Patent Application: US-201615383752-A

Abstract:
the present invention provides a method of forming a fibromodulin reprogrammed cell . this application includes the sequence listing which is identical to the sequence information in the last filed computer readable form on aug . 1 , 2014 in u . s . application ser . no . 14 / 353 , 284 , filed apr . 21 , 2014 , entitled “ 2014 - 08 - 01 384578 - 991391 st25 . tax ” and is hereby incorporated by reference in its entirety .

Description:
in one aspect of the present invention , it is provided a cell culture medium composition comprising fibromodulin ( fmod ) or a derivative or fragment thereof , wherein the composition is effective for reprogramming a cell to form a fmod reprogrammed ( frep ) cell , wherein the frep cell expresses nanog and does not form teratoma in vivo . in some embodiments of the cell culture medium , the fmod has a concentration from about 200 nm to about 800 nm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 1 nm to about 1000 μm , e . g ., from about 1 nm to about 10 nm , from about 1 nm to about 20 nm , from about 1 nm to about 50 nm , from about 1 nm to about 100 nm , from about 1 nm to about 200 nm , from about 1 nm to about 500 nm , from about 1 nm to about 1000 nm , from about 1 nm to about 2 μm , from about 1 nm to about 5 μm , from about 1 nm to about 10 μm , from about 1 nm to about 20 μm , from about 1 nm to about 50 μm , from about 1 nm to about 100 μm , from about 1 nm to about 200 μm , or from about 1 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 10 nm to about 1000 μm , e . g ., from about 10 nm to about 20 nm , from about 10 nm to about 50 nm , from about 10 nm to about 100 nm , from about 10 nm to about 200 nm , from about 10 nm to about 500 nm , from about 10 nm to about 1000 nm , from about 10 nm to about 2 μm , from about 10 nm to about 5 μm , from about 10 nm to about 10 μm , from about 10 nm to about 20 μm , from about 10 nm to about 50 μm , from about 10 nm to about 100 μm , from about 10 nm to about 200 μm , or from about 10 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 20 nm to about 1000 μm , e . g ., from about 20 nm to about 50 nm , from about 20 nm to about 100 nm , from about 20 nm to about 200 nm , from about 20 nm to about 500 nm , from about 20 nm to about 1000 nm , from about 20 nm to about 2 μm , from about 20 nm to about 5 μm , from about 20 nm to about 10 μm , from about 20 nm to about 20 μm , from about 20 nm to about 50 μm , from about 20 nm to about 100 μm , from about 20 nm to about 200 μm , or from about 20 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 50 nm to about 1000 μm , e . g ., from about 50 nm 30 to about 100 nm , from about 50 nm to about 200 nm , from about 50 nm to about 500 nm , from about 50 nm to about 1000 nm , from about 50 nm to about 2 μm , from about 50 nm to about 5 μm , from about 50 nm to about 10 μm , from about 50 nm to about 20 μm , from about 50 nm to about 50 μm , from about 50 nm to about 100 μm , from about 50 nm to about 200 μm , or from about 50 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 100 nm to about 1000 μm , e . g ., from about 100 nm to about 200 nm , from about 100 nm to about 500 nm , from about 100 nm to about 1000 nm , from about 100 nm to about 2 μm , from about 100 nm to about 5 μm , from about 100 nm to about 10 μm , from about 100 nm to about 20 μm , from about 100 nm to about 50 μm , from about 100 nm to about 100 μm , from about 100 nm to about 200 μm , or from about 100 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 200 nm to about 1000 μm , e . g ., from about 200 nm to about 500 nm , from about 200 nm to about 1000 nm , from about 200 nm to about 2 μm , from about 200 nm to about 5 μm , from about 200 nm to about 10 μm , from about 200 nm to about 20 μm , from about 200 nm to about 50 μm , from about 200 nm to about 100 μm , from about 200 nm to about 200 μm , or from about 200 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 500 nm to about 1000 μm , e . g ., from about 500 nm to about 1000 nm , from about 500 nm to about 2 μm , from about 500 nm to about 5 μm , from about 500 nm to about 10 μm , from about 500 nm to about 20 μm , from about 500 nm to about 50 μm , from about 500 nm to about 100 μm , from about 500 nm to about 200 μm , or from about 500 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 1000 nm to about 1000 μm , e . g ., from about 1000 nm to about 2 μm , from about 1000 nm to about 5 μm , from about 1000 nm to about 10 μm , from about 1000 nm to about 20 μm , from about 1000 nm to about 50 μm , from about 1000 nm to about 100 μm , from about 1000 nm to about 200 μm , or from about 1000 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 2 μm to about 1000 μm , e . g ., from about 2 μm to about 5 μm , from about 2 μm to about 10 μm , from about 2 μm to about 20 μm , from about 2 μm to about 50 μm , from about 2 μm to about 100 μm , from about 2 μm to about 200 μm , or from about 2 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 5 μm to about 1000 μm , e . g ., from about 5 μm to about 10 μm , from about 5 μm to about 20 μm , from about 5 μm to about 50 μm , from about 5 μm to about 100 μm , from about 5 μm to about 200 μm , or from about 5 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 10 μm to about 1000 μm , e . g ., from about 10 μm to about 20 μm , from about 10 μm to about 50 μm , from about 10 μm to about 100 μm , from about 10 μm to about 200 μm , or from about 10 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 20 μm to about 1000 μm , e . g ., from about 20 μm to about 50 μm , from about 20 μm to about 100 μm , from about 20 μm to about 200 μm , or from about 20 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 50 μm to about 1000 μm , e . g ., from about 50 μm to about 100 μm , from about 50 μm to about 200 μm , or from about 50 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 100 μm to about 1000 μm , e . g ., from about 100 μm to about 200 μm , or from about 100 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 500 μm to about 1000 μm . examples of the concentration of fmod protein or peptide in the culture medium can be , e . g ., about 10 nm , about 20 nm , about 50 nm , about 100 nm , about 200 nm ( e . g ., 220 nm ), about 500 nm , about 1000 nm , about 2 μm , about 5 μm , about 10 μm , about 20 μm , about 50 μm , about 100 μm , about 200 μm , or about 500 μm . in some embodiments of the cell culture medium , optionally in combination with any or all of the above various embodiments , the cell is a human cell , mouse cell , and rat cell . in some embodiments , the cell can be a bj fibroblast or primary adult normal human dermal fibroblast ( hdf ). in some embodiments of the cell culture medium , optionally in combination with any or all of the above various embodiments , reprogramming is without using a genome - integrated transcription factor . in another aspect of the present invention , it is provided a method of pluripotency reprogramming , comprising : treating a mammalian cell with a cell culture medium comprising fibromodulin ( fmod ) or a derivative or fragment thereof for a period ranging from a day to a month , and changing the cell culture medium regularly until a fmod reprogrammed ( frep ) cell forms ; wherein the frep cell expresses nanog and does not form teratoma in vivo . in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the fmod has a concentration from about 200 nm to about 800 nm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 1 nm to about 1000 μm , e . g ., from about 1 nm to about 10 nm , from about 1 