WO2019213796A1

WO2019213796A1 - INDUCED REJUVENATED MESENCHYMAL STEM CELLS (irMSCs) AND USES THEREOF

Info

Publication number: WO2019213796A1
Application number: PCT/CN2018/085752
Authority: WO
Inventors: Min Hu; Yi Sun
Original assignee: The Regents Of The University Of California
Priority date: 2018-05-05
Filing date: 2018-05-05
Publication date: 2019-11-14

Abstract

Provided a method to produce induced rejuvenated mesenchymal stem cells(irMSCs) from a primary fibroblast. Also provided are pharmaceutical compositons comprising at least one irMSC, and methods to use irMSC for treating bone injury or promoting bone repair in a subject, or for eliciting cartilage regeneration.

Description

INDUCED REJUVENATED MESENCHYMAL STEM CELLS (irMSCs) AND USES THEREOF

TECHNICAL FIELD

The present disclosure provides methods to convert aged human fibroblasts into youthful MSC-like cells with high efficiency (>95%) : these cells are referred to as “induced and rejuvenated” MSCs (irMSCs) . The methods and irMSCs are useful for rejuvenating somatic cells, allowing them to acquire stem cell activities, and thus provide a novel path to autologous tissue repair as well as age reversal. The methods and irMSCs described herein are thus useful to treat conditions associated with aging and degeneration of various tissues both in vitro and in vivo, and particularly for treating bone injury and for eliciting bone repair or cartilage regeneration.

BACKGROUND

In the following discussion, certain articles and methods are described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.

Humankind has always been intrigued by the quest for true age reversal. Stem cells are considered the “holy grail” for regenerative and anti-aging medicine. When an individual ages, all cells including stem cells in the body also age, resulting in degeneration or deterioration of multiple organs including bone, cartilage, heart, muscle, brain, skin, pancreases, liver, kidney, gastrointestinal tract, and etc. Abnormal immune system function is also associated with aging. In fact, chronic inflammation of organs and tissues is thought to significantly contribute to their degeneration. Aged cells often have accumulated damages and/or mutations to DNA, shortened telomeres, abnormal epigenetic, redox, and energy metabolic states, reduced cell growth potentials, and increased cell death. Aged stem cells appear to have one additional feature. They tend to lose the potential to differentiate into certain lineages while biasing towards others. For example, it is well known that bone-marrow-derived mesenchymal stem cells (bMSCs) from aged individuals have reduced propensity to differentiate into bone and cartilage and increased propensity towards differentiation into adipocytes. Therefore, aged bone marrow is often referred to as “yellow marrow” , which is filled with adipose tissues. Similarly, aged neural stem cells (NSCs) also decrease the potential to differentiate into neurons while increasing tendency toward astrocyte differentiation, which is believed to contribute to cognitive decline in aged individuals.

Stem cells, particularly MSCs have emerged as a promising candidate for therapeutic intervention, due to their accessibility, expandability, multipotentiality, growth factor release, and potential to modulate tissues’immune responses. MSCs have been explored for a broad spectrum of clinical conditions, including graft-versus-host disease (GVHD) , multiple sclerosis (MS) , amyotrophic lateral sclerosis (ALS) , spinal cord injury (SCI) , lupus, arthritis, and aging-related conditions. For allogeneic usages, umbilical cord-derived MSCs are widely considered. However, long-term or repetitive use of “non-self” cells is also recognized as a potential clinical risk. Autologous stem cells are considered safer, but unfortunately, when people age, their MSCs also age. Aged MSCs lose certain important functions, which preclude their clinical applications. Induced pluripotent stem cells (iPSCs) , which are youthful cells that can be derived from old individuals, had once been thought to be a good autologous cell source for therapeutic usage. However, since the iPSC induction process is a low-frequency event and often involves the introduction of exogenous genes, genetic alterations frequently occur during the iPSC induction process, creating huge safety concerns for their clinical applications. Recently genetic modification has been explored as a means of rejuvenating cells, but this method is still associated with risks including off-target effects and tumorigenicity.

Described herein is a step-wise serial treatment protocol with “cocktails” of small molecule compounds, which can convert aged human fibroblasts into youthful MSC-like cells with high efficiency (>95%) : these cells are referred to as “induced and rejuvenated” MSCs (irMSCs) . IrMSCs can self-renew, and can differentiate into bone, cartilage, and fat. More importantly, irMSCs exhibit signs of regained youth, by manifesting increased telomere length, increased proliferation potential, and regained bone and cartilage differentiation propensity, as well as decreasing expression of a battery of age-related biomarkers. In addition, irMSCs from either young or aged donors exhibit superb bone and cartilage repair functions in vivo. These cells also exert extraordinary immune regulatory functions during liver damage repair in vivo. Most strikingly, intravenous transfusion of irMSCs into old-aged NOD/SCID mice led to rejuvenation of multiple organs and prolonged lifespan. Through transcriptomic analyses, pathways leading to rejuvenation and acquisition of tri-lineage differentiation potentials from fibroblasts were identified. It has been shown that down regulation of STAT5 crucially participated in the “induction and rejuvenation” process. Comparing to iPSCs, irMSCs are less primitive and not tumorigenic, therefore they possess better safety features as therapeutic agents. The methods described herein are thus effective to rejuvenate somatic cells, allowing them to acquire stem cell activities, and providing a novel method to promote autologous tissue repair as well as age reversal.

BRIEF SUMMARY

The summary is not intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the detailed description including those aspects disclosed in the accompanying drawings and in the appended claims.

Human aging occurs at the cellular level including stem cells (SCs) , which greatly limits autologous SC applications in elderly individuals, unless youthful SCs were preserved at a young age. Here we describe in vitro effective chemical conversion of fibroblasts from young and aged donors into youthful mesenchymal SC-like cells, which are referred to herein as “induced and rejuvenated” MSCs (irMSCs) . The induction method appeared to erase aging properties and reset irMSCs from various aged donors to a juvenile state with lengthened telomeres, enhanced growth and tri-lineage differentiation potentials, reduced aging gene expression, as well as down-regulation of age-related DNA damage and epigenetic markers γH2Ax and H4K20me3. IrMSCs, but not bone-marrow-derived MSCs, from aged donors are capable of bone and cartilage repair in vivo without tumor formation. Moreover, intravenous transfusion of irMSCs into old-aged NOD/SCID mice led to rejuvenation of multiple organs and prolonged lifespan. The irMSCs of the invention are thus useful to increase youthful autologous cells and promote age-reversal as well as for repair or rejuvenation of particular tissues or organs.

The following enumerated embodiments are representative of the invention.

1. A process for preparing induced and rejuvenated mesenchymal stem cells (irMSCs) , which process comprises:

a) contacting a primary fibroblast, e.g., a dermal fibroblast, with

i) a first molecule cocktail to activate an activity of Wnt and/or cAMP/PKA, and/or to inhibit an activity of TGFβ and/or HDAC in said primary fibroblast, or

ii) a first small molecule cocktail, e.g., Mix V, comprising at least one of valproic acid (VPA) , CHIR99021, Repsox and Forskolin, e.g., 1, 2, 3, or 4 of valproic acid (VPA) , CHIR99021, Repsox and Forskolin,

for a first time to form a first treated cell;

b) contacting said first treated cell with:

i) a second molecule cocktail to activate an activity of Wnt, cAMP/PKA and/or RAR, and/or to inhibit an activity of TGFβ, HDAC, PKC and/or Rho in said first treated cell, or

ii) a second small molecule cocktail, e.g., Mix P, comprising at least one of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox,

for a second time to form a second treated cell; and

c) culturing said second treated cell in a mesenchymal stem cell (MSC) medium without adding exogenous or in the absence of bFGF, PDGF-AB and BMP4 for a third time to form irMSC.

2. The process of embodiment 1, wherein the primary fibroblast is generated from a skin biopsy specimen, urine or blood.

3. The process of embodiment 2, wherein the skin biopsy specimen is cultured for at least 6 passages to generate the primary fibroblast.

4. The process of any one of embodiments 1-3, wherein the step a) comprises contacting aprimary fibroblast with a first small molecule cocktail, e.g., Mix V, comprising at least one of valproic acid (VPA) , CHIR99021, Repsox and Forskolin, e.g., 1, 2, 3, or 4 of valproic acid (VPA) , CHIR99021, Repsox and Forskolin, for a first time to form a first treated cell. In some of these embodiments, Mix V comprises at least two compounds, or at least three compounds, or all four compounds selected from valproic acid (VPA) , CHIR99021, Repsox and Forskolin.

In some of these embodiments of embodiment 4, Mix V comprises a level of VPA up to 1 mM, typically about 0.25 mM to about 1.0 mM, or 0.1 mM to 0.7 mM, and preferably about 0.5 mM.

In some of the embodiments of embodiment 4, Mix V comprises a level of CHIR99021 up to about 6 μM, typically 0.5 μM to 5 μM; and preferably 2-4μM. In certain of these embodiments, the level of CHIR99021 is 3 μM or about 3 μM.

In some embodiments, Mix V in embodiment 4 comprises a level of Repsox up to about 5 uM, often the level is about 0.5 μM to about 5 μM, and preferably the level of Repsox is 1μM or about 1 μM.

In some embodiments within embodiment 4, Mix V comprises a level of forskolin up to about 20 μM forskolin; optionally the level of forskolin is between 5 μM to 20 μM; and preferably it is 10 μM or about 10 μM.

5. The process of embodiment 4, wherein the VPA has a level ranging from about 0.1 mM to about 0.7 mM.

6. The process of

embodiment

4 or 5, wherein the CHIR99021 has a level ranging from about 0.5 μM to about 5μM.

7. The process of any one of embodiments 4-6, wherein the Repsox has a level ranging from about 0.5 μM to about 5μM.

8. The process of any one of embodiments 4-7, wherein the Forskolin has a level ranging from about 1μM to about 20 μM.

9. The process of any one of embodiments 4-8, wherein the step a) comprises contacting a primary fibroblast with a first small molecule cocktail, e.g., Mix V, for a first time ranging from about 1 day to about 5 days to form a first treated cell.

10. The process of any one of embodiments 1-9, wherein the step b) comprises contacting said first treated cell with a second small molecule cocktail, e.g., Mix P, comprising at least one of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox, for a second time to form a second treated cell.

In some embodiments within embodiment 10, Mix P comprises at least 3, or at least 4, or at least 5, or at least 6 compounds selected from valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox.

In some embodiments within embodiment 10, Mix P comprises a level of VPA up to about 2 mM; optionally the level of VPA is between 0.2 mM and 1.0 mM; and preferably it is 0.5 mM or about 0.5 mM.

In some embodiments within embodiment 10, Mix P comprises a level of Vc up to about 1 mM; optionally the level of VPA is between 0.05 mM and 0.8 mM; and preferably it is 0.2 mM or about 0.2 mM.

In some embodiments within embodiment 10, Mix P comprises a level of CHIR99021 up to about 10μM; optionally the level is between 1μM and 8μM; and preferably it is 3μM or about 3μM.

In some embodiments within embodiment 10, Mix P comprises a level of forskolin up to about 25 μM; optionally the level is between 5 μM and 25 μM; and preferably it is 10 μM or about 10 μM.

In some embodiments within embodiment 10, Mix P comprises a level of Repsox up to about 5 μM; optionally the level is between 0.1 μM and 2 μM; and preferably it is 1 μM or about 1 μM.

In some embodiments within embodiment 10, Mix P comprises a level of SP600125 up to about 25 μM; optionally the level is between 1 μM and 25 μM; and preferably it is 10 μM or about 10 μM.

In some embodiments within embodiment 10, Mix P comprises a level of Go6983 up to about 25 μM; optionally the level is between 1 μM and 25 μM, or between 1 μM and 10 μM; and preferably it is 5 μM or about 5 μM.

In some embodiments within embodiment 10, Mix P comprises a level of Y-27632 up to about 25 μM; optionally the level is between 1 μM and 15 μM, or between 1 μM and 10 μM;and preferably it is 5 μM or about 5 μM.

In some embodiments within embodiment 10, Mix P comprises a level of TTNPB up to about 25 μM; optionally the level is between 1 μM and 20 μM, or between 1 μM and 10 μM; and preferably it is 5 μM or about 5μM.

In some embodiments within embodiment 10, Mix P comprises a level of AM580 up to about 1 mM; optionally the level is between 0.01 μM and 0.8 μM, or between 0.02 μM and 0.1μM; and preferably it is 0.05 μM or about 0.05 μM.

In some embodiments within embodiment 10, Mix P comprises a level of EPZ004777 up to about 20 μM; optionally the level is between 1 μM and 20 μM, or between 1 μM and 10 μM; and preferably it is 5 μM or about 5 μM.

11. The process of embodiment 10, wherein the VPA has a level ranging from about 0.2 mM to about 1 mM.

12. The process of

embodiment

10 or 11, wherein the Vc has a level ranging from about 0.05 mM to about 0.8 mM.

13. The process of any one of embodiments 10-12, wherein the CHIR99021 has a level ranging from about 1 μM to about 8μM.

14. The process of any one of embodiments 10-13, wherein the Forskolin has a level ranging from about 5 μM to about 25μM.

15. The process of any one of embodiments 10-14, wherein the SP600125 has a level ranging from about 1μM to about 25μM.

16. The process of any one of embodiments 10-15, wherein the Go6983 has a level ranging from about 1μM to about 25μM.

17. The process of any one of embodiments 10-16, wherein the Y-27632 has a level ranging from about 1 μM to about 15μM.

18. The process of any one of embodiments 10-17, wherein the TTNPB has a level ranging from about 1μM to about 20 μM.

19. The process of any one of embodiments 10-18, wherein the AM580 has a level ranging from about 0.01μM to about 0.8μM.

20. The process ofany one of embodiments 10-19, wherein the EPZ004777 has a level ranging from about 1μM to about 10 μM.

21. The process of any one of embodiments 10-20, wherein the RepSox has a level ranging from about 0.1μM to about 2 μM.

22. The process of any one of embodiments 10-21, wherein the step b) comprises contacting said first treated cell with a second small molecule cocktail, e.g., Mix P, for a second time ranging from about 1 day to about 7 days to form a second treated cell.

23. The process of any one of embodiments 1-22, which further comprises, before step c) , a wash step to reduce or remove components of the first molecule cocktail, the first small molecule cocktail, the second molecule cocktail or the second small molecule cocktail.

24. The process of any one of embodiments 1-23, wherein the MSC medium used in step c) is a fresh MSC medium, e.g., a MSC medium that is not conditional or that has not been used for culturing cells before.

25. The process of any one of embodiments 1-24, wherein the MSC medium used in step c) comprises serum, e.g., MSC medium from Cyagen, catalog No. HUXMA-90011, or GMP compatible serum-free MSC medium, e.g., MSC medium from Hcell, name rFib medium, catalog No. CRM0016-01.

26. The process of any one of embodiments 1-25, wherein the step c) comprises culturing said second treated cell in a MSC medium without adding exogenous or in the absence of bFGF, PDGF-AB and BMP4 for a third time that is at least about 3 days to form irMSC.

27. The process of any one of embodiments 1-26, which is used to form a single irMSC.

28. The process of any one of embodiments 1-26, which is used to form a plurality or population of irMSC.

29. The process of any one of embodiments 1-28, which is used to form a non-human mammalian irMSC, e.g., a mouse irMSC.

30. The process of embodiment 29, wherein the hon-human mammalian irMSC is formed from a primary fibroblast obtained from a non-human mammal having an age of at least about 5 weeks.

31. The process of any one of embodiments 1-28, which is used to form a human irMSC.

32. The process of embodiment 31, wherein the human irMSC is formed from a primary fibroblast obtained from a human having an age of at least about 5 years.

33. An irMSC prepared by a process of any one of embodiments 1-32.

34. The irMSC of embodiment 33, which is prepared from a primary fibroblast of a human.

35. The irMSC of embodiment 34, which is prepared from a primary fibroblast of a human having an age of at least 5 years.

36. The irMSC of any one of embodiments 33-35, wherein a fibroblast marker, e.g., fibroblast-specific protein-1 (FSP-1) , is down-regulated.

37. The irMSC of any one of embodiments 33-36, wherein a MSC-related cell surface marker, e.g., CD49a and/or CD49e, is up-regulated.

38. The irMSC of embodiment 37, wherein both CD49a and CD49e are up-regulated.

39. The irMSC of any one of embodiments 33-38, which is configured to suppress T cell proliferation, e.g., to suppress T cell proliferation in vitro.

