSCs that in normal conditions are suppressed by their senescent neighbors.
• BM-MSCs were also determined for BM-MSCs from young donors grown in growth media conditioned with secretions from subpopulations of old donor's BM- MSCs with either small cell size (Small) or large cell size (Large), secretions from old donor's BM-MSCs unsorted by size (Unsorted), and secretions from young donor BM-MSCs (young CM). See FIG. 16.
• the proliferation rate of BM-MSCs from young donors was inhibited as determined by cell count when cultured in Large and Unsorted conditioned media while culturing in Small and Young conditioned media showed little to no difference. This suggests that the environmental factors that inhibit MSC growth in old donors may be at least partially dependent on harmful factors introduced by the large MSCs.
• BM-MSCs from young donors and small size, large size, and unsorted size BM-MSCs from old donors was determined by counting the number of colony forming units (CFU) before and after culturing on tissue culture plastic (TCP) and bone marrow extracellular matrix (BM-ECM). Culturing small cells from an older donor showed an increase in stem cells rescued in comparison to large and unsorted cells from an old donor. Further, culturing in BM-ECM showed an increase in stem cells in comparison to culturing on TCP (FIG. 17).
• CFU colony forming units
• TCP tissue culture plastic
• BM-ECM bone marrow extracellular matrix
• CFUs of BM-MSCs from young donors and small size, large size, and unsorted size BM-MSCs from elderly donors were also tested to determine their ability to differentiate as demonstrated by staining for markers of differentiation into fibroblast (CFU- F), osteoblast (CFU-OB), and adipocytes (CFU-AD).
• CFU- F fibroblast
• CFU-OB osteoblast
• CFU-AD adipocytes
• the CFUs of small cells from elderly donors showed similar differentiation ability to cells from young donors (FIG. 18).
• Small cells also showed an increase in proliferation rates in comparison to large size BM-MSCs and non-separated BM-MSCs.
• Small + cells showed increased cell proliferation on both TCP and young ECM.
• BM-ECM FIG. 19B, FIG. 23, and FIG. 24.
• Small(+) cells cultured on BM-ECM actually proliferated more rapidly than young cells expanded on TCP or BM-ECM (FIG. 19B and FIG. 24).
• the results suggest that young ECM increases proliferation rate of all of the subpopulations and cells from young donors. Further small cell size also seems to increase proliferation rates.
• Elderly BM-MSCs was determined after 4 passages. The cells were passaged every 7 days in culture on TCP vs. ECM derived from BM-MSCs from young donors (young ECM). The seeding density 1500 cells/cm 2 . The number of SSEA-4 positive cells after passages of BM- MSCs from young and elderly BM-MSCs and elderly subpopulations of BM-MSCs (Small +, Small -) on TCP or young ECM increased dramatically. Serial passage on young ECM resulted in a fold change of roughly 6,000 when small+ BM-MSCs were expanded on young ECM (FIG. 32).
• SSEA-4 is a marker of early-state MSCs.
• the number of SSEA-4 positive cells after 5 passages of BM-MSCs from young donors and small size, large size, and unsorted size BM-MSCs from old donors was determined after culturing on BM-ECM and tissue culture plastic (2D).
• Culturing small cells from an older donor showed an increase in SSEA-4 positive cells after 5 passages in comparison to large and unsorted cells from an old donor (FIG. 29).
• culturing in BM-ECM showed an increase in stem cells in comparison to culturing on 2D.
• SSEA-4 expression, ROS concentration, ATP content per cell, and telomerase levels was also determined for the four subpopulations of BM-MSCs isolated from elderly donors (S+, S-, L+, L-) as well as unsorted BM-MSC cells from young and elderly donors cultured on TCP or ECM derived from BM-MSCs from young donors. It was found that Small(+) cells have high expression of SSEA-4, and in most groups culture on young ECM increased SSEA-4 expression. See FIG. 25 and 31. Further, small BM-MSCs have lower intracellular ROS than other groups, and culture on young ECM further reduces mean intracellular ROS (FIG. 26 and 31).
• small (+) BM-MSCs cultured on TCP have significantly higher ATP concentration and culture on young ECM increases ATP concentration for most groups of cells (FIG. 27).
• small(+) cells have much higher telomerase activity (FIG. 28).
• ATP levels were increased on average when BM-MSCs were culture on ECM resulted in an increase in average ATP levels relative to culture on TCP for all groups (FIG. 30B). This difference was statistically significant in 3 of the 4 groups tested. Also, small (+) BM-MSCs cultured on TCP have significantly higher ATP concentration (FIG. 30B). [00142] Cytokine profiles of young, elderly and small(+) BM-MSCs were compared (FIG. 30C).
• small BM-MSCs were immunophenotyped following expansion on TCP and ECM and compared to young and elderly MSCs (TABLE 2 and FIG. 31).
• Small(+) BM- MSCs cultured on ECM surpassed all other groups in SSEA-4 expression, while having minimal levels of intracellular reactive oxygen species (ROS). While all groups cultured on ECM had similar expression of Annexin-5, it is of note, that small(+) BM-MSCs expressed significantly lower levels on ECM relative to TCP. Together, flow cytometry shows a substantial reversal in age-related phenotypic changes in the small(+) BM-MSC group.
• Small (+) BM-MSCs cultured on ECM have higher SSEA-4 expression and lower ROS and annexin-5 relative to TCP culture.
• ROS reactive oxygen species
• the inventors predict that the differentiation capacity and efficacy for tissue repair and regeneration will be increased in Small+ cells in comparison to other subpopulations of MSCs and in comparision to unsorted MSCs. It is further predicted that cells, and especially Small+ cells cultured on ECM will have a greater differentiation capacity and efficacy for tissue repair and regeneration than cells cultured on TCP.
• Cell differentiation capacity of isolated MSCs can be determined by methods well known by one of skill in the art. It is expected that Small+ cells will show an increased cell differentiation capacity over other subpopulations of MSCs and in comparision to unsorted MSCs. Further it is expected that cells and especially Small+ cells cultured on young ECM will have a greater increase in cell differentiation capacity than Small+ cells cultured on TCP.
• Tissue repair and regeneration of isolated -MSCs can be determined by methods well known by one of skill in the art. It is expected that Small+ cells will show an increased efficacy for tissue repair and regeneration over other subpopulations of MSCs and in comparision to unsorted MSCs. Further it is expected that cells and especially Small+ cells cultured on young ECM will have a greater increase in efficacy for tissue repair and regeneration than Small+ cells cultured on TCP.

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