nm to about 20 nm , from about 1 nm to about 50 nm , from about 1 nm to about 100 nm , from about 1 nm to about 200 nm , from about 1 nm to about 500 nm , from about 1 nm to about 1000 nm , from about 1 nm to about 2 μm , from about 1 nm to about 5 μm , from about 1 nm to about 10 μm , from about 1 nm to about 20 μm , from about 1 nm to about 50 μm , from about 1 nm to about 100 μm , from about 1 nm to about 200 μm , or from about 1 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 10 nm to about 1000 μm , e . g ., from about 10 nm to about 20 nm , from about 10 nm to about 50 nm , from about 10 nm to about 100 nm , from about 10 nm to about 200 nm , from about 10 nm to about 500 nm , from about 10 nm to about 1000 nm , from about 10 nm to about 2 μm , from about 10 nm to about 5 μm , from about 10 nm to about 10 μm , from about 10 nm to about 20 μm , from about 10 nm to about 50 μm , from about 10 nm to about 100 μm , from about 10 nm to about 200 μm , or from about 10 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 20 nm to about 1000 μm , e . g ., from about 20 nm to about 50 nm , from about 20 nm to about 100 nm , from about 20 nm to about 200 nm , from about 20 nm to about 500 nm , from about 20 nm to about 1000 nm , from about 20 nm to about 2 μm , from about 20 nm to about 5 μm , from about 20 nm to about 10 μm , from about 20 nm to about 20 μm , from about 20 nm to about 50 μm , from about 20 nm to about 100 μm , from about 20 nm to about 200 μm , or from about 20 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 50 nm to about 1000 μm , e . g ., from about 50 nm to about 100 nm , from about 50 nm to about 200 nm , from about 50 nm to about 500 nm , from about 50 nm to about 1000 nm , from about 50 nm to about 2 μm , from about 50 nm to about 5 μm , from about 50 nm to about 10 μm , from about 50 nm to about 20 μm , from about 50 nm to about 50 μm , from about 50 nm to about 100 μm , from about 50 nm to about 200 μm , or from about 50 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 100 nm to about 1000 μm , e . g ., from about 100 nm to about 200 nm , from about 100 nm to about 500 nm , from about 100 nm to about 1000 nm , from about 100 nm to about 2 μm , from about 100 nm to about 5 μm , from about 100 nm to about 10 μm , from about 100 nm to about 20 μm , from about 100 nm to about 50 μm , from about 100 nm to about 100 μm , from about 100 nm to about 200 μm , or from about 100 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 200 nm to about 1000 μm , e . g ., from about 200 nm to about 500 nm , from about 200 nm to about 1000 nm , from about 200 nm to about 2 μm , from about 200 nm to about 5 μm , from about 200 nm to about 10 μm , from about 200 nm to about 20 μm , from about 200 nm to about 50 μm , from about 200 nm to about 100 μm , from about 200 nm to about 200 μm , or from about 200 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 500 nm to about 1000 μm , e . g ., from about 500 nm 25 to about 1000 nm , from about 500 nm to about 2 μm , from about 500 nm to about 5 μm , from about 500 nm to about 10 μm , from about 500 nm to about 20 μm , from about 500 nm to about 50 μm , from about 500 nm to about 100 μm , from about 500 nm to about 200 μm , or from about 500 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 1000 nm to about 1000 μm , e . g ., from about 1000 nm to about 2 μm , from about 1000 nm to about 5 μm , from about 1000 nm to about 10 μm , from about 1000 nm to about 20 μm , from about 1000 nm to about 50 μm , from about 1000 nm to about 100 μm , from about 1000 nm to about 200 μm , or from about 1000 nm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 2 μm to about 1000 μm , e . g ., from about 2 μm to about 5 μm , from about 2 μm to about 10 μm , from about 2 μm to about 20 μm , from about 2 μm to about 50 μm , from about 2 μm to about 100 μm , from about 2 μm to about 200 μm , or from about 2 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 5 μm to about 1000 μm , e . g ., from about 5 μm to about 10 μm , from about 5 μm to about 20 μm , from about 5 μm to about 50 μm , from about 5 μm to about 100 μm , from about 5 μm to about 200 μm , or from about 5 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 10 μm to about 1000 μm , e . g ., from about 10 μm to about 20 μm , from about 10 μm to about 50 μm , from about 10 μm to about 100 μm , from about 10 μm to about 200 μm , or from about 10 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 20 μm to about 1000 μm , e . g ., from about 20 μm to about 50 μm , from about 20 μm to about 100 μm , from about 20 μm to about 200 μm , or from about 20 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 50 μm to about 1000 μm , e . g ., from about 50 μm to about 100 μm , from about 50 μm to about 200 μm , or from about 50 μm to about 500 m . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 100 μm to about 1000 μm , e . g ., from about 100 μm to about 200 μm , or from about 100 μm to about 500 μm . in some embodiments , the culture medium can include a fmod protein or peptide in a concentration from about 500 μm to about 1000 μm . examples of the concentration of fmod protein or peptide in the culture medium can be , e . g ., about 10 nm , about 20 nm , about 50 nm , about 100 nm , about 200 nm ( e . g ., 220 nm ), about 500 nm , about 1000 nm , about 2 μm , about 5 μm , about 10 μm , about 20 m , about 50 μm , about 100 μm , about 200 μm , or about 500 μm . in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the cell is a human cell , mouse cell , and rat cell . examples of human cells include , e . g ., bj , mrc - 5 , hdf , keratinocytes , melanocytes , peripheral blood cells ( e . g ., cd34 +), cord blood cells or even certain stem cells ( e . g ., adipose - derived stem cells , perivascular stem cells , neural stem cells ). in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the cell is a bj fibroblast or primary adult normal human dermal fibroblast ( nhdf ). in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the method is carried out without using a genome - integrated transcription factor . in a further aspect of the present invention , it is provided a fibromodulin ( fmod ) reprogrammed ( frep ) cell , which frep cell is generated by a method comprising : treating a mammalian cell with a cell culture medium for a period ranging from a day to a month , and wherein the medium comprises fibromodulin ( fmod ) or a derivative or fragment thereof , and wherein the frep cell expresses nanog and does not form teratoma in vivo . in some embodiments of the frep cell , optionally in combination with any or all of the above various embodiments , the fmod has a concentration from about 200 nm to about 800 nm . in some embodiments of the frep cell , optionally in combination with any or all of the above various embodiments , the cell is a human cell , mouse cell , and rat cell . examples of human cells include , e . g ., bj , mrc - 5 , hdf , keratinocytes , melanocytes , peripheral blood cells ( e . g ., cd34 +), cord blood cells or even certain stem cells ( e . g ., adipose - derived stem cells , perivascular stem cells , neural stem cells ). in some embodiments of the frep cell , optionally in combination with any or all of the above various embodiments , the mammalian cell is a bj fibroblast or primary adult normal human dermal fibroblast ( nhdf ). in another aspect of the present invention , it is provided a method of treating a disorder in a mammal , which method comprising administering to the mammal a frep cell disclosed herein above and / or below . in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the