40. The irMSC of any one of embodiments 33-39, which acquires self-renewal, tri-lineage differentiation and/or immune modulation properties comparable to that of a bone-marrow-derived mesenchymal stem cell (bMSC) .

41. The irMSC of any one of embodiments 33-40, wherein an aging epigenetic marker histone, e.g., H4 lysine 20 trimethylation (H4K20me3) and/or age-related DNA damage marker, e.g., phosphorylated histone H2AX (γH2AX) is downregulated.

42. The irMSC of any one of embodiments 33-41, which has a greater proliferative capacity than its parental fibroblast.

43. The irMSC of any one of embodiments 33-42, which grows more rapidly than autologous bMSC.

44. The irMSC of any one of embodiments 33-43, which grows faster than bMSC from a younger donor.

45. The irMSC of any one of embodiments 33-44, wherein expression of an aging marker, e.g., cyclin dependent kinase inhibitor (CDKN) 1A, activating transcription factor (ATF) 3, interleukin (IL) -6, growth arrest and/or DNA damage-inducible 45 beta (GADD45B) is reduced.

46. The irMSC of embodiment 45, wherein expression of the aging marker remains after the irMSC differentiates along the osteogenic or chondrogenic lineages.

47. The irMSC of any one of embodiments 33-46, which has longer telomeres than the parental fibroblast and autologous bMSC.

48. The irMSC of embodiment 47, which maintains the telomere lengthening after its lineage differentiation.

49. The irMSC of any one of embodiments 33-48, which is capable of osteogenic lineage differentiation and has reduced adipogenesis, comparable to bMSC from a young donor.

50. The irMSC of any one of embodiments 33-49, which has a higher expression level of osterix (OSX) and/or osteoprotegerin (OPG) than its autologous bMSC.

51. The irMSC of any one of embodiments 33-50, which behaves like younger bMSC upon differentiation into chondrocyte.

52. The irMSC of any one of embodiments 33-51, which does not express a pluripotency gene, e.g., Oct4.

53. The irMSC of any one of embodiments 33-52, which does not form tumor for at least 30 days after transplantation into a mammal.

54. The irMSC of any one of embodiments 33-53, which comprises lengthened telomeres as compared to its parental fibroblast. Preferably, telomere length increases by at least 10%or at least 20%or at least 30%.

55. The irMSC of any one of embodiments 33-54, which is at stage of at least 5 passages and expresses a lower level of telomerase reverse transcriptase (TERT) as compared to the newly formed irMSC.

56. A population of irMSCs of any one of embodiments 33-55.

57. A pharmaceutical composition comprising an effective amount ofat least one irMSC of any one of embodiments 33-55 or a population of irMSCs of embodiment 56, admixed with at least one pharmaceutically acceptable carrier or excipient.

58. A method, which method comprises administering to a subject in need thereof an effective amount of an irMSC or a population of irMSCs. In these methods, the irMSC is preferably autologous, i.e., the irMSC for use in this method is obtained from the subject to be treated. In some of these embodiments, the irMSC comprises a detectable amount of at least one compound selected from valproic acid (VPA) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox. In some of these embodiments, the irMSC is administered intravenously, such as by injection or infusion. In some of these embodiments, the subject is one in need of treatment for bone injury or bone loss or cartilage damage.

59. The method of embodiment 58, wherein the subject is a non-human mammal.

60. The method of embodiment 58, wherein the subject is a human.

61. A method of any one of embodiments 58-60, which is used for treating bone injury or promoting bone repair in a subject.

62. The method of embodiment 61, wherein the irMSCs induce bone healing by cell-replacement in the subject.

63. The method of embodiment 61 or 62, wherein the irMSCs elicit bone tissue regeneration in the subject.

64. A method of any one of embodiments 61-63, wherein the irMSCs have more efficacy than its autologous bMSC.

65. A method of any one of embodiments 61-64, wherein the irMSCs are derived from an older donor and have an efficacy comparable to that of irMSCs derived from a younger donor.

66. A method of any one of embodiments 58-65, which is used for treating bone injury or eliciting cartilage regeneration in a subject.

67. The method of embodiment 66, wherein the irMSCs generate new cartilage in a subject.

68. The method of embodiment 67, wherein the new cartilage is positive for SOX9.

69. The method of any one of embodiments 66-68, wherein the irMSCs develop a proteoglycan-and type II collagen–rich extracellular matrix for cartilage, with minimal or no calcification or mineralization in a subject.

70. The method of any one of embodiments 66-69, wherein the irMSCs contribute to cartilage repair through differentiation into chondrocytes for cell replacement in a subject.

71. The method of any one of embodiments 61-70, wherein the irMSCs are administered to an injury or local site in a subject.

72. A method of any one of embodiments 58-71, which is used for slowing or reversing an aging related characteristic in a subject.

73. A method of any one of embodiments 58-72, which is used for reversing tissue degeneration and/or prolonging lifespan in a subject.

74. The method of embodiment 72 or 73, wherein the irMSCs are administered intravenously, e.g., via intravenous transfusion, to a subject.

75. The method of any one of embodiments 72-74, wherein the irMSCs are administered to a subject in a single dose.

76. The method of any one of embodiments 72-74, wherein the irMSCs are administered to a subject in multiple single doses.

77. The method of any one of embodiments 72-76, wherein the irMSCs are administered to a subject in a dose of at least from about 10 ⁶ cells to about 10 ⁸ cells.

78. The method of any one of embodiments 72-77 wherein the lifespan of the treated subject is substantially extended, e.g., extended by about 5%to about 20%compared to an average lifespan of a demographically matched subject.

79. The method of any one of embodiments 72-78 wherein the treated subject shows improvement in its appearance, e.g., improvement of the quality of its fur coat and/orreduced spine curvature as compared to a control (untreated) subject or a subject in need of such treatment.

80. The method of any one of embodiments 72-79 wherein the treated subject shows more motor activities as compared to a control (untreated) subject.

81. The method of any one of embodiments 72-80 wherein the treated subject has digestive system resembling that of a comparable younger subject or an untreated subject.

82. The method of any one of embodiments 72-81 wherein the stomach tissue of the treated subject has more regular gastric mucosa as compared to a control subject or an untreated subject.

83. The method of any one of embodiments 72-82 wherein the age-related loss of parietal cells is halted or reversedby irMSC administration, e.g., transfusion.

84. The method of any one of embodiments 72-83 wherein the treated subject shows one or more signs of rejuvenation.

85. The method of any one of embodiments 72-84 wherein the irMSC has one or more of (at least one of, and optionally two or more, or three or more of) the following properties:

1) which is prepared from a primary fibroblast of a human;

2) which is prepared from a primary fibroblast of a human having an age of at least 5 years;

3) wherein a fibroblast marker, e.g., fibroblast-specific protein-1 (FSP-1) , is down-regulated;

4) wherein a MSC-related cell surface marker, e.g., CD49a and/or CD49e, is up-regulated;

5) wherein both CD49a and CD49e are up-regulated;

6) which is configured to suppress T cell proliferation, e.g., to suppress T cell proliferation in vitro;

7) which acquires self-renewal, tri-lineage differentiation and/or immune modulation property comparable to that of a bone-marrow-derived mesenchymal stem cell (bMSC) ;

8) wherein an aging epigenetic marker histone, e.g., H4 lysine 20 trimethylation (H4K20me3) and/or age-related DNA damage marker, e.g., phosphorylated histone H2AX (γH2AX) is downregulated;

9) which has a greater proliferative capacity than its parental fibroblast;

10) which grows more rapidly than autologous bMSC;

11) which grows faster than bMSC from a younger donor;

12) wherein expression of an aging marker, e.g., cyclin dependent kinase inhibitor (CDKN) 1A, activating transcription factor (ATF) 3, interleukin (IL) -6, growth arrest and/or DNA damage-inducible 45 beta (GADD45B) is reduced;

13) wherein expression of the aging marker remains after the irMSC differentiates along the osteogenic or chondrogenic lineages;

14) which has longer telomeres than the parental fibroblast and autologous bMSC;

15) which maintains the telomere lengthening after its lineage differentiation;

16) which is capable of osteogenic lineage differentiation and has reduced adipogenesis, comparable to bMSC from a young donor;

17) which has a higher expression level of osterix (OSX) and/or osteoprotegerin (OPG) than its autologous bMSC.

18) which behaves like younger bMSC upon differentiation into chondrocyte;

19) which does not express a pluripotency gene, e.g., Oct4;

20) which does not form tumor for at least 30 days after transplanted into a mammal;

21) which comprises lengthened telomeres as compared to its parental fibroblast; and/or

22) which is at stage of at least 5 passages and expresses a lower level of telomerase reverse transcriptase (TERT) as compared to the newly formed irMSC. In some embodiments, the irMSC possesses or exhibits at least two, or preferably at least three of the listed characteristics.

86. The method of any one of embodiments 72-85 wherein the irMSC of any of claims 33-55 or a population of irMSCs of embodiment 56 are administered to a subject.

87. Use of the irMSC of any of embodiments 33-55 or a population of irMSCs of embodiment 56 for the manufacture of a medicament.

88. A method for facilitating or promoting fibroblasts to differentiate into bone and cartilage, and/or for preparing induced and rejuvenated mesenchymal stem cell (irMSC) , which method comprises deleting or inactivating STAT5A gene, reducing or blocking expression of STAT5A gene, and/or removing or inactivating STAT5A gene product, e.g., protein encoded by said STAT5A gene, in fibroblasts.

89. The method of embodiment 88, which is conducted in vitro.

90. The method of embodiment 88, which is conducted in vivo.

91. The method of any one of embodiments 88-90 which comprises knocking out or reducing activity ofSTAT5A in fibroblasts, e.g., by homologous recombination, site-specific nuclease cleavage, Zinc-finger nuclease (ZFN) cleavage, transcription activator-like effector nucleases (TALEN) cleavage, and/or clustered regularly interspaced short palindromic repeats (CRISPR) technology.

92. A kit comprising at least one irMSC packaged with means to administer the irMSC to a subject in need thereof, e.g., a hypodermic syringe, and/or instructions for administration of the irMSC. Preferably, the irMSC is produced by the method of any of embodiments 1-33.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Induction of IrMSCs from Skin Fibroblasts.

1. FIG. 1A. Schematic representation of the protocol used for irMSC conversion from skin fibroblasts.

2. FIG. 1B. Growth curve of irMSCs during long-term expansion relative to parental fibroblasts.

3. FIG. 1C. Histochemical analysis of tri-lineage differentiation of various cells before and after conversion (parental fibroblasts were at P8 from a 39-year-old donor) .

4. FIG. 1D. q-RT-PCR analysis of ALP levels of various cells upon osteogenic differentiation.

5. FIG. 1E. Mixed lymphocyte reaction assay.

6. FIG. 1F. Absolute numbers of T lymphocytes in liver tissues determined by flow cytometry.

7. FIG. 1G. tSNE analysis of global gene expression in parental fibroblasts, D5 cells, D12 cells, D15 irMSCs, andbMSCs, as well as hESCs and iPSCs.

8. FIGs 1H-1I. Tri-lineage differentiation potential: tri-lineage differentiation of the cells induced by mix V and mix P without addition of three growth factors by Day 15 (FIG. 1H) and generation of osteoblasts, chondrocytes, and adipocytes from irMSCs from a 38-year-old donor (FIG. 1I) .

9. FIG. 1J. Cell Expansion During Conversion of Fibroblasts into IrMSCs.

10. FIGs. 1 K-1M. Immunohistochemical and Flow Cytometric Analyses of D0 (Fib) , D5, D12, D15 (irMSCs) Cells and after Passaging. Immunohistochemical analysis showing the absence of fibroblast-specific protein (FSP) -1 expression in irMSCs (FIG. 1K) . Flow cytometric analysis showing the progressive expression of CD49e and CD49a by fibroblast treatment with small molecule cocktails. Representative density plots are shown (FIG. 1L) . The proportion of CD49a+ and CD49e+ cells at passages 9 and 13 determined by flow cytometry (FIG. 1M) .

11. FIG. 1N. Flow Cytometric Analysis of Fibroblasts, bMSCs and IrMSCs.

12. FIGs. 1O-1P. Compound Mix V and Mix P are Toxic to bMSCs and IrMSCs, but not Fibroblasts. β-Galactosidase activity in bMSCs, irMSCs, and fibroblasts before and after (Mix V + Mix P) treatment for 12 days (FIG. 1O) . Cell counts of Day 0 (untreated) , Day 12 after (Mix V+Mix P) treatment cultures (FIG. 1P) .

13. FIGs. 1Q-1T. Immune Suppression by IrMSCs.

14. FIGs. 1U-1W. Immune Suppression of IrMSCs in Vivo and Liver Protection. Liver morphology and H&E staining (FIG. 1P) . Serum levels of ALT and AST in ConA-treated and untreated mice (FIGs. 1V-1W) .

Figure 2. Rejuvenated Features of IrMSCs Compared to Fibroblasts and bMSCs.

1. FIG. 2A. Immunofluorescence of H3K9me3 and H4K20me3 levels in D0 (Fib, parental fibroblasts at P11) and D15 (irMSCs) .

2. FIG. 2B and 2C. Immunofluorescence detection and quantification of γH2AX foci at D0 (Fib, P11) and D15 (irMSCs) .

3. FIG. 2D. Growth curve of fibroblasts, bMSCs and irMSCs during long-term expansion.

4. FIGs. 2E. to 2G: q-RT-PCR analysis of CDKN1A, ATF3 and IL-6 expression in D0 (Fib) .

5. FIG. 2H. Relative telomere length.

6. FIG. 2I. Growth Curve of IrMSC from Different aged donors.

7. FIG. 2J. IrMSCs do not Senescence after Long-term Passage.

8. FIG. 2K. Gene Expression of GADD45B in Fibroblasts, bMSCs and IrMSCs.

9. FIG. 2L. q-PCR Analysis of OSX and OPG in Osteoblasts Derived from bMSCs and IrMSCs.

10. FIG. 2M. ESCs, but not Parental Fibroblasts, bMSCs or IrMSCs Express the Pluripotent Gene Oct4.

11. FIG. 2N. IrMSCs Remain Stable Through Long-term Passaging.

Figure 3. Aged bMSCs were Worse at Bone and Cartilage Differentiation than Autologous IrMSCs, which are not Tumorigenic.

1. FIG. 3A. Alizarin Red staining of osteoblasts derived from bMSCs and irMSCs from different aged donors.

2. FIG. 3B: Oil Red O staining of adipocytes derived from bMSCs and irMSCs from donors of different ages.

3. FIGs. 3C and 3D: q-RT-PCR analysis of ALP (osteogenic marker) and PPARG (adipogenic marker) expression, respectively, in osteoblasts and adipocytes derived from bMSCs and irMSCs from donors with different ages.

4. FIG. 3E. Immunofluorescence analysis of Col2a1 and MMP13 expression in chondrocytes derived from bMSCs and irMSCs from donors of different ages.

5. FIGs. 3F and 3G. q-RT-PCR analysis of Col2a1 and MMP13 in chondrocytes derived from bMSCs and irMSCs.

6. FIG. 3H. Typical Karyotypes of irMSCs at Passages 9 and 13 and of Parental Fibroblasts at Passages 6.

7. FIG. 3I. Teratoma formation assay of irMSCs showing negative results.

8. FIG. 3J. Analysis of telomere length and TERT gene expression in the same cultures from various conditions and passages.

Figure 4. IrMSCs Promote Bone Repair in Vivo. FIG. 4A.

1. FIG. 4A. Aschema for femur bone injury used in the study.

2. FIG. 4B. Macroscopic images and HE staining indicating femur bone injury and repair.

3. FIG. 4C. IrMSCs (from a 39 year old donor, at passage 6) with Hoechst 33342-stained. FIG. 4D. Micro-CT imaging of femur injury and repair after irMSC transplantation.

4. FIG. 4E. BV/TV, Tb. N, and BMD were determined from micro-CT measurements.

5. FIGs. 4F-G. Human Specific Antigen Detection of Human Implants. IrMSCs with Hoechst 33342-stained nuclei (FIG. 4F) . New bone formed by irMSC (FIG. 4G) .

Figure 5. In Vivo Cartilage Formation by IrMSC.