mammal is a human being . in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the disorder is a neurodegenerative disorder . in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the disorder is a central nervous system ( cns ) disease , cardiovascular disease , blood diseases , crohn &# 39 ; s disease , bone disease , muscle disease , or chondrocyte disease . in some embodiments of the method , optionally in combination with any or all of the above various embodiments , the disorder is a retina disease , a trauma and injury to a tissue , a skeletal disorder , an organ disease or an injury to skin , muscle , cartilage , tendon , peripheral nerve , spinal cord , blood vessels , or bone . in a further aspect of the present invention , it is provided a supematant , comprising a cell culture medium disclosed above or below . in some embodiments of the supematant , the supematant can be included in a composition . in some embodiments , such composition can be , for example , a pharmaceutical or cosmetic composition . in a further aspect of the present invention , it is provided a method of treating or ameliorate a disorder , comprising administering to a mammalian subject a supematant or a composition disclosed above or below . in further aspect of the present invention , it is provided a method or inhibiting tumor growth , comprising adding fmod directly to tumorigenic , or tumor cells to inhibit their growth . for example , one can adminster to a subject ( e . g ., a cancer patient ) in need thereof a composition comprising an effective amount of fibromodulin ( fmod ) to a site having tumorigenic or tumor cells in the subject to cause the tumorigenic cells or tumor cells to stop growth or growing at a slower rate . as used herein , the term effective amount shall mean an amount fmod effective to stop or slow the growth of tumor cells in the subject using a dosage regime prescribed by a medical practictioner . as used herein , the term “ grow at a slower rate ” shall mean a rate of growth of the tumorigenic or tumor cells slower than their rate of growth without receiving a composition comprising fmod described herein . in addition to the fmod protein or peptide or a derivative or fragment thereof , the culture medium can be any cell culture medium commonly used in the art . for example , the culture medium generally includes saline . an example of cell culture medium includes , e . g ., saline , a ph of 7 . 4 pbs , dmem medium , or fibroblast basic medium ( fbm , lonza ). in some embodiments , the culture medium can include additional components or agents , e . g ., transforming growth factor ( tgf )- β . as used herein , the term “ sufficient time ” shall mean a period sufficiently long to reprogram the mammalian cell by the culture medium disclosed herein . in some embodiments , the term “ sufficient time ” ranges from hours to about 180 days , e . g ., from 8 hrs to about 12 hrs , from about 8 hrs to about 24 hrs , from about 8 hrs to about 2 days , from about 8 hrs to about 7 days , from about 8 hrs to about 14 days , from about 8 hrs to about 21 days , from about 8 hrs to about 30 days , from about 8 hrs to about 45 days , from about 8 hrs to about 60 days , from about 8 hrs to about 90 days , from about 8 hrs to about 120 days , from about 8 hrs to about 150 days , or from about 8 hrs to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 1 day to about 180 days , e . g ., from about 1 day to about 2 days , from about 1 day to about 7 days , from about 1 day to about 14 days , from about 1 day to about 21 days , from about 1 day to about 30 days , from about 1 day to about 45 days , from about 1 day to about 60 days , from about 1 day to about 90 days , from about 1 day to about 120 days , from about 1 day to about 150 days , or from about 1 day to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 2 days to about 180 days , e . g ., from about 2 days to about 7 days , from about 2 days to about 14 days , from about 2 days to about 21 days , from about 2 days to about 30 days , from about 2 days to about 45 days , from about 2 days to about 60 days , from about 2 days to about 90 days , from about 2 days to about 120 days , from about 2 days to about 150 days , or from about 2 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 7 days to about 180 days , e . g ., from about 7 days to about 14 days , from about 7 days to about 21 days , from about 7 days to about 30 days , from about 7 days to about 45 days , from about 7 days to about 60 days , from about 7 days to about 90 days , from about 7 days to about 120 days , from about 7 days to about 150 days , or from about 7 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 14 days to about 180 days , e . g ., from about 14 days to about 21 days , from about 14 days to about 30 days , from about 14 days to about 45 days , from about 14 days to about 60 days , from about 14 days to about 90 days , from about 14 days to about 120 days , from about 14 days to about 150 days , or from about 14 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 21 days to about 180 days , e . g ., from about 21 days to about 30 days , from about 21 days to about 45 days , from about 21 days to about 60 days , from about 21 days to about 90 days , from about 21 days to about 120 days , from about 21 days to about 150 days , or from about 21 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 30 days to about 180 days , e . g ., from about 30 days to about 45 days , from about 30 days to about 60 days , from about 30 days to about 90 days , from about 30 days to about 120 days , from about 30 days to about 150 days , or from about 30 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 45 days to about 180 days , e . g ., from about 45 days to about 60 days , from about 45 days to about 90 days , from about 45 days to about 120 days , from about 45 days to about 150 days , or from about 45 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 60 days to about 180 days , e . g ., from about 60 days to about 90 days , from about 60 days to about 120 days , from about 60 days to about 150 days , or from about 60 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 90 days to about 180 days , e . g ., from about 90 days to about 120 days , from about 90 days to about 150 days , or from about 90 days to about 180 days . in some embodiments , the term “ sufficient time ” ranges from 120 days to about 180 days , e . g ., from about 12 days to about 150 days , or from about 60 days to about 180 days . in some embodiments , the method provided herein further includes changing culture medium with fresh culture medium regularly . the term “ regularly ” shall mean changing culture medium hourly , bi - hourly , four times a day , twice a day , daily , once per two - day , bi - weekly , weekly , bi - monthly , or monthly . in another aspect of the present invention , it is provided a supernatant of the frep cell disclosed herein . the supernatant includes the culture medium of the present invention and also growth factors and / or transcriptional factors excreted by the frep cells or clones provided herein . the supematant disclosed herein is effective for treating or ameliorating a disorder as is the frep cell disclosed herein . in some embodiments , the supernatant can form a composition , optionally with a carrier . the composition can be applied to a mammalian subject for treating or ameliorating a disorder . in some embodiments , the composition can be a cosmetic composition or a pharmaceutical composition . as used herein , the term frep cell shall mean a cell reprogrammed by exposure to a culture medium comprising fmod that is not a pluripotent stem cell (“ psc ”) but possesses at least one of the characteristics of psc . the frep cell disclosed herein has ability to differentiate into a desired tissue cell in a physiological condition of a tissue . an attribute of the frep disclosed herein is that it expresses nanog . in some embodiments , the frep cell disclosed herein does not form teratoma . the characteristics or attributes of psc are generally known in the art , some features of which are described as follows . generally recognized characteristics of psc include its ability to differentiate into different tissue cells under proper conditions . other attributes of a psc include , e . g ., expression of transcriptional regulators such as oct4 , sox2 , or nanog or antigens such as ssea - 4 , tra - 1 - 60 , or tra - 1 - 81 , as well as high expression of alkaline phosphatase ( ap ). in some embodiments , the frep cell disclosed herein has all the attributes or a pluripotent stem cell . in these embodiments , the frep cell is presumably a psc . in some embodiments , the frep cell disclosed herein has one or more , but not all , of the attributes or a pluripotent stem cell . in these embodiments , the frep cell disclosed herein does not amount to a psc . these transcriptional regulators or antigens can be readily recognized by antibodies against these regulators or antigens in , e . g ., immunofluorescent staining . as used herein , the term psc shall also encompass pluripotent germ cells . generally , a frep cell can be generated by a method comprising the steps of : treating a mammalian cell with a cell culture medium for a period ranging from a day to a month , and changing the cell culture medium regularly until the frep cell forms . wo 2011 / 143400 discloses a method of forming a pluripotent stem cell like ( pscl ) cell and method of making thereof . the teaching in wo 2011 / 143400 is incorporated herein in its entirety . the frep cell can be used in medicine as is a psc to treat or ameliorate a disorder in a mammal ( e . g ., a human being or an animal ). generally , the method includes administering to a subject having a disorder a frep cell ( s ) or clone ( s ) so as to treat or ameliorate the disorder . methods of using pluripotent stem cell to treat a disorder is generally established and known in the art as it will closely resemble the protocols used for embryonic stem cells ( see , e . g ., sun , et al ., cell cycle 9 : 5 , 880 - 885 ( 2010 )). although , there are no approved products yet , there are a lot of potential applications , such as transplantation , gene repair and cell replacement therapy for a variety of genetic disorders ( see , e . g ., gunaseelie , et al ., curr med chem . ; 17 ( 8 ): 759 - 766 ( 2010 )). the disorder can be any disorder that can be treated or ameliorated by a pluripotent stem cell . in some embodiments , the disorder can be a degenerative disease such as a neurodegenerative disorder or cardiac degenerative disease . in some embodiments , the disorder can be a central nervous system ( cns ) disease , cardiovascular disease , blood diseases , crohn &# 39 ; s disease , bone disease , muscle disease , baldness , cancer , infertility , or chondrocyte disease , such as adenosine deaminase deficiency - related severe combined immunodeficiency ( ada - scid ), shwachman - bodian - diamond syndrome ( sbds ), gaucher disease ( gd ) type iii , duchenne ( dmd ) and becher muscular dystrophy ( bmd ), parkinson disease ( pd ), huntington disease ( hd ), juvenile - onset , type 1 diabetes mellitus ( jdm ), down syndrome ( ds )/ trisomy 21 , the carrier state of lesch - nyhan syndroms , alzheimer &# 39 ; s disease , or ischemic heart diseases ( see , e . g ., gunaseelie , et al ., curr med chem . ; 17 ( 8 ): 759 - 766 ( 2010 )). in some embodiments , the disorder can be a trauma and injury to a tissue . examples of such tissue can be skin , muscle , cartilage , tendon , peripheral nerve , spinal cord , blood vessels , or bone . examples of trauma can be trauma inflicted by physical impact or trauma by a procedure in medicine , e . g ., removal of tissue in treating cancer , etc . in further aspect of the present invention , it is provided a method or inhibiting tumor growth , comprising adding fmod directly to tumorigenic , or tumor cells to inhibit their growth . for example , one can adminster to a subject ( e . g ., a cancer patient ) in need thereof a composition comprising an effective amount of fibromodulin ( fmod ) to a site having tumorigenic or tumor cells in the subject to cause the tumorigenic cells or tumor cells to stop growth or growing at a slower rate , which is defined above . administration of an effective amount of fmod or a composition comprising an effective amount of fmod can be achieved by systemic or local administration . systemic administration and local adminstration are well understood in the art . examples of systemic administration is parenteral injection or iv injection . examples of local administration include e . g ., injection to the local site or implantation . the following examples illustrate , rather than limit , embodiments of the present invention . studies on reprogramming human fibroblasts to pluripotency using a single protein , fibromodulin pluripotent and / or multipotent stem cell - based therapeutics are a vital component of tissue engineering and regenerative medicine . the generation or isolation of safer and readily available stem cell sources will significantly aid clinical applications . we report here a technique using a single molecule , recombinant human fibromodulin protein ( fmod ), to reprogram human fibroblasts into multipotent cells . like virally - induced pluripotent stem ( ips ) cells , fmod reprogrammed ( frep ) cells express pluripotency markers , form embryoid bodies ( ebs ), and differentiate into ectoderm , mesoderm , and endoderm derivatives in vitro . notably , frep cells regenerate muscle and bone tissues but do not generate teratomas in vivo . unlike ips cells , undifferentiated frep cells proliferate slowly and express low proto - oncogene c - myc and unexpectedly high levels of cyclin - dependent kinase inhibitors p15 ink4b and p21 waf1 / cip1 . remarkably , in a fashion reminiscent of quiescent stem cells , the slow replicative phenotype of undifferentiated frep cells reverses after differentiation induction , with differentiating frep cells proliferating faster and expressing less p15 ink4b and p21 waf1 / cip1 than differentiating ips cells . overall , single protein , fmod - based , cell reprogramming bypasses the risks of mutation , gene instability , and malignancy associated with genetically - modified ips cells and provides an alternative strategy for engineering patient - specific multipotent cells for basic research and therapeutic applications . patient — specific stem cells bypass many ethical and immunologic concerns and may be created by reprogramming somatic cells into a pluripotent state . previous studies show that a mammalian somatic cell can be reprogrammed by transferring its nucleus into an oocyte [ 1 - 3 ] or by fusion with an embryonic stem ( es ) cell [ 4 , 5 ]. recently , somatic cells were reprogrammed to gain pluripotency utilizing viral - mediated genomic integration of yamanaka factors ( oct4 , sox2 , klf4 , and c - myc ) in mouse cells , or thomason factors ( oct4 , sox2 , nanog , and lin28 ) in human cells [ 6 , 7 ]. these virally - induced pluripotent stem ( ips ) cells resemble natural pluripotent stem cells such as