1. FIG. 5A. Macroscopic images of gross patellar groove 28 days post-implantation.

2. FIG. 5B. Histological analysis of sections through knee cartilage by Safranin (Saf-) O and Fast Green staining.

3. FIG. 5C. Cartilage repair.

4. FIG. 5D. IrMSCs with Hoechst 33342-stained nuclei forming hyaline cartilage in the patellar groove.

5. FIGs. 15E-15G. Characteristics of Cartilage Formed by IrMSCs. The repaired patellar groove area (FIG. 15E) . Immunofluorescence analysis of Col2a1 and aggrecan (ACAN) expression in newly formed cartilage in irMSC implanted group (FIGs. 15F-15G) .

6. FIGs. 15H-15I. Cartilage Repair by IrMSCs. New chondrogenic tissue formed by irMSCs (FIG. 15H) . The repaired area of the patellar groove (FIG. 15I) .

Figure 6. IrMSCs Extend the Lifespan of Aged NOD/SCID Mice and Improve Bone Function.

1. FIG. 6A. Survival of aged NOD/SCID mice.

2. FIG. 6B. Representative images of aged mice treated with DMEM or irMSCs (39Y, P13) .

3. FIG. 6C. Necropsy of young and aged NOD-SCID mice before or after irMSC injections.

4. FIG. 6D. H&E staining of stomach tissues from young and aged NOD/SCID mice.

5. FIG. 6E. Micro-CT imaging of bone microarchitecture in the lumbar spine.

6. FIG. 6F. BMD, BV/TV, Tb. N, Tb. Sp and Tb. Th which were determined from micro-CT measurements.

7. FIG. 6G. q-RT-PCR analysis of p16 ^Ink4a expression.

8. FIG. 6H. ALP and TRAP staining of lumbar spine in irMSC-treated and DMEM treated aged mice for detection of osteoblasts and osteoclasts (age > 43 weeks) .

9. FIG. 6I. Osteoblast numbers per bone perimeter (N. ob/B. Pm; /mm) .

10. FIG. 6J. Osteoclast numbers per bone perimeter (N. oc/B. Pm; /mm) .

11. FIG. 6K. Injected irMSCs were detected by immunohistochemical analysis.

12. FIG. 6L. Representative Photograph of Aged Mice Treated with DMEM or IrMSCs.

13. FIG. 6M. Necropsy of Young and Aged Mice Treated with DMEM or IrMSCs.

14. FIGs. 6N-6Q. Analyses of Distribution of Human IrMSCs in NOD/SCID Mice after Transfusion. Hoechst 33422-stained cells were distributed in the stomach, spleen, lung, and liver of aged SCID mice treated with irMSCs (FIG. 6N) . The human-specific actin gene in different organs was detected by PCR (FIG. 6O) . The human-specific actin gene was detected in mouse bones (lumbar spines) by gPCRs (FIG. 6P) . Injected irMSCs detected by immunohistochemical analysis (FIG. 6Q) .

Figure 7. Signaling Pathways Involved in Fibroblast Conversion into IrMSCs.

1. FIG. 7A. Four representative modules of KEGG pathways enrichment were determined by WGCNA of 12, 036 genes.

2. FIG. 7B. Box plots of changes in expression of 4 gene modules across each sample types.

3. FIG. 7C. q-RT-PCR analysis of STAT5 in fibroblasts, irMSCs, and bMSCs from different aged donors.

4. FIG. 7D. Alizarin Red and Alcian blue staining of fibroblasts with and without STAT5 knockout.

5. FIG. 7E. Expression of ALP (osteogenic marker) and COL2A1 (chondrogenic marker) in fibroblasts with and without STAT5 knockout.

6. FIG. 7F. Immunofluorescence detection of H4K20me3 after STAT5 knockout in fibroblasts.

7. FIG. 7G. q-RT-PCR analysis of STAT5, CDKN1A, and GADD45B levels after STAT5 knockout in fibroblasts.

8. FIG. 7H. Schematic representation of somatic cell rejuvenation during conversion.

9. FIG. 7I-7J. Transcriptomic Analysis during the Fibroblast-to-IrMSC Conversion. WGCNA of 12, 036 genes whose expression was altered during the conversion process (FIG. 7I) . Heat map showing the correlation between modules and cell types (FIG. 7J) .

10. FIG. 7K. Enriched KEGG Pathways of Each Modules.

11. FIG. 7L. Expression of Genes Related to the JAK-STAT Pathway Based on RNA-seq Analysis.

12. FIGs. 7M-7P. JAK-STAT Signaling in Aged MSC. Histochemistry showing adipogenesis (oil red staining) and osteogenesis (alizarin red staining) from MSCs at passage 9 and passage 22 respectively (FIG. 7M) . Barplots showing KEGG pathways enriched in genes up regulated in P10 and P21 MSCs, respectively (FIG. 7N) . Eexpression profiles of JAK-STAT members, which are upregulated in P21 MSCs (FIG. 7O) . Schematic diagram of JAK-STAT signaling pathway, which could be involved in the aging process of MSCs (FIG. 7P) .

13. FIGs. 7Q-7R. Changes in STAT5A and H3K9me3 Levels Following STAT5A Knockout. STAT5A expression (FIG. 7Q) . H3K9me3 expression in fibroblasts with or without STAT5A knockout (FIG. 7R) .

14. FIGs. 7S-7U. Telomere Length and TERT, IL-6 and ATF3 Expression after STAT5 Knockout in Fibroblasts. Telomere length, TERT, and IL6 or ATF3, with or without STAT5A knockout are shown, respectively.

DETAILED DESCRIPTION

Numerous specific details are set forth in the following description in order to provide a thorough understanding of the present disclosure. These details are provided for the purpose of example and the claimed subject matter may be practiced according to the claims without some or all of these specific details. It is to be understood that other embodiments can be used and structural changes can be made without departing from the scope of the claimed subject matter. It should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described. They instead can be applied, alone or in some combination, to one or more of the other embodiments of the disclosure, whether or not such embodiments are described, and whether or not such features are presented as being a part of a described embodiment. For the purpose of clarity, technical material that is known in the technical fields related to the claimed subject matter has not been described in detail so that the claimed subject matter is not unnecessarily obscured.

All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entireties for all purposes to the same extent as if each individual publication were individually incorporated by reference. Citation of the publications or documents is not intended as an admission that any of them is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

The practice of the provided embodiments will employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques) , cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polypeptide and protein synthesis and modification, polynucleotide synthesis and modification, polymer array synthesis, hybridization and ligation of polynucleotides, detection of hybridization, and nucleotide sequencing. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999) ; Weiner, Gabriel, Stephens, Eds., Genetic Variation: A Laboratory Manual (2007) ; Dieffenbach, Dveksler, Eds., PCR Primer: A Laboratory Manual (2003) ; Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003) ; Mount, Bioinformatics: Sequence and Genome Analysis (2004) ; Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006) ; and Sambrook and Russell, Molecular Cloning: A Laboratory Manual (2002) (all from Cold Spring Harbor Laboratory Press) ; Ausubel et al. eds., Current Protocols in Molecular Biology (1987) ; T. Brown ed., Essential Molecular Biology (1991) , IRL Press; Goeddel ed., Gene Expression Technology (1991) , Academic Press; A. Bothwell et al. eds., Methods for Cloning and Analysis of Eukaryotic Genes (1990) , Bartlett Publ.; M. Kriegler, Gene Transfer and Expression (1990) , Stockton Press; R. Wu et al. eds., Recombinant DNA Methodology (1989) , Academic Press; M. McPherson et al., PCR: A Practical Approach (1991) , IRL Press at Oxford University Press; Stryer, Biochemistry (4th Ed. ) (1995) , W.H. Freeman, New York N.Y.; Gait, Oligonucleotide Synthesis: A Practical Approach (2002) , IRL Press, London; Nelson and Cox, Lehninger, Principles of Biochemistry (2000) 3rd Ed., W.H. Freeman Pub., New York, N.Y.; Berg, et al., Biochemistry (2002) 5th Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entireties by reference for all purposes.

A. Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which the present disclosure belongs. If a definition set forth in this section is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth in this section prevails over the definition that is incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more. ” As used herein, the singular forms “a, ” “an, ” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, reference to “a stem cell” , for example, refers to one or more stem cells, and reference to “the method” includes reference to equivalent steps and methods disclosed herein and/or known to those skilled in the art, and so forth.

It is understood that aspects and embodiments of the disclosure described herein as ‘comprising’ also include “consisting” and/or “consisting essentially of” aspects and embodiments.

Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X. ” Additionally, use of “about” preceding any series of numbers includes “about” each of the recited numbers in that series. For example, description referring to “about X, Y, or Z” is intended to describe “about X, about Y, or about Z. ”

The term “average” as used herein refers to either a mean or a median, or any value used to approximate the mean or the median, unless the context clearly indicates otherwise.

A “subject” as used herein refers to an organism, or a part or component of the organism, to which the provided compositions, methods, kits, devices, and systems can be administered or applied. For example, the term “subject” may refer to a patient, and frequently a human patient. However, this term is not limited to humans and thus encompasses a variety of mammalian species. The subject can be a mammal or a cell, a tissue, an organ, or a part of the mammal. As used herein, “mammal” refers to any of the mammalian class of species, preferably human (including humans, human subjects, or human patients) . Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, and rodents such as mice and rats.

As used herein, “an effective amount of a compound” for treating a particular disease, or effecting a particular result, is an amount that is sufficient to ameliorate, or in some manner reduce the symptoms associated with the disease or to produce the designated result. Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective. The amount may cure the disease but, typically, is administered in order to ameliorate the symptoms of the disease. Repeated administration may be required to achieve the desired amelioration of symptoms.

As used herein, “treatment” means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein.

As used herein, “amelioration” of the symptoms of a particular disorder by administration of a particular pharmaceutical composition refers to any lessening, whether permanent or temporary, lasting or transient that can be attributed to or associated with administration of the composition.

As used herein, “molecular cocktail” refers to a composition containing at least one of the designated compounds or materials. Preferably, the composition is an aqueous pharmaceutically acceptable composition, and the designated compound can be present as a pharmaceutically-acceptable salt.

As used herein, “activate an activity” refers to the effect of measurably increasing the specified activity, e.g., causing the activity to increase by at least 20%, typically at least 50%, as compared to an untreated control or to baseline activity prior to the treatment being described.

As used herein, a “level of X” in a molecule cocktail refers to the concentration of compound X in the molecule cocktail.

As used herein, “production by recombinant means” refers to production methods that use recombinant nucleic acid methods that rely on well known methods of molecular biology for expressing proteins encoded by cloned nucleic acids.

As used herein the term “sample” refers to anything which may contain a target molecule for which analysis is desired, including a biological sample. As used herein, a “biological sample” can refer to any sample obtained from a living or viral (or prion) source or other source of macromolecules and biomolecules, and includes any cell type or tissue of a subject from which nucleic acid, protein and/or other macromolecule can be obtained. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. For example, isolated nucleic acids that are amplified constitute a biological sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, sweat, semen, stool, sputum, tears, mucus, amniotic fluid or the like, an effusion, abone marrow sample, ascitic fluid, pelvic wash fluid, pleural fluid, spinal fluid, lymph, ocular fluid, extract of nasal, throat or genital swab, cell suspension from digested tissue, or extract of fecal material, and tissue and organ samples from animals and plants and processed samples derived therefrom.

As used herein, “pharmaceutically acceptable salts, esters or other derivatives” include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects and that either are pharmaceutically active or are prodrugs. For example, compounds described or discussed herein may be used or administered as a pharmaceutically acceptable salt, and suitable pharmaceutically acceptable salts are well known in the art.

As used herein, a “prodrug” is a compound that, upon in vivo administration, is metabolized or otherwise converted to the biologically, pharmaceutically or therapeutically active form of the compound. To produce a prodrug, the pharmaceutically active compound is modified such that the active compound will be regenerated by metabolic processes. The prodrug may be designed to alter the metabolic stability or the transport characteristics of a drug, to mask side effects or toxicity, to improve the flavor of a drug or to alter other characteristics or properties of a drug. By virtue of knowledge of pharmacodynamic processes and drug metabolism in vivo, those of skill in this art, once a pharmaceutically active compound is known, can design prodrugs of the compound (see, e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pages 388-392) .

As used herein, “test substance (or candidate compound) ” refers to a chemically defined compound (e.g., organic molecules, inorganic molecules, organic/inorganic molecules, proteins, peptides, nucleic acids, oligonucleotides, lipids, polysaccharides, saccharides, or hybrids among these molecules such as glycoproteins, etc. ) or mixtures of compounds (e.g., a library of test compounds, natural extracts or culture supernatants, etc. ) .

As used herein, high-throughput screening (HTS) refers to processes that test a large number of samples, such as samples of diverse chemical structures against disease targets to identify “hits” (see, e.g., Broach, et al., High throughput screening for drug discovery, Nature, 384: 14-16 (1996) ; Janzen, et al., High throughput screening as a discovery tool in the pharmaceutical industry, Lab Robotics Automation: 8261-265 (1996) ; Fernandes, P. B., Letter from the society president, J. Biomol. Screening, 2: 1 (1997) ; Burbaum, et al., New technologies for high-throughput screening, Curr. Opin. Chem. Biol., 1: 72-78 (1997) ) . HTS operations are highly automated and computerized to handle sample preparation, assay procedures and the subsequent processing of large volumes of data.

The terms “polynucleotide, ” “oligonucleotide, ” “nucleic acid” and “nucleic acid molecule” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and comprise ribonucleotides, deoxyribonucleotides, and analogs or mixtures thereof. The terms include triple-, double-and single-stranded deoxyribonucleic acid ( “DNA” ) , as well as triple-, double-and single-stranded ribonucleic acid ( “RNA” ) . It also includes modified, for example by alkylation, and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide, ” “oligonucleotide, ” “nucleic acid, ” and “nucleic acid molecule” include polydeoxyribonucleotides (containing 2-deoxy-D-ribose) , polyribonucleotides (containing D-ribose) , including tRNA, rRNA, hRNA, and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N-or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids ( “PNAs” ) ) and polymorpholino (commercially available from the Anti-Virals, Inc., Corvallis, OR, as Neugene) polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. Thus, these terms include, for example, 3'-deoxy-2', 5'-DNA, oligodeoxyribonucleotide N3'to P5'phosphoramidates, 2'-O-alkyl-substituted RNA, hybrids between DNA and RNA or between PNAs and DNA or RNA, and also include known types of modifications, for example, labels, alkylation, “caps, ” substitution of one or more of the nucleotides with an analog, inter-nucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc. ) , with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc. ) , and with positively charged linkages (e.g., aminoalkylphosphoramidates, aminoalkylphosphotriesters) , those containing pendant moieties, such as, for example, proteins (including enzymes (e.g. nucleases) , toxins, antibodies, signal peptides, poly-L-lysine, etc. ) , those with intercalators (e.g., acridine, psoralen, etc. ) , those containing chelates (of, e.g., metals, radioactive metals, boron, oxidative metals, etc. ) , those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc. ) , as well as unmodified forms of the polynucleotide or oligonucleotide. A nucleic acid generally will contain phosphodiester bonds, although in some cases nucleic acid analogs may be included that have alternative backbones such as phosphoramidite, phosphorodithioate, or methylphophoroamidite linkages; or peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with bicyclic structures including locked nucleic acids, positive backbones, non-ionic backbones and non-ribose backbones. Modifications of the ribose-phosphate backbone may be done to increase the stability of the molecules; for example, PNA: DNA hybrids can exhibit higher stability in some environments. The terms “polynucleotide, ” “oligonucleotide, ” “nucleic acid” and “nucleic acid molecule” can comprise any suitable length, such as at least 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1,000 or more nucleotides.

It will be appreciated that, as used herein, the terms “nucleoside” and “nucleotide” include those moieties which contain not only the known purine and pyrimidine bases, but also other heterocyclic bases which have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Modified nucleosides or nucleotides can also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen, aliphatic groups, or are functionalized as ethers, amines, or the like. The term “nucleotidic unit” is intended to encompass nucleosides and nucleotides.

The terms “complementary” and “substantially complementary” include the hybridization or base pairing or the formation of a duplex between nucleotides or nucleic acids, for instance, between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid. Complementary nucleotides are, generally, A and T (or A and U) , or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared and with appropriate nucleotide insertions or deletions, pair with at least about 80%of the other strand, usually at least about 90%to about 95%, and even about 98%to about 100%. In one aspect, two complementary sequences of nucleotides are capable of hybridizing, preferably with less than 25%, more preferably with less than 15%, even more preferably with less than 5%, most preferably with no mismatches between opposed nucleotides. Preferably the two molecules will hybridize under conditions of high stringency.