es cells in many aspects including stem cell surface markers and transcription factor expression profiles , doubling time , embryoid body ( eb ) formation , teratoma formation , and the capacity to differentiate into the three germ layers : endoderm , mesoderm , and ectoderm [ 6 - 10 ]. despite significant promise for patient - specific cell therapy , cumbersome ips cell reprogramming protocols as well as safety concerns over genome integrative approaches that increase ips cell tumorigenicity have impeded clinical application [ 11 , 12 ]. viral or dna - based methodologies all exhibit varying degrees of genomic integration and insertional mutagenesis risks , making them potentially unsafe for human use [ 11 - 13 ]. in this study , we describe a technically straightforward method to create induced multipotent cells from somatic cells based on extracellular delivery of a single extracellular matrix ( ecm ) component — fibromodulin ( fmod ). these fmod reprogrammed ( frep ) cells have the potential to differentiate into various therapeutic cells , but unlike ips cells , they do not impose a risk of tumor formation from undifferentiated cells . cdna of human fmod transcript ( genebank accessory number : m 002023 ) was subcloned into commercially available vector psectag2a ( invitrogen ) with c - terminal his - tag and transfected into cho - k1 cells . after establishing a stable expression clone , fmod was harvested from the conditioned medium and purified via probond ™ purification system ( invitrogen ) as previously described [ 14 ]. human newborn foreskin fibroblast bj ( atcc crl - 2522 ) and primary adult normal human dermal fibroblast hdf ( lonza biosciences ) cells were maintained in clonetics ® normal human fibroblast cell systems ( fgm - 2 ; lonza biosciences ). viral vector - mediated human ips ( clone ips2 ) [ 15 ] cells were maintained on irradiated mouse embryonic fibroblast ( mef ) feeder cells ( globalstem inc .) in es - dmem / f12 ( optimized for human es cells ; globalstem inc .) supplemented with 20 % knockout serum replacement ( invitrogen ) and 10 ng / ml human recombinant fibroblast growth factor ( fgf )- 2 ( globalstem inc ). viral vector harboring enhanced green fluorescent protein gene ( egfp ) derived by nanog promoter ( nanog :: egfp reporter ) was produced and infected as previously described [ 16 ]. 4 × 10 5 / well human fibroblasts cultured in fgm - 2 medium were seeded in 6 - well cell culture plates overnight to confluence . serum - free , growth factor - free fibroblast basal medium ( fbm ; lonza biosciences ) supplied with 0 . 4 mg / ml recombinant human fmod , 2 mm l - glutamine ( invitrogen ), and 1 % penicillin / streptomycin ( ps ; invitrogen ) was used to treat human fibroblasts daily for three to four weeks ( fig1 ). 1 × 10 4 / cm 2 cells were seeded into 12 - well culture plates for proliferation assay . after 3 days incubation , cell proliferation was analyzed by click - it ® edu microplate assay ( invitrogen ). following manufacturer &# 39 ; s instructions , aggrewell ™ 800 plates and aggrewell ™ medium ( stemcell technologies ) were used for formation of ebs in suspension culture . established protocols for direct differentiation of human es and ips cells were used for the differentiation of frep cells with some modification [ 17 - 23 ]. for neuron differentiation , ebs were transferred into 6 - well ultra - low plates with knockout dmem medium ( invitrogen ) supplied with 10 % knockout serum replacement , 2 mm l - glutamine , 1 % ps , 10 μm all - trans retinoic acid ( ra ; sigma - aldrich ), and 100 nm of the n - terminal active fragment of human sonic hedgehog ( shh ; r & amp ; d systems ) to generate spheres . fresh retinoic acid ( ra ) was added every day , and the medium and supplements , including shh , were replaced every 72 h . after 8 - day suspension culture , these induced spheres were transferred onto poly - omithine / fibronectin ( sigma - aldrich ) coated plates with dmem f12 medium ( invitrogen ) supplied with 2 % fetal bovine serum ( fbs ; invitrogen ), n2 supplement ( invitrogen ), 20 ng / ml glial - derived neurotrophic factor ( gd f ; invitrogen ), 20 ng / ml brain - derived neurotrophic factor ( bd f ; invitrogen ), 20 ng / ml ciliary neurotrophic factor ( cntf ; invitrogen ), and 1 × b27 serum - free supplement ( invitrogen ) [ 17 , 18 ]. after 3 - 4 days , cultures were fixed for immunofluore scent ( if ) staining . cells were cultured in rpmi 1640 medium ( invitrogen ) supplied with 2 % fbs , 2 mm l - glutamine , 1 % ps , and 100 ng / ml recombinant activin a ( r & amp ; d systems ) for 4 days for differentiation into endoderm derivative . for further induction of pancreatic lineage cells , cells with 4 days of activin a treatment were cultured for another 8 days without activin a [ 19 , 20 ]. for cardiac differentiation , colonies were detached by dispase ( invitrogen ) and transferred into 6 - well ultra - low plates with dmem f12 medium supplied with 20 % fbs , 1 mm nonessential amino acids ( neaa ; invitrogen ), and 0 . 1 mm β - mercaptoethanol ( sigma - aldrich ) to initiate cardiac differentiation . during suspension culture , the medium was changed at day 1 followed by culture for another 3 days . afterwards , the spheres were plated on af solution ( invitrogen ) coated plates for another 10 days before if staining . medium was changed every day [ 19 , 21 ]. for skeletal myogenesis , colonies were transferred onto af solution coated plates with myogenic medium i [ dmem medium supplied with 10 % fbs , 10 % horse serum ( hs ; invitrogen ), 1 % chicken embryo extract ( cee ; accurate ), and 1 % ps ] for 7 days , and then for 7 - 10 days in myogenic medium ii [ dmem medium supplied with 1 % fbs , 1 % hs , 0 . 5 %) cee , and 1 %& gt ; ps ]. half of the medium was renewed every 4 days [ 22 ]. for osteogenesis , colonies were transferred onto af solution coated plates in a - mem medium ( invitrogen ) supplied with 10 % fbs , 50 μg / ml ascorbic acid ( sigma - aldrich ), 10 mm β - glycerophosphate ( sigma - aldrich ), and 1 % ps for 28 days . mineralization was detected by alizarin red staining as previously described [ 23 ]. hmsc mesenchymal stem cell adipogenic differentiation medium ( lonza biosciences ) was used for adipogenesis in vitro . oil red o staining was used to identify adipocytes . 8 - week old scid mice were anaesthetized by isoflurane / 02 inhalation . for osteogenesis in vivo , a demineralized bone matrix ( dbx ; musculoskeletal transplant foundation ) was used as scaffold [ 23 ]. after 3 - day pre - induction in a - mem medium supplied with 10 % fbs , 50 μg / ml ascorbic acid , 10 mm β - glycerophosphate , and 1 % ps , 5 × 10 5 cells were harvested and suspended in 150 μî pbs and mixed with dbx before implantation into a pocket in the gluteofemoral muscle of scid mice . mice were sacrificed 8 weeks post procedure , and tissues were harvested and fixed in 10 % formalin [ 23 ]. for myogenesis in vivo , 5 × 10 5 cells were cultured in myogenic medium i for 3 days , and slowly injected into a pocket of gluteofemoral muscle of scid mice ; specimen were harvested at 8 weeks post injection . alternatively , cardiotoxin ( 15 μg ; sigma ) was injected into the gastrocnemius muscle 3 hours prior to transplantation of 5 × 10 5 frep cells . mice were re - anaesthetized , and cells suspended in 35 μl pbs , or the same volume of pbs as a control , were then slowly injected into the injured muscle . mice were sacrificed 3 weeks post - procedure and muscle tissue was harvested and fixed in 10 % formalin [ 22 ]. karyotyping and tumor formation assays were performed by applied stemcell inc . briefly , cells were collected by collagenase iv treatment and injected into male scid - beige mice kidney and testis . tissues were harvested after 3 - 4 months and processed for paraffin embedding and hematoxylin and eosin ( h & amp ; e ) staining following standard procedures . cells were harvested with ripa buffer ( pierce ) supplemented with halt ™ protease and phosphatase inhibitor cocktail ( pierce ). 15 μg of protein from each sample was loaded onto sds - page and transferred to nitrocellulose membranes as previously described [ 24 ]. western blotting was performed using the following primary antibodies : glyceraldehyde - 3 - phosphate dehydrogenase ( gapdh ; santa cruz biotechnology , inc . ), nanog ( cell signaling technology ), oct4a ( cell signaling technology ), smad3 ( abeam inc . ), phosphorylated smad3 ( psmad3 , abeam inc . ), and sox2 ( cell signaling technology ). immu - star ™ westernc ™ kit ( biorad ) was used for development . bands on western blot were quantified using quantityone ® ( biorad ), and values were expressed in relative arbitrary densitometry units . rnas were extracted using rneasy ® mini kit ( qiagen ) with dnase ( qiagen ) followed by reverse transcription with superscript ™ iii first - strand synthesis system for rt - pcr ( invitrogen ). pcr was performed with taq dna polymerase ( invitrogen ). primers used in this study are listed in table 1 [ 25 ]. qrt - pcr was performed on a 7300 real - time pcr system ( applied biosystems inc .) with taqman ® gene expression assays ( applied biosystems inc .). concomitant gapdh was also performed in separate tubes for each rt reaction . for each gene , at least three separate sets of qrt - pcr analyses were performed from different cdna templates . the δc t levels of gapdh did not differ significantly between treatment conditions ; thus , they were used as housekeeping standard ( data not shown ). qbiomarker ™ screening pcr arrays for ipsc colony screening ( sabiosciences corp .) were used for rna expression profile as well . villa - diza lg , nandivada h , ding j , nogueira - de - souza nc , krebsbach ph , o &# 39 ; shea ks , et al . synthetic polymer coatings for long - term growth of human embryonic stem ap staining was performed with the vector red substrate kit from vector laboratories . samples for if were fixed in pre - chilled acetone , and 4 ′, 6 - diamidino - 2 - phenylindole ( dap i ) was used for count staining . meanwhile , samples for ihc were fixed in 10 % formalin . after fixation , samples for ihc were dehydrated , paraffin - embedded , and sectioned at 5 μm for h & amp ; e , masson &# 39 ; s trichrome , and ihc staining . if and ihc staining were performed using the following primary antibodies : cardiac troponin t ( ctnt ; abeam inc . ), α1 - fetoprotein ( afp ; abeam inc . ), flt - 1 ( abeam inc . ), ampa glutamate receptor 1 ( glur1 ; abeam inc . ), krt - 18 ( abeam inc . ), human major histocompatibility complex ( mhc ) class 1 ( clone h - 300 , santa cruz biotechnology inc . ; clone ep1395y , abeam inc . ), myosin ( skeletal , slow ) ( sigma - aldrich ), osteocalcin ( ocn , santa cruz biotechnology ), nanog ( cell signaling technology ), nestin ( abeam inc . ), nkx2 . 5 ( abeam inc . ), oct4a ( cell signaling technology ), pancrease / duodenum homoeobox 1 ( pdx1 ; abeam inc . ), runt - related transcription factor 2 ( runx2 , santa cruz biotechnology , inc . ), sox2 ( cell signaling technology ), ssea4 ( cell signaling technology ), tra - 1 - 60 ( cell signaling technology ), tra - 1 - 81 ( cell signaling technology ), neuron specific βiπ - tubulin ( abeam inc . ), ve - cadherin ( abeam inc . ), and alexa fluor ® 594 - phalloidin ( invitrogen ). results were graphically depicted as the mean ± standard error of mean ( s . e . m ). statistical significance was computed using anova and tukey - fisher lsc criterion based on the post hoc t statistics . independent - samples t - test was used to analyze experiments of two groups . all statistical analyses in this manuscript were as per consultation with the ucla statistical biomathematical consulting clinic ( sbcc ). human newborn foreskin fibroblast bj cells exposed to continuous recombinant human fmod under serum - free conditions formed ap - positive , es cell - like colonies on mef feeders at an average frequency of 0 . 03 % ( 32 ± 2 . 6 es cell - like colonies from 100 , 000 bj fibroblasts , n = 16 ) ( fig1 ). the 0 . 03 % fmod - mediated reprogramming efficiency is comparable to original retroviral - mediated ips cell reprogramming rates [ 6 , 7 ]. fmod - induced es cell - like colonies expressed several es / ips cell pluripotency markers , including core transcriptional regulators of pluripotent cells ( nanog , sox2 , and oct4 ), and common pluripotent cell surface antigen ( ssea4 , tra - 1 - 60 , and tra - 1 - 81 ) ( fig2 a and b ). besides bj human fibroblasts , adult normal human dermal fibroblast ( nhdf ) cells similarly exposed to fmod also formed es cell - like colonies on mef feeders with positive ap activity and pluripotency marker expression ( fig9 ). to confirm induction of nanog expression , a nanog :: egfp reporter was introduced into bj fibroblasts . as expected , nanog :: egfp reporter was highly activated in fmod - reprogrammed bj ( bj - frep ) colonies ( fig2 c ). in addition , western blotting and qrt - pcr revealed that besides nanog , which is essential for embryonic and induced pluripotency [ 26 , 27 ], sox2 was also significantly upregulated starting at 2 weeks after fmod treatment ( fig2 d - f ). by week 3 , nanog , sox2 , and oct4 were all markedly upregulated in fully - reprogrammed bj - frep cells ( fig2 d - h ). meanwhile , continuous fmod exposure substantially increased phosphorylated smad3 ( psmad3 ) levels at weeks 2 and 3 ( fig2 i and j ). interestingly , persistent transforming growth factor ( tgf )- β / activin / nodal - responsive smad3 phosphorylation is also associated with maintenance of human es cells in an undifferentiated state [ 28 - 33 ]. additionally , karyotyping was normal in all tested frep cells ( fig3 and fig1 ). therefore , fmod reprogrammed ( frep ) human fibroblasts resembled es cells with respect to colony morphology , pluripotency marker expression , and sustained psmad3 signaling . similar to human es and ips cells , bj - frep cells formed ebs ( fig4 a ) and strongly expressed ectoderm and mesoderm markers , but differed in the expression time course of key reprogramming genes ( fig2 b ). notably , bj - frep - ebs , but not ips - ebs , expressed nanog and sox2 at day 2 and 14 ( fig2 b ). to confirm the multipotency of established frep cells , we assessed their ability to differentiate into ectoderm , endoderm or mesoderm derivatives following protocols established for human es and ips cells [ 17 - 19 , 21 - 23 ]. in the presence of differentiation media for neurons ( ectoderm derivatives ), the bj - frep - ebs dissociated and expanded , and most of the cells (˜ 90 %) ultimately differentiated into neurons characterized by typical neuronal morphology and expression of neuron specific βiii - tubulin ( a broadly used ectodermal differentiation marker ) and glur1 ( an excitatory neurotransmitter receptor in the mammalian brain , and activated in a variety of normal neurophysiologic processes ) ( fig4 b ). in differentiation medium with activin a , bj - frep cells stained positive for an early endodermal lineage marker afp by day 4 ( fig4 c ). with another 8 days of further differentiation without activin a , these afp - positive bj - frep cells further differentiated into pancreatic lineage cells , characterized by staining of pdx1 , a transcription factor necessary for pancreatic development and β - cell maturation ( fig4 d ). similarly , following differentiation protocols described above , we also differentiated bj - frep cells into several mesoderm derivatives , including cardiomyocytes [ fig4 e ; characterized by double staining of ctnt ( a cardiomyocyte differentiation marker ) and nkx2 . 