As used herein, for a reference sequence, the reverse complementary sequence is the complementary sequence of the reference sequence in the reverse order. For example, for 5’-ATCG-3’, the complementary sequence is 3’-TAGC-5’, and the reverse-complementary sequence is 5’-CGAT-3’.

“Hybridization” as used herein may refer to the process in which two single-stranded polynucleotides bind non-covalently to form a stable double-stranded polynucleotide. In one aspect, the resulting double-stranded polynucleotide can be a “hybrid” or “duplex. ” “Hybridization conditions” typically include salt concentrations of approximately less than 1 M, often less than about 500 mM and may be less than about 200 mM. A “hybridization buffer” includes a buffered salt solution such as 5%SSPE, or other such buffers known in the art. Hybridization temperatures can be as low as 5℃, but are typically greater than 22℃, and more typically greater than about 30℃, and typically in excess of 37℃. Hybridizations are often performed under stringent conditions, i.e., conditions under which a sequence will hybridize to its target sequence but will not hybridize to other, non-complementary sequences. Stringent conditions are sequence-dependent and are different in different circumstances. For example, longer fragments may require higher hybridization temperatures for specific hybridization than short fragments. As other factors may affect the stringency of hybridization, including base composition and length of the complementary strands, presence of organic solvents, and the extent of base mismatching, the combination ofparameters is more important than the absolute measure of any one parameter alone. Generally stringent conditions are selected to be about 5℃ lower than the T _m for the specific sequence at a defined ionic strength and pH. The melting temperature T _m can be the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the T _m of nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the T _m value may be calculated by the equation, T _m=81.5 + 0.41 (%G + C) , when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985) ) . Other references (e.g., Allawi and SantaLucia, Jr., Biochemistry, 36: 10581-94 (1997) ) include alternative methods of computation which take structural and environmental, as well as sequence characteristics into account for the calculation of T _m.

In general, the stability of a hybrid is a function of the ion concentration and temperature. Typically, a hybridization reaction is performed under conditions of lower stringency, followed by washes of varying, but higher, stringency. Exemplary stringent conditions include a salt concentration of at least 0.01 M to no more than 1 M sodium ion concentration (or other salt) at a pH of about 7.0 to about 8.3 and a temperature of at least 25℃. For example, conditions of 5 × SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA at pH 7.4) and a temperature of approximately 30℃ are suitable for allele-specific hybridizations, though a suitable temperature depends on the length and/or GC content of the region hybridized. In one aspect, “stringency of hybridization” in determining percentage mismatch can be as follows: 1) high stringency: 0.1 × SSPE, 0.1%SDS, 65℃; 2) medium stringency: 0.2 × SSPE, 0.1%SDS, 50℃ (also referred to as moderate stringency) ; and 3) low stringency: 1.0 × SSPE, 0.1%SDS, 50℃. It is understood that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. For example, moderately stringent hybridization can refer to conditions that permit a nucleic acid molecule such as a probe to bind a complementary nucleic acid molecule. The hybridized nucleic acid molecules generally have at least 60%identity, including for example at least any of 70%, 75%, 80%, 85%, 90%, or 95%identity. Moderately stringent conditions can be conditions equivalent to hybridization in 50%formamide, 5 × Denhardt’s solution, 5x SSPE, 0.2%SDS at 42℃, followed by washing in 0.2 × SSPE, 0.2%SDS, at 42℃. High stringency conditions can be provided, for example, by hybridization in 50%formamide, 5 × Denhardt’s solution, 5 × SSPE, 0.2%SDS at 42℃, followed by washing in 0.1 × SSPE, and 0.1%SDS at 65℃. Low stringency hybridization can refer to conditions equivalent to hybridization in 10%formamide, 5 × Denhardt’s solution, 6 ×SSPE, 0.2%SDS at 22℃, followed by washing in 1x SSPE, 0.2%SDS, at 37℃. Denhardt’s solution contains 1%Ficoll, 1%polyvinylpyrolidone, and 1%bovine serum albumin (BSA) . 20 × SSPE (sodium chloride, sodium phosphate, EDTA) contains 3 M sodium chloride, 0.2 M sodium phosphate, and 0.025 M EDTA. Other suitable moderate stringency and high stringency hybridization buffers and conditions are well known to those of skill in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y. (1989) ; and Ausubel et al., Short Protocols in Molecular Biology, 4th ed., John Wiley &Sons (1999) .

Alternatively, substantial complementarity exists when an RNA or DNA strand will hybridize under selective hybridization conditions to its complement. Typically, selective hybridization will occur when there is at least about 65%complementary over a stretch of at least 14 to 25 nucleotides, preferably at least about 75%, more preferably at least about 90%complementary. See M. Kanehisa, Nucleic Acids Res. 12: 203 (1984) .

A “primer” used herein can be an oligonucleotide, either natural or synthetic, that is capable, upon forming a duplex with a polynucleotide template, of acting as a point of initiation of nucleic acid synthesis and being extended from its 3'end along the template so that an extended duplex is formed. The sequence of nucleotides added during the extension process is determined by the sequence of the template polynucleotide. Primers usually are extended by a polymerase, for example, a DNA polymerase.

“Ligation” may refer to the formation of a covalent bond or linkage between the termini of two or more nucleic acids, e.g., oligonucleotides and/or polynucleotides, in a template-driven reaction. The nature of the bond or linkage may vary widely and the ligation may be carried out enzymatically. As used herein, ligations are usually carried out enzymatically to form a phosphodiester linkage between a 5'carbon terminal nucleotide of one oligonucleotide with a 3'carbon of another nucleotide.

“Amplification, ” as used herein, generally refers to the process of producing multiple copies of a desired sequence. “Multiple copies” means at least 2 copies. A “copy” does not necessarily mean perfect sequence complementarity or identity to the template sequence. For example, copies can include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced through a primer comprising a sequence that is hybridizable, but not complementary, to the template) , and/or sequence errors that occur during amplification.

“Sequence determination” and the like include determination of information relating to the nucleotide base sequence of a nucleic acid. Such information may include the identification or determination of partial as well as full sequence information of the nucleic acid. Sequence information may be determined with varying degrees of statistical reliability or confidence. In one aspect, the term includes the determination of the identity and ordering of a plurality of contiguous nucleotides in a nucleic acid.

The term “Sequencing, ” “High throughput sequencing, ” or “next generation sequencing” includes sequence determination using methods that determine many (typically thousands to billions) of nucleic acid sequences in an intrinsically parallel manner, i.e. where DNA templates are prepared for sequencing not one at a time, but in a bulk process, and where many sequences are read out preferably in parallel, or alternatively using an ultra-high throughput serial process that itself may be parallelized. Such methods include but are not limited to pyrosequencing (for example, as commercialized by 454 Life Sciences, Inc., Branford, CT) ; sequencing by ligation (for example, as commercialized in the SOLiD ^TM technology, Life Technologies, Inc., Carlsbad, CA) ; sequencing by synthesis using modified nucleotides (such as commercialized in TruSeq ^TM and HiSeq ^TM technology by Illumina, Inc., San Diego, CA; HeliScope ^TM by Helicos Biosciences Corporation, Cambridge, MA; and PacBio RS by Pacific Biosciences of California, Inc., Menlo Park, CA) , sequencing by ion detection technologies (such as Ion Torrent ^TM technology, Life Technologies, Carlsbad, CA) ; sequencing of DNA nanoballs (Complete Genomics, Inc., Mountain View, CA) ; nanopore-based sequencing technologies (for example, as developed by Oxford Nanopore Technologies, LTD, Oxford, UK) , and like highly parallelized sequencing methods.

“SNP” or “single nucleotide polymorphism” may include a genetic variation between individuals; e.g., a single nitrogenous base position in the DNA of organisms that is variable. SNPs are found across the genome; much of the genetic variation between individuals is due to variation at SNP loci, and often this genetic variation results in phenotypic variation between individuals. SNPs for use in the present disclosure and their respective alleles may be derived from any number of sources, such as public databases (U.C. Santa Cruz Human Genome Browser Gateway (genome. ucsc. edu/cgi-bin/hgGateway) or the NCBI dbSNP website (ncbi. nlm. nih gov/SNP/) , or may be experimentally determined as described in U.S. Pat. No. 6,969,589; and US Pub. No. 2006/0188875 entitled “Human Genomic Polymorphisms. ” Although the use of SNPs is described in some of the embodiments presented herein, it will be understood that other biallelic or multi-allelic genetic markers may also be used. A biallelic genetic marker is one that has two polymorphic forms, or alleles. As mentioned above, for a biallelic genetic marker that is associated with a trait, the allele that is more abundant in the genetic composition of a case group as compared to a control group is termed the “associated allele, ” and the other allele may be referred to as the “unassociated allele. ” Thus, for each biallelic polymorphism that is associated with a given trait (e.g., a disease or drug response) , there is a corresponding associated allele. Other biallelic polymorphisms that may be used with the methods presented herein include, but are not limited to multinucleotide changes, insertions, deletions, and translocations.

It will be further appreciated that references to DNA herein may include genomic DNA, mitochondrial DNA, episomal DNA, and/or derivatives of DNA such as amplicons, RNA transcripts, cDNA, DNA analogs, etc. The polymorphic loci that are screened in an association study may be in a diploid or a haploid state and, ideally, would be from sites across the genome. Sequencing technologies are available for SNP sequencing, such as the BeadArray platform (GOLDENGATE ^TM assay) (Illumina, Inc., San Diego, CA) (see Fan, et al., Cold Spring Symp. Quant. Biol., 68: 69-78 (2003) ) , may be employed.

“Multiplexing” or “multiplex assay” herein may refer to an assay or other analytical method in which the presence and/or amount of multiple targets, e.g., multiple nucleic acid sequences, can be assayed simultaneously by using more than one markers, each of which has at least one different detection characteristic, e.g., fluorescence characteristic (for example excitation wavelength, emission wavelength, emission intensity, FWHM (full width at half maximum peak height) , or fluorescence lifetime) or a unique nucleic acid or protein sequence characteristic.

As used herein, “disease or disorder” refers to apathological condition in an organism resulting from, e.g., infection or genetic defect, and characterized by identifiable symptoms.

DISCUSSION

Described herein are methods forthe successful conversion of skin fibroblasts from young and aged donors into youthful irMSCs that exhibit excellent capacity for bone and cartilage repair and age reversal in vivo. Compared to parental fibroblasts and bMSCs from old aged donors, irMSCs displayed heightened proliferative potential without being tumorigenic. Fibroblasts from a small patch of skin (less than 1 cm ²) can generate irMSCs in large quantities to meet the needs of autologous cell therapy or tissue engineering without ethical issues or clinical concerns associated with usage of “non-self” cells. Based on telomere length, growth potential, and marker expression, are youthful cells that canbe derived from elderly individuals. Moreover, osteoblasts and chondrocytes derived from irMSCs also exhibit signs of rejuvenation as compared to parental fibroblasts or autologous bMSC-derived cells. IrMSCs from elderly donors exhibit excellent in vivo bone and cartilage repair properties. Accordingly, intravenous transfusions of irMSCs have thepotential to prolong life and improve a series of age-related phenotypes, as well as reducing the age-related degerative characteristics of tissue either in vitro or in vivo.

A detailed analysis of bone tissue reveals that spine trabecular bone had an increased BMD and better microarchitecture comparable to younger mice of 25-week-old; this was accompanied by increased activities of ALP and reduced activities of TRAP similar to that in young mice. A subset of human irMSCs in aged mice bone tissue was detected to differentiate into osteoblasts, which would contribute to enhanced bone formation. IrMSCs may have potential capability to revitalize aged tissues by producing new youthful cells to replace aged cells, as demonstrated by irMSC cell integration into bone and cartilage during in vivo repair. Shen et al. reported that transplantation of allogeneic MSCs from young mice into irradiated aged Balb/C mice could restore their bone density and prolong lifespan (Shen et al., 2011) . Golpanian et al. also demonstrated that allogeneic human MSCs from young donors could improve immunologic status of frail persons with ameliorating physical and mental aging symptoms within 6 months (Golpanian et al., 2017) . These allogeneic young cells including those from ESC and iPSCs can revitalize stem cell niches in the old body (Kfoury et al., 2015) but can not replace feeble endogenous tissue due to immune rejections. Reduced endogenous stem cells in elderly individuals and their loss in differentiation capacity contribute to a decline in physiologic regeneration of multiple organs. When such regeneration fails, stress response mechanisms such as apoptosis and cellular senescence are activated and the resultant senescent cells accumulate over time, further accelerating aging (de Keizer, 2016) . In our study, although we still do not know its mechanism, irMSCs injected intravenously reduced bone resorption and decreased osteoclast activity, which may prevent age-related bone loss. Therefore, irMSC may hold great promise as a regenerative agent not only to modulate microenvironment but also directly participate in youthful cell-replacement based tissue repair.

The high conversion rate (>90%) of irMSCs suggests that there is no selection pressure during conversion, which is a good feature to avoid selection of growth advantageous cancerous cells. Moreover, transient induction of TERT expression also distinguishes irMSCs from cancerous cells. From the large amount of transplantation experiments with irMSCs into NOD/SCID mice, we concluded that our cells were not tumorigenic, which disclosed some safety features of irMSCs for future therapeutic applications.

The mechanism by which irMSCs counteract aging remains unclear; MSCs may release anti-aging factors or through immune modulation to curb aging related chronic inflammation (Boomsma and Geenen, 2012; Golpanian et al., 2017; Schwerk et al., 2015) . A more thorough analysis of factors (proteins and RNAs, perhaps exosomes) secreted by irMSCs and their effects on various tissues remain to be determined in the near future.

A variety of small molecules with specific targets are known to regulate intracellular signaling pathways, some of which are used in different reprogramming protocols. For example, VPA, CHIR99021, Repsox and Forskolin were used repeatedly to promote epithelial-to-mesenchymal transition of mouse fibroblasts into pluripotent cells or to convert mouse fibroblasts into cardiomyocytes: the retinoic acid receptor ligands, TTNPB and AM580, could enhance chemical reprogramming of mouse fibroblasts to cardiomyocytes or iPSCs, and could also induce human iPSCs/ESCs into nephrogenic intermediate mesoderm; Go6983 improved establishment of

human pluripotent stem cells and facilitated fibroblast to neuron conversion; Y-27632, EPZ004777 and Vc reduce cell senescence in vitro. We selected from these compounds to formulate our induction method. Compound mix V was tried first but failed to enable cells for tri-lineage differentiation. Compound mix P is mix V plus 7 additional small molecules. However, adding mix P directly to parental fibroblasts caused massive cell death. Therefore, we used compound mix V to alter the state of fibroblasts so that they become competent to receive induction by compound mix P. Transcriptome analyses revealed such step wise alterations. Prolonged treatment with Mix V or Mix P was also detrimental to cells, so that three extra days of washout period was necessary to complete the induction. We concluded that cells needed to go through two transitory states to achieve successful conversion, but the transitory states were not stable cellular states, therefore could only exist for limited time windows. Interestingly, JAK-STAT pathway was found to be involved in such conversion while none of the molecules included in our small molecule cocktails specifically targeted this pathway, but a combination of the molecules led to inhibition of this pathway. Our study revealed that genes like STAT5A, may represent a significant hurdle for fibroblasts to acquire osteogenic and chondrogenic differentiation potentials as well as some features of youthful cells. Obviously, there may still be room for optimization of the induction methods, but induction via multiple steps could be key to our success.

In summary, our study describes a method for allowing skin fibroblasts to acquire stem cell properties and youth. We demonstrated that irMSCs function in tissue repair and age reversal like magic bullets. Given irMSCs could be obtained from young and aged donors with erased aging properties, they could be excellent autologous cells for tissue repair, age reversal, and extension of life expectancy. In a sense, these cells could be considered as the “Fountain of Youth” and provide tremendous new opportunities for future regenerative and anti-aging medicine.

Disclosed in another aspect herein is a kit, comprising at least one irMSC, or a population of irMSCs, preferably produced by the methods described herein. Typically, the irMSC is provided in combination with at least one means for administration of the irMSC to a subject in need thereof, such as a hypodermic syringe. Optionally, the kit comprising an irMSC also comprises instructions for administration of the irMSC.