5 ( the earliest transcription factor expressed in cardiac lineage cells )], skeletal myocytes ( fig4 f ; characterized by staining of myosin ), osteoblasts ( fig4 g ), and adipocytes ( fig4 h ). besides bj - frep cells , fmod reprogrammed nhdf ( nhdf - frep ) cells also demonstrated analogous multipotency in vitro ( fig1 ). overall , frep cell responses to ectoderm , endoderm , and mesoderm differentiation signals appeared similar to es / ips cells in vitro . to study their in vivo osteogenic potential , bj - frep cells were pre - differentiated in osteogenic medium for 3 days , loaded onto dbx scaffolds , and then implanted into the gluteofemoral muscle pocket of immunodeficient scid mice ( fig5 a ). at 8 weeks post - implantation , regeneration by bj - frep cells was tracked by ihc with antibodies against human major mhc class i ( fig5 a ). to further confirm the relative contribution of engrafted bj - frep cells to osteoblastogenesis , we immunostained for runx2 — the master transcription factor specifying osteoblastic lineage commitment [ 34 ] and ocn — a mature osteoblast differentiation marker [ 35 ]. the spatial co - localization of human mhc class i with either runx2 or ocn immunostaining ( fig5 a ) confirmed the engraftment , persistence , and differentiation of bj - frep cells into new bone tissue . in a separate study , to assess in vivo myogenic potential , bj - frep cells underwent 3 - day myogenic pre - differentiation in vitro before injection into the gluteofemoral muscle pocket of scid mice . at 8 weeks post - injection , robust co - localization of human mhc class i and human slow skeletal myosin confirmed bj - frep cell differentiation into skeletal muscle in vivo ( fig5 b ). to more rigorously assess muscle regeneration , we injured the recipient muscle with cardiotoxin [ 22 , 36 ]. identically pre - differentiated bj - frep cells were transplanted into scid mice gastrocnemius muscle 3 hours after injury by intramuscular cardiotoxin injection . at 3 weeks post - transplant , bj - frep cell treated animals revealed significantly less scar tissue and muscle degeneration ( fig5 c ). ihc confirmed human mhc class i - expressing myofibers , indicating also the engraftment , persistence and functional differentiation of bj - frep cells , this time into skeletal muscle tissue ( fig5 c ). taken together , these data conclusively demonstrated the ready feasibility and ease of using frep cells to regenerate different functional tissues in vivo using simple fmod - based reprogramming and relatively brief ( 3 - day ) in vitro pre - differentiation protocols . these data demonstrate the in vivo multipotency of frep cells and highlight their potential usefulness for cell - based tissue engineering . to test frep cell pluripotency and safety in vivo , we used a standard , scid - beige mouse teratoma model injecting bj - frep cells or positive control human h9 es cells into the kidney or testis . instead of forming tumors , injected undifferentiated bj - frep cells developed into small , calcified nodules after 3 months ( fig6 ). nhdf - frep cells also did not form teratoma in a separate study ( supplementary fig4 ). cell proliferation ratios of untreated bj fibroblasts , undifferentiated vs . differentiating bj - frep cells , and undifferentiated vs . differentiating ips cells were compared . our studies showed that undifferentiated bj - frep cells proliferated minimally , while differentiating bj - frep cells proliferated rapidly ( fig7 ). in contrast , consistent with the requirement for increased cell cycle rates during ips cell reprogramming [ 26 , 37 ], undifferentiated ips cells proliferated significantly more rapidly than differentiating ips cells or undifferentiated bj - frep cells ( fig7 ). overall , the absence of frep - associated teratoma and low rates of undifferentiated frep proliferation suggest fundamental biologic differences between frep and ips cells . frep and ips cells also showed different gene expression signatures ( fig2 h and fig8 ). in particular , when compared to ips cells , undifferentiated frep cells exhibited relatively lower oct4 ( fig2 a and 2 h ) and expressed ectoderm and mesoderm markers ( e . g . krt - 18 , nestin , and flt - 1 ) not detected in ips cells ( fig2 b ). in addition , non - fully reprogrammed “ pre - frep ” cells expressed nanog ( fig2 d and e ), while fully reprogrammed frep cells retained nanog , sox2 , and oct4 expression during eb formation ( fig2 b ). in contrast , pre - ips cells do not express nanog [ 26 ]; and lose nanog , sox2 , and oct4 expression during ips cell eb formation ( fig2 b ). remarkably , expression of the proto - oncogene c - myc , which critically regulates cell proliferation and transformation [ 38 ] and can induce teratoma formation [ 8 , 39 ], was considerably lower in bj - frep than undifferentiated ips cells by 0 . 18 - fold ( fig8 a ). in contrast , cyclin - dependent kinase inhibitors , p15 ink4b and p21 waf1 / cip1 which mediate tgf - β induced cell cycle arrest [ 40 ], were significantly upregulated ( 83 . 1 - and 3 . 8 - fold , respectively ) in bj - frep but not in ips cells ( fig8 b and c ). collectively , frep and ips cells have distinctly different proliferative , tumorigenic , and molecular phenotypes ( table 2 ). tumorigenesis remains a significant obstacle to safe clinical application of pluripotent stem cells , such as ips and es cells [ 13 , 41 ]. at the genetic level , one crucial difference between ips cells and es cells is the introduction and integration of genes , which is essential for the reprogramming process , into the genome of somatic cells . as such , ips cells are likely to carry a higher tumorigenicity risk than es cells due to gene activation or interruption from viral integration during reprogramming [ 12 , 13 , 42 - 44 ]. additionally , undesirable transgene reactivation is another problem of using viral ips cells . for example , c - myc reactivation has shown to significantly increase tumor formation in chimeric mice generated from ips cells [ 8 ]. while c - myc is dispensable for ips cells generation , reactivation of the other reprogramming factors may also cause tumors [ 8 , 12 , 13 , 19 , 39 , 44 ]. meanwhile , apart from the extremely low efficiency [ 12 , 44 - 46 ], using adenoviruses , plasmids and chemicals to generate ips cells does not exclude the risk of teratoma formation since genetic alterations from integration of small virus / plasmid dna fragments or chemically induced mutations can still occur [ 12 , 44 ]. moreover , non - integrative rna - and protein - based ips - generation approaches are complicated by the need for cell - penetrating , cytosolic delivery [ 11 ]. although the minimal criteria for demonstrating successful human pluripotent stem cell reprogramming is teratoma formation , teratoma formation in and of itself does not guarantee pluripotency as there are instances of mouse es - like cells that form teratomas but do not produce germline chimeras [ 44 ]. concurrently , significant issues impede food and drug administration ( fda ) approval of ips cells for human use , including ( i ) undesirable use of oncogenes for reprogramming , ( ii ) undesirable