EXAMPLES

Generation of IrMSCs from Dermal Fibroblasts

Fresh skin biopsy specimens (1 cm ² in size) were used to generate primary fibroblast cultures for at least 6 passages (P6) . Between P6 to P13, fibroblast cells were exposed to small molecule cocktails along with Mix V (0.5 mM VPA; 3 μM CHIR99021; 1 μM Repsox; 10 μM Forskolin) for 5 days, followed by Mix P (0.5mM VPA, 0.2mM Vc, 3 μM CHIR99021, 10 μM Forskolin, 10 μM SP600125, 5 μM Go6983, 5 μM Y-27632, 5 μM TTNPB, 0.05 μM AM580, 5 μM EPZ004777, 1 μM RepSox) for another 7 days. On day 12, cells were washed to remove any residual small molecules and cultured in fresh MSC medium with bFGF, PDGF-AB and BMP4 for an additional 3 days, at which point (day 15, D15) cells became irMSCs (FIG. 1A) . Fibroblasts close to senescence (P13) exhibitedtremendous growth potential (expansion for another 19 passages) after conversion. Once D15 irMSC cells were generated, they grew faster and could be expanded extensively for at least another 19 passages (FIG. 1B) . The resulting irMSCs could self renew and acquire the potential to differentiate into bone, cartilage, and fat. Even at P9 or P16, irMSCs still have robust tri-lineage differentiation potentials (FIG. 1C-1D) . Samples at D0 (fibroblasts before treatment) , D5, D12, D15 (irMSCs) , and after serial passaging were examined for tri-lineage differentiation potentials; osteogenesis, adipogenesis, and chondrogenesis were evaluated on day 21 upon differentiation. Col2a1 levels in chondrogenic micromass cultures after 14 days, and PPARGlevels in adipogenic cultures after 21 days are compared to D0 (Fib) in FIG. 1D. *p< 0.05, **p<0.01, ***p ＜ 0.001, n≥3. Further experiments demonstratedthat without the addition of three growth factors from D12-D15, by D15, cells still acquired tri-lineage differentiation potentials (FIG. 1H) .

Compared to irMSCs, the parental fibroblasts did not have the capacity for tri-lineage differentiation; neither do D5 or D12 cells, which were in transition to be converted to irMSCs (FIG. 1C) . This method was highly reproducible and highly efficient for generation of osteoblasts, chondrocytes and adipocytes from irMSCs, as detected by Alizarin Red, Alcian Blue, and Oil Red O staining, respectively, after 21 days (FIG. 1I) . Results are consistent with the multipotency of irMSCs demonstrated above.

It is well known that fibroblasts would senesce upon culturing in vitro after 14-20 passages. However, if fibroblasts at passage 13 were converted to irMSCs, at least another 19 passages can be added to the lifespan of the cells. To assess the extent of cell expansion, we counted cells (FIG. 1A and FIG. 1J) . From a piece of 1x1cm ² skin tissue specimen, we derived fibroblast cultures. By passage 3, we followed a cohort of cells in the amount of 10 ⁵. If we further expanded them to passage 6, we would obtain about 10 ⁷ cells. During the subsequent 15-day conversion process, there was no big change in the cell number. Therefore, by D15, we obtained about 10 ⁷ irMSCs, which was considered as passage 0 for the irMSC phase. These irMSCs were passaged to P3, when another cohort of 10 ⁵ cells was tracked quantitatively afterwards. Upon expansion, at P13 of the irMSC phase, we calculated the cell number, which was about 10 ¹⁷, suggesting that after cells entered the irMSC phase, one cell can produce at least 10 ¹² cells from P3 to P13. If we induced fibroblasts at P9, a total of a 10 ¹⁸ fold of expansion can be achieved from the initial fibroblast cohort at P3. We knew that even at P19 irMSCs still did not show signs of senescence. Taken together, conversion of fibroblasts into irMSCs extensively prolonged the lifespan of the cells.

In the first 5 days during Mix V treatment, a fibroblast marker fibroblast-specific protein-1 (FSP-1) was down-regulated, whereas MSC-related cell surface markers CD49a and CD49e (Deschaseaux et al., 2003; Meirelles and Nardi, 2003) were up-regulated and continued to be expressed during irMSC expansion (FIGs. 1K-1L) . IrMSCs were also positive for the widely used human MSC markers CD90, CD105, CD73, CD44, and were negative for CD14, CD34, CD45, CD19, and human leukocyte antigen-DR (HLA-DR) (FIG. 1N) . In this figure, gray and black represent specific antibody isotype control and indicated cells staining, respectively. Fibroblasts (D0 denotes the start of induction) , bMSCs, and irMSCs (D15 denotes day 15 of the induction process) showed similar CD markers expression profiles. Surprisingly, the parental fibroblast cultures are also positive for

CDs

90, 105, 73, 44 andnegative for

CDs

14, 34, 45, 19 and HLA-DR, but are incapable of tri-lineage differentiation. We therefore concluded that these sets of cell surface markers should not be used to define MSCs. To estimate conversion efficiency, 1 × 10 ⁵ fibroblasts (negative for CD49a and CD49e) were subjected to the small molecular treatment. By D15, there were around 1 × 10 ⁵ cells (irMSCs at birth) , of which 97%were double positive for CD49a and CD49e. There might be a low rate of cell death and/or cell proliferation, but the total number of cells before and after the 15 day treatment was about the same. Therefore, there was no high “selection” pressure during the conversion process. To exclude the possibility that there might be some MSCs hidden in our fibroblasts cultures, which winded up being selected and expanded from the small molecular induction process, we subjected bone-marrow-derived MSCs (bMSCs) as well as irMSCs to the same cocktail treatment. As shown in FIG. 1A and FIGs. 1O-1P, neither bMSCs nor irMSCs could survive the initial induction process, which further demonstrated that parental cells that went through the conversion were not MSCs but fibroblasts (FIG. 1A and FIGs. 1O-1P) . FIG. 1O shows β-Galactosidase activity in bMSCs, irMSCs, and fibroblasts before and after (Mix V + Mix P) treatment for 12 days: while many cells died, those remaining in bMSC and irMSC groups exhibited senescence. FIG. 1P shows cell counts at Day 0 (untreated) andDay 12 after (Mix V+Mix P) treatment cultures. The third group is D12 culture plus 18 additional days of culturing after washing out of the compounds. Only fibroblasts survived the conversion process; bMSCs or irMSCs did not survive.

To investigate the immunomodulatory function of irMSCs, we co-cultured human peripheral blood lymphocytes with parental fibroblasts, irMSCs, or bMSCs and evaluated T cell proliferation rate by carboxyfluorescein succinimidyl ester (CFSE) labeling with or without phytohemagglutinin (PHA) stimulation. Similar to control bMSCs, irMSCs suppressed T cell proliferation in vitro, and both cell types also suppressed both CD4+ and CD8+ T cell subtypes (FIG. 1E and FIGs. 1Q-1T) . FIG. 1E demonstrates immune suppression of T cell allo-reactivity after co-culturing for 5 days with parental fibroblasts, irMSCs, and bMSCs. PBMCs from normal donor blood were labeled with CFSE. *p < 0.05, **p<0.01, ***p < 0.001, n=3, when samples were compared to the “T+PHA” group. FIGs. 1Q-1T provide a mixed lymphocyte reaction assay showing immune suppression by irMSCs in vitro. CD8+ (A-B) and CD4+ (C-D) T cells from normal donors’blood were labeled with CFSE and then co-cultured for 5 days with parental fibroblasts, bMSCs, or irMSCs. PHA stimulation was used to activate T cell proliferations. Significance was determined relative to the “T + PHA” group, *p < 0.05, **p<0.01, ***p < 0.001, n=3.

To further examine the immune modulatory functions of irMSC, we injected C57BL/6 mice with concanavalin A (ConA) to induce acute liver injury. It is well known that MSCs may reverse acute liver damage by their immune-suppressive function. Here we showed that injections of concentrated conditioned medium (CM) from cultured bMSCs or irMSCs, but not CM from parental fibroblasts, reduced liver injury as shown by liver biopsy as well as H&E staining of liver tissues 8.5 h after ConA-induced injury and transfusion with concentrated conditioned medium of fibroblasts, irMSCs, and bMSCs or with PBS (control) . “M” , concentrated conditioned medium (n = 6 animals per group) (FIG. 1U) . Consistently, CM from cultures of bMSCs and irMSCs decreased CD3+ T cell infiltration into the liver (FIG. 1F) . Absolute numbers of T lymphocytes in liver tissues were determined by flow cytometry 8.5 h after intravenous injection of conditioned medium (CM, conditioned for 72h before ultracentrifugation to obtain supernatant) . *p < 0.05, **p<0.01, ***p < 0.001, n=6, when samples were compared to the “ConA + PBS” group.

To evaluate the severity of hepatic injury, we also measured the levels of the enzymes aspartate transaminase (AST) and alanine transaminase (ALT) in mouse serum 8.5 hours after ConA-induced injury. AST and ALT expression induced by liver damage were significantly suppressed by the administration of CM from irMSCs or bMSCs (FIGs. 1V-1W) .

To objectively examine the true identities of the cells, we performed transcriptome analyses of parental fibroblast cells (D0) , D5, D12 cells, and D15 (irMSCs) , together with bMSCs. We also downloaded data from the public database for pluripotent human embryonic stem cells (hESCs) and iPSCs. tSNE analyses of whole transcriptome demonstrated that fibroblasts and bMSCs were already closely related but still distinct cell types based on their transcriptome (FIG. 1G) . Interestingly, upon first induction, large-scale transcription changes occurred at day 5 and day 12 as the transcriptome of cells drastically deviated from that of fibroblasts. By day15, however, transcriptome reverted back to one that was similar to fibroblasts and bMSCs, yet cells now acquired self-renewal, tri-lineage differentiation and immune modulation properties, similar to bMSCs.

Rejuvenation of Skin Fibroblasts after Conversion into IrMSCs

Comparisons of irMSCs immediately after conversion (D15) with parental fibroblasts from a 39-year-old individual prior to small molecular induction (D0 Fib) revealed that an aging epigenetic marker histone H4 lysine 20 trimethylation (H4K20me3) and age-related DNA damage marker phosphorylated histone H2AX (γH2AX) were downregulated (FIG. 2A-2C) . Statistical significance was determined relative to D0 (Fib, 39Y) , ***p<0.001; 100 cells were counted to calculate the number of γH2AX foci per cell. We also examined the growth potential of cells from different aged donors and found that fibroblast-derived irMSCs had greater proliferative capacity than their parental fibroblasts (FIG. 2D) . In FIG. 2D, cells from the same donors are shown in the same color. Triangles represent fibroblasts, squares represent bMSCs, and circles represent irMSCs. Notably, irMSCs from aged individuals (62, 63, and 74 years old) grew more rapidly than autologous bMSCs as shown in FIG. 2D and FIG. 2I (Growth curve of fibroblasts, bMSCs and irMSCs during long-term expansion: cells from the same donors are shown in the same color. ) Note that irMSCs from aged donors grew even faster than bMSCs from much younger donors (31 and 33 years old) .

Fibroblasts and bMSCs from aged donor (62Y) further aged after in vitro culturing. By passage 13, both cultures became senescence-associated β-galactosidase positive, while irMSCs from the same donor at P13 did not. This is illustrated in FIG. 2J, where representative images show the typical morphology of cells at passages 9 and 13 of parental fibroblasts, irMSCs, and bMSCs from a 62-year-old donor. Based on SA-β-Gal staining, both parental fibroblasts and bMSCs senesced at P13, but not irMSCs from the same old age donor. Expressions of aging markers including cyclin dependent kinase inhibitor (CDKN) 1A, activating transcription factor (ATF) 3, interleukin (IL) -6, as well as growth arrest and DNA damage-inducible 45 beta (GADD45B) did increase in parental fibroblasts or bMSCs depending on the age of the donors, while expression of these genes all reduced in irMSCs regardless of the age of the donors (FIG. 2 E-G and FIG. 2K) . FIG. 2E-2G show q-RT-PCR analysis of CDKN1A, ATF3 andIL-6 expression in D0 (Fib) at passage 6, D15 (irMSCs) , autologous or allogeneic bMSCs, before and after differentiation into osteoblasts (OB) or chondrocytes (CH) . “12W” sample stands for embryonic fibroblasts from an aborted embryo at 12 weeks of gestation. FIG. 2K shows that expression of GADD45B was down-regulated in irMSCs from donors of different ages compared to homologous fibroblasts and bMSCs. Stars denote significance compared to autologous fibroblasts, and pound symbols denote significance compared to autologous bMSCs. Moreover, lowered expression of these aging markers remained after irMSCs differentiate along the osteogenic or chondrogenic lineages (FIG. 2E-2G) . FIG. 2H shows relative telomere length, expressed as a T/Sratio determined by qPCR, in Fib, irMSCs, autologous or allogeneic bMSCs, before and after differentiation into osteoblasts (OB) or chondrocytes (CH) . Cells from the same individual are shown in the same color in panels 2E–2H. Significance was determined when samples were compared to their own parental fibroblasts in all autologous cells (*) , *p<0.05, **p<0.01, ***p<0.001, n=3, and relative to their own bMSCs before and after differentiation into osteoblasts and chondrocytes (#) , #p<0.05, ##p<0.01, ###p<0.001, n=3.

Analysis of relative telomere length showed that irMSCs had longer telomeres than the parental fibroblasts and autologous bMSCs, and such telomere lengthening maintained after lineage differentiation of irMSCs (FIG. 2H) . Together, these data indicated that our induction method appeared to erase aging properties and reset irMSCs to a juvenile state regardless of the age of the donors.

Aged IrMSCs Regain Osteogenic and Chondrogenic Potentials

Aged bMSCs have a reduced capacity to differentiate into osteoblasts and are more likely to differentiate into adipocytes not only in vitro but also in vivo, resulting in yellow bone marrow filled with adipose tissues. Indeed, when bMSCs at P9 from 31-, 33-, 62-, 63-, and 82-year-old individuals were induced to differentiate, cells showed reduced and enhanced capacities to differentiate into osteo-and adipo-lineages, respectively, depending on the age of the donors See FIGs. 3A-3D. FIG. 3A shows that aged bMSCs attenuate osteogenic potential, while irMSCs from same aged donors reversed the phenotype. FIG. 3B shows that aged bMSCs biased toward adipogenesis, while irMSCs from same aged donors also reversed the phenotype. FIGs. 3C and 3D show q-RT-PCR analysis of ALP (osteogenic marker) and PPARG (adipogenic marker) expression, respectively, in osteoblasts and adipocytes derived from bMSCs and irMSCs from donors with different ages. This was further supported by the expression of lineage-specific markers alkaline phosphatase (ALP) for bone and peroxisome proliferator-activated receptor gamma (PPARG) for fat. In contrast, at P9 irMSCs from 38-, 39-, 62-, 63-, and 74-year-old individuals were all fully capable of osteogenic lineage differentiation and had reduced adipogenesis, similar to bMSCs from young (31-, 33-year-old) donors. We also examined the levels of osterix (OSX) and osteoprotegerin (OPG) , which promoted bone formation, and found that while aged bMSCs expressed low levels of these two genes, irMSCs expressed these genes at high levels regardless of the age of the donors, upon differentiation (FIG. 2L) . As shown by this experiment, osteoblasts derived from different irMSC (irMSC-OB) exhibit heightened expression of osteogenic genes (OSX and OPG) from all aged donors, while osteoblasts derived from bMSCs from aged donors had reduced osteogenic gene expression. *p<0.05, **p<0.01, ***p < 0.001, n=3, colored stars denote significance compared to left most bar (31Y) of the same group (bMSC-OB) .

These observations further support the notion that irMSCs, but not bMSCs, from aged donors behave similarly to those from young donors.

Previous studies have shown that the matrix biosynthesis activity of chondrocytes, such as type-II collagen (COL2A1) production declined with age, while senescence-associated matrix metalloproteinase (MMP) 13 (Mcculloch et al., 2017; Pestka et al., 2011) , which degrades the cartilage type II collagen lattice, was up-regulated. Adult MSCs undergo premature hypertrophy with heightened expression of MMP13 after transplantation in vivo (Steinert et al., 2007) , which undermines hyaline articular cartilage repair. Here we found that bMSCs from aged donor decrease COL2A1 and increased MMP13 expression when undergoing chondrogenesis (FIGs. 3E-3G) . *p < 0.05, **p<0.01, ***p < 0.001, n=3, colored stars denote comparisons to the left-most bars (youngest samples) within each colored groups; black starts denote comparisons between autologous cells as indicated. However, aged irMSCs, behaved like young bMSCs upon differentiation into chondrocytes.

Safety Profiles of IrMSCs

To address whether youthful irMSCs, like iPSCs, are prone to tumor formation, we first studied expression of a hESCs/iPSC marker, Oct 4, which is also frequently expressed in cancerous cells. Our results indicated that unlike iPSCs, irMSCs do not express the pluripotency gene, Oct4 (FIG. 2M) . We found that irMSCs were genetically stable and carried the same karyotype as parental fibroblasts even after long-term passaging (at P9 or even at P13) (FIG. 3H) . In addition, cellular morphology at P9 is indistinguishable from that at P13 (FIG. 2N) , showing that irMSCs maintained a normal morphology after long-term expansion at P9 and P13.

) . Unlike hESCs, which can form teratoma after subcutaneous injection into immune compromised NOD/SCID mice, irMSCs did not form tumors (FIG. 3I) . We transplanted, in NOD/SCID mice, various irMSCs from different donors and at different passages for 30 days, 60 days, and up to 250 days, and never found tumor formation from irMSCs (Table 1) .

Table 1. A Summary of Teratoma Formation Rates from IrMSCs in NOD-SCID mice, Related to FIG. 3.

Tumor-formation rate was shown as number of mice with tumor /total number in one set of experiment.

During irMSC induction, relative telomere lengths in D5 and D12 cells were not lengthened, however by D15 a sudden increase in telomere length was observed. Such lengthened telomeres remained relatively stable during passaging of irMSCs up to 13 passages (FIG. 3J) . In fact, we found that telomere length is in general stable in fibroblast cultures as well. Only after really long-term passaging, did we start to observe a statistically significant small decline (FIG. 3J) . *p＜0.05, **p＜0.01, ***p＜0.001, n≥3, compared to D0 (Fib, 39Y at P6) . Notice that TERT induction precedes telomere lengthening, and TERT elevation is transient, during the induction and in early irMSC passages. Unlike cancerous H293T cells, where telomerase reverse transcriptase (TERT) expression remains high, irMSCs at late passages as well as fibroblasts all have relatively low expression of TERT, therefore should not be cancerous. During the induction process, TERT expression was heightened at D5, remained high at D12, D15, and passage 2 of irMSC, but then declined at passage 5 and on-ward. Interestingly there is a delay between increased TERT mRNA expression and telomere lengthening. This could be due to the possibility that it takes time to assemble functional telomerase complex with additional components. We view this transient induction of telomerase activity a good safety feature. Long-term passaged irMSCs with low TERT expression but lengthened telomeres should represent good safety features.

IrMSCs Promote Bone Repair after Transplantation In Vivo

To determine whether human irMSCs can enhance bone regeneration, we transplanted matrigel, human parental fibroblasts, human bMSCs, or human irMSCs into femurs of NOD/SCID mice with severe surgical lesions. Bone repair was assessed by macroscopic, radiographic, and histologic examinations 28 days after transplantation. Macroscopic images and histological analysis by hematoxylin and eosin (H&E) staining revealed that bridging callus formation at lesion site was most extensive in irMSC-transplanted femurs (FIG. 4A-4B) . To assess bone matrix formation by cell grafts, nuclei of human irMSCs were stained with Hoechst 33342 before transplantation into the bone lesion site (FIG. 4C and FIG. 4F) . An abundance of fluorescently labeled irMSC nuclei were observed under ultraviolet light in the callus and bone marrow cavity at the lesion site. Accordingly, H&E-stained tissues revealed parallel collagen bundles of the lamellar structure in new bone tissue under polarized light and highly mineralized new bone matrix under ultraviolet light (FIG. 4F) . Thus, the number and location of Hoechst-positive human cells corresponded to newly formed bone cells highlighted by H&E staining, indicating that transplantation of irMSCs induced bone healing by a cell-replacement strategy. This conclusion was further supported by immune staining using an antibody against human-specific antigen CD29 (FIG. 4G) , as new bone formed by irMSC showed obvious positive response with human-specific CD29 staining. IBSP is another marker for the osteogenic lineage. Moreover, there was no abnormal tissue around the regenerated area, indicative of a lack of tumor formation by transplanted irMSCs. Micro-computed tomography (micro-CT) revealed that matrigel, parental fibroblasts, and bMSCs from a 62-year-old donor had a minimal, if any, effect on bone repair, whereas bMSCs from a 31-year-old donor and irMSCs from both 39-and 62-year old donors elicited significant bone tissue regeneration (FIG. 4D-E) . For FIG. 4D, NOC/SCID mice were sacrificed on day 28 after transplantation of irMSCs, and micro-CT imaging of the femoral diaphysis was performed. 10-μm serial slices are shown. Arrows indicate bone lesions and arrowheads represent regenerated bone tissue. Y = year. For FIG. 4E, BV/TV, Tb.N, and BMD were determined from micro-CT measurements. BV/TV, Bone volume/Total volume; Tb. N, Trabecular Number; BMD, bone mineral density. *p<0.05, **p<0.01, ***p<0.001, n=5, compared to Neg (no implant) control. Y=year.

The extent of bone-repair was quantified by measuring bone volume (BV) /total volume (TV) , trabecular number (Tb. N) , and bone mineral density (BMD) . IrMSCs derived from donors of all ages were rather effective for bone repair.

IrMSCs Elicits In Vivo Cartilage Regeneration

We evaluated the regenerative potential of irMSCs using an articular cartilage injury model. Matrigel, parental fibroblasts, bMSCs, or irMSCs were transplanted into the patellar groove of NOC/SCID mice. By 4 weeks there was little evidence of cartilage repair in the first two groups or with bMSCs from the 62-year-old donor (FIG. 5A-C) . For FIG. 5B, slices are 10 μm in thickness. Red staining shows hyaline cartilage and green shows fibrocartilage or bone. Arrowheads denote the injured/regenerated area. In FIG. 5C, cartilage repair was scored according to Pineda’s system, from 0 (best) to 14 (worst) . Results represent mean ± SD (n =5 animals per group; 30 animals in total) . Significance was determined relative to Neg (no implant) , *p<0.05, **p<0.01, ***p<0.001, n=5. In contrast, histological assessment and Pineda’s histologic grading indicated that irMSCs from both young and old donors and bMSCs from a young donor had generated new cartilage (FIG. 5A) . Grafts from irMSC developed a proteoglycan-and type II collagen–rich extracellular matrix for cartilage, with no evidence of calcification or mineralization (FIG. 5E-5G) . FIG. 5E shows a repaired patellar groove area stained with Safranin O, von Kossa, and Toluidine Blue to visualize proteoglycans (Safranin O and Toluidine Blue) and calcium deposition (von Kossa) at the lesion site by grafted irMSCs. FIG. 5F and FIG. 5G show mmunofluorescence analysis of Col2a1 and aggrecan (ACAN) expression in newly formed cartilage in irMSC implanted group.

There was no abnormal tissue around the regenerated area, again indicative of a lack of tumor formation from irMSCs. Viable human cells with Hoechst 33342-stained nuclei or stained by antibodies against a human-specific antigen CD29 were detected in regenerated cartilage tissue (FIG. 5D and FIG. 5H) , which corresponded to newly formed cartilage cells that were positive for SOX9 (FIG. 5I) . As shown in FIG. 5H, new chondrogenic tissue formed by irMSCs showed obvious positivity for human-specific CD29 staining; while in FIG. 5I, the patellar groove was labeled with an antibody against (sex-determining region Y) -box (SOX) 9. Hoechst 33342-prestained human nuclei were found to co-label with SOX9, indicative of human irMSC-derived chondrocytes involved in cartilage repair.

Together, these observations indicate that human irMSCs contribute to cartilage repair through differentiation into chondrocytes for cell replacement.

IrMSCs Reverse Tissue Degeneration and Prolong Lifespan of Aged NOD/SCID Mice

Given that irMSCs from both young and aged donors have similarly robust regenerative capacity in vivo, but not their parental fibroblasts or autologous bMSCs from aged donors, we investigated whether rejuvenated cells administered by intravenous transfusion could reverse age-related phenotypes and extend lifespan. Passage 13 irMSCs from a 39-year-old donor labeled with Hoechst 33342 were injected into 43-week old, chronologically aged NOD/SCID mice via tail veins at a dose of 106 cells/animal. Only one injection was given. Lifespan was substantially extended in mice treated with irMSCs (FIG. 6A) as compared to animals injected with vehicle (DMEM) (n = 5/group) . Moreover, these animals showed great improvement in their appearance based on their fur coats and reduced spine curvature (FIG. 6B and FIG. 6L) as compared to DMEM medium (vehicle) -treated animals. Mice (8 animals) in FIG. 6L were followed starting from 10-week-old: when these animals reached 43-week-old, they were either injected with 10 ⁶ irMSCs or DMEM (fresh culture medium/non conditioned) via tail veins. Their pictures were taken at 47W, 4 weeks after cell injection. In the case of DMEM injection, pictures were taken when animals die before reaching 47 weeks of age, with one exception.

IrMSC-injected animals also showed more motor activities. When the aged animals died by natural causes, we observed their internal organs, and found that irMSC injected animals had digestive systems resembling those of young (25W) untreated animals, while DMEM injected animals had digestive systems that appeared discolored and malfunctioning (FIG. 6C and FIG. 6M) . The necropsy images in FIG. 6M are for the treated and untreated animals in the study described for FIG. 6L above. Consistently, stomach tissue in irMSC-treated animal had more regular gastric mucosa and age-related loss of parietal cells was recovered by irMSC transfusions, indicative of rejuvenation (see necropsy images in FIG. 6D of H&E staining of stomach tissues from young and aged NOD/SCID mice before and after irJSC (39Y, P13) injections) . After the aged animals had died naturally, their tissues were also analyzed for the presence of the injected human cells by genomic PCR detection of human-specific actin sequence and by fluorescence microscopic visualization of Hoechst 33342-positive nuclei in frozen tissue sections. IrMSCs were detected in stomach, liver, lung, and spleen (FIGs. 6N-6O) . FIG. 6N shows that Hoechst 33422-stained cells were distributed in the stomach, spleen, lung, and liver of aged SCID mice treated with irMSCs. This representative animal was injected with human irMSCs at 43W and analyzed at 58W when the animal died. FIG. 6O shows where human-specific actin gene in different organs was detected by PCR of genomic DNAs (gPCRs) , using a human-specific actin sequence. This representative animal was injected with human irMSCs at 43W and analyzed at 58W when the animal died.

In skeletal tissues, we also detected human DNA within mice lumbar spine (FIG. 6P: human-specific actin gene was detected by gPCR after the animals died of natural causes) . Since osteoporosis is a medical condition associated with aging, we carried out more detailed analyses on aged skeleton following irMSC transfusion. As expected, 43-week-old NOD/SCID mice after irMSC intravenous injection displayed improved lumbar spine trabecular bone microarchitecture (FIG. 6E-6F) . For FIG. 6F, BMD (%) , bone volume fraction; Tb. N (1/mm) , trabecular number; Tb.Th (mm) , trabecular thickness; Tb. Sp (mm) , and trabecular separation were determined by micro-CT. Significance was determined relative to Control (10W) , *p<0.05, **p<0.01, ***p<0.001, n=5. Various micro-CT parameters all revealed that irMSC injected NOD/SCID mice were rejuvenated with bone structures resembling their 25-week-old counterparts (FIG. 6E-6F) . This result is in agreement with decreased expression of another aging marker p16Ink4a with irMSC-administration (FIG. 6G: statistical significance was determined relative to Control (10W) , *p<0.05, **p<0.01, ***p<0.001, n=5. ) . Additionally, both bone formation and bone resorption activities, manifested by bone ALP and tartrate-resistant acid phosphatase (TRAP) staining in the lumbar spine of irMSC-injected mice were restored to the level of younger mice (FIG. 6H-6J: statistical significance in FIG 6I-6J was determined relative to Control (25W) , *p<0.05, **p<0.01, ***p<0.001, n=5.

) . Accompanied by the increased activity of the osteoblasts, we identified injected irMSCs in bone and bone marrow by immunodetection of human-specific CD29 and found that a subset of the cells had differentiated into osteoblasts, as confirmed by co-labeling with anti- osteocalcin antibodies. Injected irMSCs were detected by immunohistochemical analysis using antibodies against human-specific CD29 (green) and osteocalcin (red) in lumbar spines of two NOD/SCID mice injected with human irMSCs via tail veins at 43W and analyzed at 58W (FIG. 6K) or 47W and 49W, respectively (FIG. 6Q) .

Transcriptomic Analysis of Signaling Pathways Involved in the Conversion of Fibroblasts to IrMSCs

To explore potential underlying molecular mechanisms involved in the conversion of fibroblasts to irMSCs, we performed Weighted Gene Coexpression Network Analyses (WGCNA) on transcriptome of parental fibroblasts, D5, D12 transitional cells, D15 irMSCs and bMSCs: FIG. 7I shows WGCNA data of 12, 036 genes whose expression was altered during the conversion process, and reveals 12 co-expressed gene modules. FIG. 7J is a heat map showing correlation between modules and cell types for this data: numbers of each square represent correlation of modules and cell states; the P value of each correlation value is shown in parenthesis. Red, positive correlation; Green, negative correlation; White, no correlation. Using this unbiased, objective “big-data” processing approach, 12 gene modules (clusters) were identified (FIGs. 7I-7J) . Based on the “module-trait” relationship plot, “darkred” , “salmon” , and “cyan” modules are highly expressed in (positively correlated with) parental fibroblasts. Genes enriched in these three modules are related to "TGFβ signaling pathway" , "ECM-receptor interaction" , “Focal adhesion” (darkred) , “JAK-STAT signaling pathway” (salmon, cyan) , “Notch signaling pathway” (cyan) , “NOD-like receptor signaling” , “Gap junction” , and “p53 signaling pathway” (cyan) , all of which decreased expression upon the conversion process (FIG. 7A-7B and FIG. 7K) . The bar plots in FIG. 7K show KEGG pathways enriched in eight modules (the other 4 modules are shown in FIG. 7) ; representative genes for each KEGG pathways were shown in the order of gene’s module membership. Box plots show distribution of averaged expression level of genes in each module.

No gene modules are highly enriched in D5 transitional cells, however, “cyan” and “darkgrey” (focal adhesion and cell adherence junction) gene modules are most negatively correlated, indicative of decreased expression in D5 samples. The “blue” and “grey” modules are highly expressed in D12 samples, and related genes are in the “Notch signaling” (blue) , “Wnt signaling” (grey) , as well as “extracellular matrix (ECM) ” (grey) . D12 samples had profound reduction in “darkred” , “darkgrey” , and “turquoise” (cell cycle, DNA mismatch repair) gene expression, indicative of reduced cell cycling at D12, which was also observed in actual cell cultures. For D15 samples, when irMSCs were finally generated and cells acquired “tri-lineage” differentiation potentials, only “turquoise” and “skyblue” genes were relatively highly expressed, indicative of regained and enhanced cell cycling potential (turquoise) and increased TCA cycle and metabolic pathways (skyblue) . bMSC samples have profound high expression of “greenyellow” genes, which were involved in “Hedgehog and Wnt” signaling, and at the same time profound low expression of “salmon” , “blue” , and “skyblue” genes. Based on WGCNA, the data demonstrated that regulation of JAK-STAT, TGFβ signaling, ECM-receptor interactions, cell adhesions, actin cytoskeleton, Wnt, Hedgehog, and Notch signaling, p53, cell cycle, and cell metabolic processes all seemed to be involved in the final conversion of fibroblasts to irMSCs. Mix V is designed to activate Wnt, cAMP/PKA, inhibit TGFβ, and HDAC, while compound Mix P is to activate Wnt, cAMP/PKA, RAR, but inhibit TGFβ, HDAC, PKC and Rho. In parallel, these molecular effects were confirmed by RNA-seq data or q-RT-PCR analysis of their target genes (Table 2) . This objective analysis is consistent with the nature of small molecules used in the process, demonstrating the power of unbiased transcriptomic analyses. WGCNA of the transcriptome showed expression of age-related pathway genes increased with aging, including those in p53 signaling (Gorgoulis and Halazonetis, 2010; Serrano et al., 1997) , NOD-like receptor pathway (Ebersole et al., 2016) , JAK-STAT signaling (Ming et al., 2015) . These genes decreased expression during the fibroblast-to-irMSC conversion at D5 and D12, and remained low at D15. These results confirmed that treatment with Mix V and Mix P rejuvenated cells.

Given its appearance in multiple gene modules and the fact that it was not directly targeted by our small molecular treatment, the JAK-STAT pathway was analyzed, and the level of STAT5A was markedly reduced at D5, D12, and D15 (FIG. 7L) . This pathway was thought to be associated with aging, and FIGs. 7M-7P shows that MSCs with time in culture exhibited lower propensity to differentiate into the osteocyte lineage, and increased propensity for differentiation into adipocytes. Inhibition of JAK-STAT pathway genes belonging to the cyan and salmon modules such as SOS1, STAT5B, JAK1, JAK3, SOCS3, IL6ST, and etc., and other JAK-STAT members such as STAT1, STAT5A and STAT6, were found to be associated with fibroblast cell rejuvenation and acquisition of tri-lineage-particularly bone and cartilage-differentiation potential. Among these, STAT5A declined significantly during conversion and also appeared to be an aging marker (FIG. 7C, a heat map of JAK-STAT members that are upregulated in P21 MSCs, and are thus age-related) . To determine whether inhibition of the JAK-STAT pathway is sufficient for the conversion of fibroblasts into irMSCs and rejuvenation, we knocked out STAT5A in fibroblasts from 39-and 63-year-old individuals using the CRISPR/Cas9 system. FIG. 7Q shows STAT5A expression was not detected in parental fibroblasts after its knockout, and also was not observed in irMSCs derived from parental fibroblasts without STAT5A knockout. STAT5A expression was detected in fibroblasts treated with control lentivirus. Notably, simply knocking out of STAT5A was sufficient to allow fibroblasts to differentiate into bone and cartilage (FIG. 7D-E) . Alizarin Red and Alcian blue staining of fibroblasts with and without STAT5 knockout is shown in FIG. 7D. Fib-Control, fibroblasts treated with empty lentivirus for CRSPR-cas9-mediated STAT5 knockout; Fib-STAT5-KO, fibroblasts with STAT5 knockout. Y, year. FIG. 7E shows Expression of ALP (osteogenic marker) and COL2A1 (chondrogenic marker) in fibroblasts with and without STAT5 knockout. *p<0.05, **p<0.01, ***p<0.001, n=3. STAT5A-knockout decreased expression of an epigenetic aging marker H4K20me3, and downregulated GADD45B and CDKN1A expression without changing H3K9me3 (FIG. 7F-7G (*p < 0.05, **p<0.01, ***p < 0.001, n=3) and FIG. 7R) . However, simply knockout STAT5A was not able to lengthen the telomeres of STAT5A knockout fibroblasts, even after a 55-day culturing time, whereas it only took 15 days for our induction method to lengthen telomeres from parental fibroblasts (FIG. 7S-7T) . Moreover, STAT5A knockout was also not capable ofreversing expression of two other aging markers IL6 and ATF3 (FIG. 7U) . Together these observations indicated that inhibition of STAT5A could be partially involved in the “induction and rejuvenation” process by small molecular mixes. In the future, more biological validation experiments could be carried out to test how Wnt signaling, TGFβ signaling, changes in cells adhesions, and actin-cytoskeletons participate in rejuvenating skin fibroblasts and generating irMSCs, which harbor huge therapeutic potentials (FIG. 7H) .

Table 2. Targets of the Chemical Compounds during IrMSCs Conversion

EXPERIMENTAL PROCEDURES

IrMSC Conversion from Skin Fibroblasts

Primary fibroblasts cultures were generated from skin specimens donated by volunteers. All subjects signed informed consent, which was approved by the Medical Ethics Committee of the Department of Medicine in Kunming University. This study was conducted in accordance with the recommendations from the aforementioned Medical Ethics committee. Conversion of fibroblasts into irMSCs was initiated by seeding cells at 150,000 cells per well of a 6-well plate. At the next day (day 0) culture medium was changed to Mix V medium containing 0.5 mM VPA, 3 μM CHIR99021, 1 μM RepSox, and 10 μM Forskolin. At day 5, culture medium was again switched to Mix P medium containing 0.5mM VPA, 0.2mM Vc, 3 μM CHIR99021, 10 μM Forskolin, 10 μM SP600125, 5 μM Go6983, 5 μM Y-27632, 5 μM TTNPB, 0.05 μM AM580, 5 μM EPZ004777, 1 μM RepSox. At day 12, MixP medium was washed out and culture medium was switched to MSC basal medium with 10ng/ml bFGF, 100ng/ml PDGF-AB, 10ng/ml BMP4 for 3 days, when irMSCs were generated. For long-term expansion, irMSCs were cultured in MSC basic medium. Medium was changed every 2 days. Stepwise conversion protocols as well as detailed information about the medium used in this study are provided in herein.

Cell Transplantation

All experiments were conducted in accordance with Guidelines for Use and Care of Laboratory Animals and approved by the Medical Ethics Committee of the Department of Medicine of Kunming University. All efforts were made to minimize animal suffering as well as total numbers of rodents utilized in the study. Liver repair was conducted using C57BL/6 mice with ConA-induced liver injury. Conditioned Medium from various cells including irMSCs were concentrated and injected via tail veins. For cell implantation in vivo assays, NOD-SCID mice were used. Femur bone and cartilage repairs by irMSCs are described in details in the Experimental Procedures.

Intravenous irMSC injections

106 irMSCs from a 39-year-old donor labeled with Hoechst 33342, were harvested at passage 13 and diluted in 200μL DMEM. IrMSCs (10 ⁶/animal) were then injected into chronologically aged NOD-SCID mice (43 weeks old) via tail veins. In control group, 200μL DMEM were injected.

Transcriptome Analysis

Counts of each gene were estimated by HTSeq (0.6.0) and normalized to log transformed Counts per million mapped reads (logCPM) via R package edgeR. tSNE Analysis was performed on whole transcriptome without gene selections, but genes with logCPM >1 in at least 5 samples were selected for further analysis. DESeq2 was used to detect differentially expressed genes between any 2 conditions. 12036 Genes with adjust P <0.05 were passed to weighted gene co-expression network analysis (WGCNA) . WGCNA was performed as described in (Luo et al., 2015) . Specifically, power of 26 is interpreted as a soft-threshold of the correlation matrix. Modules whose eigengenes were highly correlated (correlation > 0.6) were merged. Function enrichment analysis and KEGG pathway analysis of each module were performed byTopGO.

Cell Culture

Primary fibroblasts were harvested from leg skin of healthy donors with various ages at 38, 39, 62, 63, 74 years old. Fibroblasts from healthy individual of an 8-year-old was harvested from foreskin. Fibroblast from 12-week embryos were isolated from aborted fetus at 12 weeks of gestation. Bone marrow mesenchymal stem cells were isolated from healthy individuals, with various ages at 26, 31, 33, 62, 63 and 82 years old. All sample collection procedures were approved by the Medical Ethics Committee of the Department of Medicine at Kunming University. This study was conducted in accordance with guidelines from the Medical Ethics Committee of the Department of Medicine.

Fibroblasts were expanded in growth medium consisting of High Glucose DMEM (HG-DMEM) (HYCLONE) , 10%fetal bovine serum (FBS) (BI) , 100U/ml penicillin and 100μg/ml streptomycin antibiotics (sigma) ; bMSCs were cultured in basic MSC medium (Cyagen, HUXMA-90011) ; All other cell culture supplements and reagents were acquired from Sigma.

Reagents: VPA, 0.5 mM (Selleck, 1 M stock in H2O) ; CHIR99021, 3 μM (MCE, 10 mM stock in DMSO) ; Repsox, 1 μM (MCE, 10 mM stock in DMSO) ; Forskolin, 10 μM (MCE, 50 mM stock in DMSO) ; SP600125, 10 μM (MCE, 25 mM stock in DMSO) ; Go6983, 5 μM (Selleck, 10 mM stock in DMSO) ; Y-27632, 5 μM (MCE, 20 mM stock in DMSO) ; TTNPB, 5 μM (Selleck, 8mM stock in DMSO) ; AM580, 0.05 μM (Selleck, 0.05mM stock in DMSO ) ; EPZ004777, 5 μM (Selleck, 50mM stock in DMSO) ; Vc, 0.2mM (Sigma, 0.2M stock in H2O) ; bFGF (Peprotech) , PDGF-AB (Peprotech) , BMP4 (Peprotech)

Medium:

Fibroblast medium: HG-DMEM supplemented with 10%fetal bovine serum (FBS) ;

Inducing medium: Mix V and Mix P

Mix V: HG-DMEM supplemented with 10%FBS, containing VPA, 0.5 mM; CHIR99021, 3μM; Repsox, 1 μM; Forskolin, 10 μM;

Mix P: HG-DMEM supplemented with 10%FBS, containing VPA, 0.5 mM; CHIR99021, 3 μM; Repsox, 1 μM; Forskolin, 10 μM; SP600125, 10 μM; Go 6983, 5 μM; Y-27632, 5 μM; AM580 0.05μM; EPZ004777 5μM; Vc, 0.2mM; TTNPB, 5μM

Cell Conversion

Procedure: 1. Initial fibroblasts were seeded onto 6-well culture plates (100,000-150,000 cells/well in 6-well plates) and cultured in fibroblast medium for 24 hours. 2. Cells were then transferred to irMSC induction medium with chemical cocktails Mix V. Medium containing chemical compounds was changed every two days. 3. Induction medium was changed after 5 days with chemical cocktails Mix P for another 7 days; 4. On day12, medium was changed to HG-DMEM supplemented with 10%FBS with bFGF 10ng/ml, PDGFAB 100ng/ml, BMP4 10ng/ml. 5. For long-term expansion, irMSCs were cultured in basic MSC medium.

Immunofluoresent Microscopy and Flow Cytometric Analysis

Cells plated on 24-well were fixed by 4%PFA solution for 10 min and then changed to PBS at room temperature. Cells were then treated with 0.1%Triton X-100 for 10 min, followed by incubation in blocking buffer (3%bovine serum albumin in PBS) for 30 min. Afterwards, samples were incubated with primary antibodies at 4℃ overnight and then with appropriate fluorescent probe-conjugated with secondary antibodies for 2 h at RT. Nuclei were counter-stained with DAPI. Images were captured with fluorescence microscope (Nikon) . The primary antibody is including FSP-1 (abcam) , Oct4 (abcam) , STAT5A (abcam) , human specific CD29 (abcam) , osteocalcin (abcam) , IBSP (abcam) , aggrecan (abcam) , COL2A1 (abcam) , MMP13 (abcam) , γH2AX (abcam) , H3K9me3 (abcam) , H4K20me3 (abcan) and SOX9 (abcam) .

For flow cytometric analysis, samples were stained with antibodies for 30 min at 4℃ in staining buffer (PBS, 3%FCS) , and analyzed by flow cytometry on BD FACSJazz (BD biosciences) . The antibodies including CD49a-PE, CD49e-FITC, CD90-PerCP-Cy5.5, CD105-eFluor450, CD73-FITC, CD44-APC, CD14-FITC, CD34-FITC, CD45-PE, CD19-APC and HLA-DR-eFluor450, all were acquired from BD biosciences.

PCR for ordinary genes

Total RNA was extracted with TRIzol reagent (Takara Bio) according to manufacturer’s instructions. The RNA (1.0 μg) was reverse-transcribed to cDNA using Primescript RT reagent Kit (Takara Bio) . For qRT-PCR, the cDNA was used as a template along with specific primers and SYBR Green using SYBR Premix EX TaqTM II (Takara Bio) . Cycling conditions were according to manufacturer’s instructions. The relative expression levels were normalized to that of the internal control (ACTIN) . For gPCR, genomic DNA was used as the template for human specific primer of ACTIN using Premix Taq (Takara Bio) . Primers used are listed in Table 3.

Table 3. Primer List Related to Experimental Procedures

Quantification of γH2AX

For quantification of immunofluorescence microscopy images, a total of 100 cells from multiple culture repeats per condition were counted to determine the number of γH2AX foci per cell. To ensure reliable quantification, image recording was performed under the same scanning conditions for all the samples.

Quantitative Real-time (qRT-) PCR for Telomere Measurement

Average telomere length was measured using total genomic DNA by qRT-PCR on a 7900HT Fast Real-Time PCR system (Applied Biosystems) using telomeric primers according to Cawthon’s method (Cawthon, 2002) . The thermal cycling profile for telomere signal TEL (T) in telomeric PCR was as follows: 95℃ for 30s; and 25 cycles of 95℃ for 5 s, 56℃ for 30 s and 72℃ for 30 s. The single-copy gene 36B4 (S) was used as an internal reference, thermal cycling profile of which was 95℃ for 30s, and 40 cycles of 95℃ for 5 s, 60℃ for 30s. The telomere signal (T) was normalized to the single-copy gene signal (S) to obtain the T/Sratio reflecting relative telomere length. Primers used are listed in Table 3.

Immune Modulation Assay In Vitro

Handled with Mitomycin C for 2.5h, digestion and counting, cells were seeded on 24-well plate with 1×10 ⁵ cells/well. Lymphocytes were stained with Carboxyfluoresce indiacetate succinimidyl ester (CFDA-SE) at 37℃ for 30min, and seeded on this plate with a density of 2×10 ⁵ cells/well. PHA at the final concentration 2μg/ml was used to stimulate proliferation of lymphocytes. 5 groups were designed: bMSC + lymphocytes + PHA contact culture group, irMSC + lymphocytes + PHA contact culture group, fibroblast + lymphocytes +PHA contact culture group, lymphocytes + PHA positive control group, lymphocytes-only negative control group. After co-culturing for 5 days, lymphocytes from each well were collected and washed with PBS three times. Proliferation of lymphocytes was assessed by the flow cytometer using antibodies against CD3, CD4 and CD8, which were all acquired from BD biosciences.

Immune Modulation Assay In Vivo

The culture medium from bMSCs, irMSCs, fibroblasts at a density of 1×10 ⁶ were filtered through the 0.22μm filter (Millipore) to remove residual cells or cell debris, and were concentrated for about 100 times using ultrafiltration centrifugal concentration tube (Millipore) . C57BL/6 mice (8-12 weeks old) were injected with ConA in PBS at 25 mg/kg via tail veins to induce acute inflammatory liver injury and injected PBS as control (Han et al., 2014) . Six mice per group were used. After 30min, mice were injected via tail veins with various concentrated conditioned medium or PBS. 8.5h after Con A injection, mice were euthanized. Blood and liverwere harvested. The liver stained with HE, CD3+T flow cytometry assay and blood was tested for ALT/AST.

The accumulation of AST and ALT in the serum was quantified using ELISA kits (Shanghai enzyme association) according to manufacturer’s instructions. Samples collected from three independent experiments were measured, and data were shown as means ± SD.

Femoral Defect Model

NOD/SCID mice aged 8-10 weeks weighing at 18-24g were used, according to local ethical committee’s approval. All efforts were made to minimize animal suffering as well as the number of rodents utilized. 5 animals per group were used. A femoral defect model was used as previously reported (Yamamoto et al., 2015) . Briefly, under pentobarbital anesthesia, incision of skin and subcutaneous tissues was performed to expose the muscle white line, which provided sufficient exposure of femurmid-diaphysis by blunt separation in rectus femoris and semitendinous. Centered proximal intersection of the right side of the femur was performed. The operation created a 4mm x 1.5mm successional bony defect. Human fibroblasts (Fibs) , bMSCs, and irMSCs were stained with Hoechst 33342 (Thermo, NucBlue live cell) and then mixed with Matrigel and transplanted into the lesion site at a dose of 6×10 ⁵ cells/mouse.

28 days after transplantation, mice were euthanized with a lethal dose of pentobarbital. Thighs were dissected, fixed with 4%PFA, and subjected to μCT imaging (SkyScan 1272, Bruker microCT) , and then the data was analyzed by CT Analyzer.

Articular Cartilage Defect Model and Cells Transplantation

NOD/SCID mice (8–10 weeks old, weighing 18–24 g) were used according to guidelines from the Medical Ethics Committee of the Department of Medicine at Kunming University for animal use and care. A modified articular cartilage deficit model was used to assess therapeutic potentials of irMSCs (Cheng et al., 2014) . Articular cartilage defects (2 × 1 mm wide and 0.8 mm deep) were created in the trochlear groove of the distal femur using a biopsy punch. Cells (2.5 × 10 ⁵ in 35 μl of Matrigel) were labeled with Hoechst 33342 using NucBlue Live Cell Stain Ready Probes reagent (Life Technologies) was implanted into the lesion site. Matrigel without cells was implanted as controls.

Teratoma Formation Assay in NOD-/SCID Mice

Male immunodeficient NOD-/SCID mice aged 8-10 weeks were used. Undifferentiated ESCs or irMSCs were harvested at 2x10 ⁶, mixed with Matrigel in a 1: 1 ratio, and injected at a volume of 200μL subcutaneously. Mice were euthanized at different sampling time point and excised tumors or implants were fixed in PFA (Sigma-Aldrich) .

Histomorphometric Analyses

Bone tissue was isolated and were fixed in 4%PFA, decalcified for 1～ 2 weeks in 10%EDTA and were paraffin-embedded or frozen sectioned. Or the fixed tissue was non-decalcified and then sectioned. Sections were stained with Hematoxylin and Eosin, toluidine blue, von Kossa, or safranin O (counterstained with fast green) , as indicated.

The histomorphometric analyses were performed in a blinded fashion. The lumbar vertebrae was stained with H&E to assess osteoblast numbers per bone perimeter (N. Ob/B. Pm; /mm) , or stained for TRAP activity to assess osteoclast numbers per bone perimeter (N. Oc/B. Pm; /mm) (Bian et al., 2011; Farr et al., 2017) .

Statistical Analysis

Student T test was used for simple two sample comparisons. One-way ANOVA plus posthoc Tukey HSD Test was used for multiple comparisons between different sample groups. p<0.05 was regarded as statistically significant changes. Tests were performed by IBM SPSS Statistics (v22) .

Claims

A process for preparing induced and rejuvenated mesenchymal stem cell (irMSC) , which process comprises:

a) contacting a primary fibroblast, e.g., dermal fibroblast, with

i) a first molecule cocktail to activate an activity of Wnt and/or cAMP/PKA, and/or to inhibit an activity of TGFβ and/or HDAC in said primary fibroblast, or

ii) a first small molecule cocktail, e.g., Mix V, comprising at least one of valproic acid (VPA) , CHIR99021, Repsox and Forskolin, e.g., 1, 2, 3, or 4 of valproic acid (VPA) , CHIR99021, Repsox and Forskolin,

for a first time to form a first treated cell;

b) contacting said first treated cell with:

i) a second molecule cocktail to activate an activity of Wnt, cAMP/PKA and/or RAR, and/or to inhibit an activity of TGFβ, HDAC, PKC and/or Rho in said first treated cell, or

ii) a second small molecule cocktail, e.g., Mix P, comprising at least one of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox,

for a second time to form a second treated cell; and

c) culturing said second treated cell in a mesenchymal stem cell (MSC) medium without adding exogenous or in the absence of bFGF, PDGF-AB and BMP4 for a third time to form irMSC.
The process of claim 1, wherein the primary fibroblast is generated from a skin biopsy specimen, urine or blood.
The process of claim 2, wherein the skin biopsy specimen is cultured for at least 6 passages to generate the primary fibroblast.
The process of any one of claims 1-3, wherein the step a) comprises contacting a primary fibroblast with a first small molecule cocktail, e.g., Mix V, comprising at least one of valproic acid (VPA) , CHIR99021, Repsox and Forskolin, e.g., 1, 2, 3, or 4 of valproic acid (VPA) , CHIR99021, Repsox and Forskolin, for a first time to form a first treated cell.
The process of claim 4, wherein the VPA in Mix V has a level ranging from about 0.1 mM to about 0.7 mM.
The process of claim 4 or 5, wherein the CHIR99021 in Mix V has a level ranging from about 0.5 μM to about 5μM.
The process of any one of claims 4-6, wherein the Repsox in Mix V has a level ranging from about 0.5 μM to about 5μM.
The process of any one of claims 4-7, wherein the Forskolin in Mix V has a level ranging from about 1μM to about 20 μM.
The process of any one of claims 4-8, wherein the step a) comprises contacting a primary fibroblast with a first small molecule cocktail, e.g., Mix V, for a first time ranging from about 1 day to about 5 days to form a first treated cell.
The process of any one of claims 1-9, wherein the step b) comprises contacting said first treated cell with a second small molecule cocktail, e.g., Mix P, comprising at least one of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 of valproic acid (VPA) , vitamin C (Vc) , CHIR99021, Forskolin, SP600125, Go6983, Y-27632, TTNPB, AM580, EPZ004777 and RepSox, for a second time to form a second treated cell.
The process of claim 10, wherein the VPA in Mix P has a level ranging from about 0.2 mM to about 1 mM.
The process of claim 10 or 11, wherein the Vc in Mix P has a level ranging from about 0.05 mM to about 0.8 mM.
The process of any one of claims 10-12, wherein the CHIR99021 in Mix P has a level ranging from about 1μM to about 8μM.
The process of any one of claims 10-13, wherein the Forskolin in Mix P has a level ranging from about 5 μM to about 20 μM.
The process of any one of claims 10-14, wherein the SP600125 in Mix P has a level ranging from about 1μM to about 25μM.
The process of any one of claims 10-15, wherein the Go6983 in Mix P has a level ranging from about 1μM to about 25μM.
The process of any one of claims 10-16, wherein the Y-27632 in Mix P has a level ranging from about 1 μM to about 15μM.
The process of any one of claims 10-17, wherein the TTNPB in Mix P has a level ranging from about 1 μM to about 20 μM.
The process of any one of claims 10-18, wherein the AM580 in Mix P has a level ranging from about 0.01 μM to about 0.8μM.
The process of any one of claims 10-19, wherein the EPZ004777 in Mix P has a level ranging from about 1μM to about 10 μM.
The process of any one of claims 10-20, wherein the RepSox in Mix P has a level ranging from about 0.1μM to about 2 μM.
The process of any one of claims 10-21, wherein the step b) comprises contacting said first treated cell with a second small molecule cocktail, e.g., Mix P, for a second time ranging from about 1 day to about 7 days to form a second treated cell.
The process of any one of claims 1-22, which further comprises, before step c) , a wash step to reduce or remove components of the first molecule cocktail, the first small molecule cocktail, the second molecule cocktail or the second small molecule cocktail.
The process of any one of claims 1-23, wherein the MSC medium used in step c) is a fresh MSC medium, e.g., a MSC medium that is not conditional or that has not been used for culturing cells before.
The process of any one of claims 1-24, wherein the MSC medium used in step c) comprises serum, e.g., MSC medium from Cyagen, catalog No.HUXMA-90011, or GMP compatible serum-free MSC medium, e.g., MSC medium from Hcell, name rFib medium, catalog No. CRM0016-01.
The process of any one of claims 1-25, wherein the step c) comprises culturing said second treated cell in a MSC medium without adding exogenous or in the absence of bFGF, PDGF-AB and BMP4 for a third time that is at least about 3 days to form irMSC.
The process of any one of claims 1-26, which is used to form a single irMSC.
The process of any one of claims 1-26, which is used to form a plurality or population of irMSC.
The process of any one of claims 1-28, which is used to form a non-human mammalian irMSC, e.g., a mouse irMSC.
The process of claim 29, wherein the hon-human mammalian irMSC is formed from a primary fibroblast obtained from a non-human mammal having an age of at least about 5 weeks.
The process of any one of claims 1-28, which is used to form a human irMSC.
The process of claim 31, wherein the human irMSC is formed from a primary fibroblast obtained from a human having an age of at least about 5 years.
An irMSC prepared by a process of any one of claims 1-32.
The irMSC of claim 33, which is prepared from a primary fibroblast of a human.
The irMSC of claim 34, which is prepared from a primary fibroblast of a human having an age of at least 5 years.
The irMSC of any one of claims 33-35, wherein a fibroblast marker, e.g., fibroblast-specific protein-1 (FSP-1) , is down-regulated.
The irMSC of any one of claims 33-36, wherein a MSC-related cell surface marker, e.g., CD49a and/or CD49e, is up-regulated.
The irMSC of claim 37, wherein both CD49a and CD49e are up-regulated.
The irMSC of any one of claims 33-38, which is configured to suppress T cell proliferation, e.g., to suppress T cell proliferation in vitro.
The irMSC of any one of claims 33-39, which acquires self-renewal, tri-lineage differentiation and/or immune modulation property comparable to that of a bone-marrow-derived mesenchymal stem cell (bMSC) .
The irMSC of any one of claims 33-40, wherein an aging epigenetic marker histone, e.g., H4 lysine 20 trimethylation (H4K20me3) and/or age-related DNA damage marker, e.g., phosphorylated histone H2AX (γH2AX) is downregulated.
The irMSC of any one of claims 33-41, which has a greater proliferative capacity than its parental fibroblast.
The irMSC of any one of claims 33-42, which grows more rapidly than autologous bMSC.
The irMSC of any one of claims 33-43, which grows faster than bMSC from a younger donor.
The irMSC of any one of claims 33-44, wherein expression of an aging marker, e.g., cyclin dependent kinase inhibitor (CDKN) 1A, activating transcription factor (ATF) 3, interleukin (IL) -6, growth arrest and/or DNA damage-inducible 45 beta (GADD45B) is reduced.
The irMSC of claim 45, wherein expression of the aging marker remains after the irMSC differentiates along the osteogenic or chondrogenic lineages.
The irMSC of any one of claims 33-46, which has longer telomeres than the parental fibroblast and autologous bMSC.
The irMSC of claim 47, which maintains the telomere lengthening after its lineage differentiation.
The irMSC of any one of claims 33-48, which is capable of osteogenic lineage differentiation and has reduced adipogenesis, comparable to bMSC from a young donor.
The irMSC of any one of claims 33-49, which has a higher expression level of osterix (OSX) and/or osteoprotegerin (OPG) than its autologous bMSC.
The irMSC of any one of claims 33-50, which behaves like younger bMSC upon differentiation into chondrocyte.
The irMSC of any one of claims 33-51, which does not express a pluripotency gene, e.g., Oct4.
The irMSC of any one of claims 33-52, which does not form tumor for at least 30 days after transplanted into a mammal.
The irMSC of any one of claims 33-53, which comprises lengthened telomeres as compared to its parental fibroblast.
The irMSC of any one of claims 33-54, which is at stage of at least 5 passages and expresses a lower level of telomerase reverse transcriptase (TERT) as compared to the newly formed irMSC.
A population of irMSCs of any one of claims 33-55.
A pharmaceutical composition comprising an effective amount of irMSC of any one of claims 33-55 or a population of irMSCs of claim 56, admixed with at least one pharmaceutically acceptable carrier or excipient.
A method, which method comprises administering to a subject in need thereof an effective amount of an irMSC or a population of irMSCs.
The method of claim 58, wherein the subject is a non-human mammal.
The method of claim 58, wherein the subject is a human.
A method of any one of claims 58-60, which is used for treating bone injury or promoting bone repair in a subject.
The method of claim 61, wherein the irMSCs induce bone healing by cell-replacement in the subject.
The method of claim 61 or 62, wherein the irMSCs elicit bone tissue regeneration in the subject.
A method of any one of claims 61-63, wherein the irMSCs have more efficacy than its autologous bMSC.
A method of any one of claims 61-64, wherein the irMSCs are derived from an older donor and have an efficacy comparable to that of irMSCs derived from a younger donor.
A method of any one of claims 58-65, which is used for treating bone injury or eliciting cartilage regeneration in a subject.
The method of claim 66, wherein the irMSCs generate new cartilage in a subject.
The method of claim 67, wherein the new cartilage is positive for SOX9.
The method of any one of claims 66-68, wherein the irMSCs develop a proteoglycan-and type II collagen–rich extracellular matrix for cartilage, with minimal or no calcification or mineralization in a subject.
The method of any one of claims 66-69, wherein the irMSCs contribute to cartilage repair through differentiation into chondrocytes for cell replacement in a subject.
The method of any one of claims 61-70, wherein the irMSCs are administered to an injury or local site in a subject.
A method of any one of claims 58-71, which is used for slowing or reversing an aging related character in a subject.
A method of any one of claims 58-72, which is used for reversing tissue degeneration and/or prolong lifespan in a subject.
The method of claim 72 or 73, wherein the irMSCs are administered intravenously, e.g., via intravenous transfusion, to a subject.
The method of any one of claims 72-74, wherein the irMSCs are administered to a subject in a single dose.
The method of any one of claims 72-74, wherein the irMSCs are administered to a subject in multiple single doses.
The method of any one of claims 72-76, wherein the irMSCs are administered to a subject in a dose of at least from about 10 ⁶ cells to about 10 ⁸ cells.
The method of any one of claims 72-77 wherein the lifespan of the treated subject is substantially extended, e.g., extended for about 5%to about 20%of average lifespan of the subject.
The method of any one of claims 72-78 wherein the treated subject shows improvement in its appearance, e.g., improvement on its fur coats and reduced spine curvature as compared to the control subject.
The method of any one of claims 72-79 wherein the treated subject shows more motor activities as compared to the control subject.
The method of any one of claims 72-80 wherein the treated subject has digestive system resembling of a comparable younger subject.
The method of any one of claims 72-81 wherein the stomach tissue of the treated subject has more regular gastric mucosa as compared to the control subject.
The method of any one of claims 72-82 wherein the age-related loss of parietal cells are recovered by irMSC administration, e.g., transfusion.
The method of any one of claims 72-83 wherein the treated subject shows one or more signs of rejuvenation.
The method of any one of claims 72-84 wherein the irMSC has one or more of the following property:

1) which is prepared from a primary fibroblast of a human;

2) which is prepared from a primary fibroblast of a human having an age of at least 5 years;

3) wherein a fibroblast marker, e.g., fibroblast-specific protein-1 (FSP-1) , is down-regulated;

4) wherein a MSC-related cell surface marker, e.g., CD49a and/or CD49e, is up-regulated;

5) wherein both CD49a and CD49e are up-regulated;

6) which is configured to suppress T cell proliferation, e.g., to suppress T cell proliferation in vitro;

7) which acquires self-renewal, tri-lineage differentiation and/or immune modulation property comparable to that of a bone-marrow-derived mesenchymal stem cell (bMSC) ;

8) wherein an aging epigenetic marker histone, e.g., H4 lysine 20 trimethylation (H4K20me3) and/or age-related DNA damage marker, e.g., phosphorylated histone H2AX (γH2AX) is downregulated;

9) which has a greater proliferative capacity than its parental fibroblast;

10) which grows more rapidly than autologous bMSC;

11) which grows faster than bMSC from a younger donor;

12) wherein expression of an aging marker, e.g., cyclin dependent kinase inhibitor (CDKN) 1A, activating transcription factor (ATF) 3, interleukin (IL) -6, growth arrest and/or DNA damage-inducible 45 beta (GADD45B) is reduced;

13) wherein expression of the aging marker remains after the irMSC differentiates along the osteogenic or chondrogenic lineages;

14) which has longer telomeres than the parental fibroblast and autologous bMSC;

15) which maintains the telomere lengthening after its lineage differentiation;

16) which is capable of osteogenic lineage differentiation and has reduced adipogenesis, comparable to bMSC from a young donor;

17) which has a higher expression level of osterix (OSX) and/or osteoprotegerin (OPG) than its autologous bMSC.

18) which behaves like younger bMSC upon differentiation into chondrocyte;

19) which does not express a pluripotency gene, e.g., Oct4;

20) which does not form tumor for at least 30 days after transplanted into a mammal;

21) which comprises lengthened telomeres as compared to its parental fibroblast; and/or

22) which is at stage of at least 5 passages and expresses a lower level of telomerase reverse transcriptase (TERT) as compared to the newly formed irMSC.
The method of any one of claims 72-85 wherein the irMSC of any of claims 33-55 or a population of irMSCs of claim 56 are administered to a subject.
Use of the irMSC of any of claims 33-55 or a population of irMSCs of claim 56 for the manufacture of a medicament.
A method for facilitating fibroblasts to differentiate into bone and cartilage, and/or for preparing induced and rejuvenated mesenchymal stem cell (irMSC) , which method comprises deleting or inactivating STAT5A gene, reducing or blocking expression of STAT5A gene, and/or removing or inactivating STAT5A gene product, e.g., protein encoded by said STAT5A gene, in fibroblasts.
The method of claim 88, which is conducted in vitro.
The method of claim 88, which is conducted in vivo.
The method of any one of claims 88-90 which comprises knocking out of STAT5A in fibroblasts, e.g., by homologous recombination, site-specific nuclease cleavage, Zinc-finger nuclease (ZFN) cleavage, transcription activator-like effector nucleases (TALEN) cleavage, and/or clustered regularly interspaced short palindromic repeats (CRISPR) technology.
A kit comprising at least one irMSC packaged with or accompanied by means to administer the irMSC to a subject in need thereof, e.g., a hypodermic syringe, or instructions for administration of the irMSC.
The kit of claim 92, wherein the irMSC is produced by the method of any of claims 1-33.