use of retroviral or genome integrative approaches , and ( iii ) undesirable teratoma formation . thus , reprogramming approaches that specifically concentrate on fda safety issues are required [ 12 , 47 - 49 ]. to begin addressing fda concerns , several groups have successfully reprogrammed fibroblasts into neurons and pancreatic exocrine cells into insulin - producing beta cells without an intermediate , potentially tumorigenic , pluripotent stage [ 50 , 51 ]. this reflects a recent focus on safer cellular reprogramming methodologies that generate curative cell types without teratoma formation [ 12 , 44 , 48 ]. in this respect , frep cells may fulfill both goals of functional tissue generation and teratoma prevention . in this example , we disclose an approach that uses a single extracellular matrix ( ecm ) proteoglycan — fmod , instead of genetic modifications [ 6 , 7 , 11 , 45 , 46 ], undefined embryonic stem cell extracts [ 4 , 52 ], or animal oocyte extracts [ 53 , 54 ] to generate multipotent cells useful for replacing , restoring , and / or regenerating tissue where disease or injury has caused irreparable damage to native tissues . after simple exposure to fmod , human skin fibroblasts become at least partially reprogrammed as demonstrated by alterations in gene expression profiles ( fig2 a , 2 b , 2 h , and fig9 b ). notably , a set of genes characteristic of undifferentiated es cells , nanog , sox2 , and oct4 [ 12 , 44 , 55 - 59 ] is activated during the fmod reprogramming ( fig2 d - g ). nanog , sox2 , and oct4 regulate the es potency network by acting on promoters of thousands of es genes and serve as the major transcription factors that collectively define es cell identity [ 12 , 57 , 59 - 61 ]. we have demonstrated that the frep cells are able to be differentiated into multiple functional cell types , including neurons , pancreatic lineage cells , cardiomyocytes , skeletal myocytes , osteoblasts , and adipocytes in vitro ( fig4 b - h and fig1 b - g ), suggesting a potential for multiple lineage differentiation and functional tissue regeneration . thus far , frep cells have generated functional tissues such as bone and skeletal muscle in vivo ( fig5 ), confirming that the observed in vitro multipotency is at least partially translated in vivo . overall reprogramming by fmod on upregulating nanog , sox2 , oc4 gene associated on es pluripotency may mimic a functional transcriptional network towards cell multipotency [ 62 - 65 ]. the fact that extracellular approaches can reprogram cells lends further support to our ability to reprogram fibroblasts using a single extracellular protein , fmod [ 54 ]. the success of generating multipotent frep cells using an extracellular protein approach has led us to explore the possible mechanism underlying fmod - based reprogramming . theoretically , only small molecules can freely penetrate into the cell for reprogramming . as a 59 kd ecm proteoglycan , passive entry of fmod is unlikely . alternatively , fmod has been found to be a critical component for maintenance of endogenous stem cell niches [ 66 ]. specific cellular niches or microenvironments are able to significantly modify epigenetic expression and lead to the altered cell fate [ 67 , 68 ], while fmod is known to modulate expression , organization , and function of various growth factors , cytokines , and ecm components [ 14 , 69 - 75 ]. it is thus possible that fmod significantly affects ecm microenvironment to promote signal pathways involved in cell reprogramming . for instance , fmod induces a specific , biphasic tgf - β / activin / nodal signaling response characterized by significantly reduced psmad3 levels at week 1 followed by markedly increased psmad3 at weeks 2 and 3 ( fig2 i ). previous studies has shown that fmod presence or absence profoundly alters tgf - β bioactivity in a fetal scarless wound repair model [ 14 ], and that tgf - β - stimulated smad signaling can directly upregulate nanog expression [ 29 ]. while direct smad transactivation of oct4 or sox2 has not been described , nanog itself can induce oct4 or sox2 expression [ 60 , 76 ]. thus , fmod modulation of smad signaling may explain the unique molecular phenotype of frep cells wherein by week 2 of reprogramming , pluripotency genes nanog , oct4 , and sox2 are concurrently upregulated ( fig2 d - g ). theunissen et al . have demonstrated that constitutive nanog expression , although not sufficient for pluripotency induction , does promote transition of both mef - derived pre - ips and neural stem ( ns ) cells to pluripotency under serum - free conditions with leukemia inhibitory factor ( lif ); this result suggests that nanog is able to overcome reprogramming barriers and induce pluripotency in minimal conditions [ 77 ]. hence , increased nanog expression through stimulation of tgf - β / activin / odal - responsive smad signaling is likely a significant permissive pathway for fmod - based reprogramming . in addition , early combined chemical inhibition of tgf - β and mek signaling between weeks 1 and 2 improves human cell reprogramming efficiency [ 78 ]. thus , it is also possible that the initial fmod - mediated psmad3 reduction facilitates early reprogramming . unlike undifferentiated ips cells , undifferentiated frep cells exhibit minimal proliferation rates ( fig7 ) and c - myc expression levels ( fig8 a ) in vitro and do not form teratoma in vivo ( fig6 and fig1 ). it is noteworthy that tgf - β - stimulated smad signaling can directly upregulate p15 ink4b and p21 waf1 / cip1 expression [ 40 ]. thus , fmod modulation of smad signaling may explain the increase of p15 ink4b and p21 waf1 / cip1 in undifferentiated frep cells ( fig8 b and c ). meanwhile , active smad suppression of c - myc is essential for tgf - β induced cytostasis since c - myc interacts directly with smad2 / 3 to block smad and sp1 dependent transcription oip15ink4b and p21waf1 / cip1 [ 40 ]. since low c - myc and high p15 ink4b and p21 waf1 / cip1 levels promote cytostasis [ 40 ], the expression profile of these genes in undifferentiated frep cells may explain the absence of frep - associated teratoma relative to undifferentiated ips cells in which high c - myc and lowp15 ink4b and p21 waf1 / cip1 conditions favor teratoma formation . however , further studies are needed to address the specific mechanism underlying cell reprogramming by this simple exposure to fmod . remarkably , the slow proliferative rate of undifferentiated frep cells is significantly reversed by culture in differentiation media , and differentiating frep cells proliferate faster overall than differentiating ips cells ( fig7 ). in addition , p15 ink4b and p21 waf1 / cip1 , which are critical in suppressing tumorigenesis and maintaining stem cell quiescence [ 79 - 82 ], are increased in undifferentiated frep cells ( fig8 b and c ). phenotypically , concomitant molecular multipotency and cytostasis marker expression coupled with low proliferation rates ( unless stimulated to differentiate ) as seen in the frep cells are more typical of quiescent stem cells than ips cells that require induction of cell proliferation for reprogramming [ 26 , 37 , 40 ]. moreover , the lack of teratoma formation by frep cells suggests that from a tumor avoidance perspective , undifferentiated or partially differentiated frep cells do not need to be selected out before in vivo therapeutic implantation . since incompletely differentiated es and ips cells are known to be tumorigenic and have to be removed before implantation [ 44 , 83 ], this would make frep cells potentially safer and less cumbersome than es or ips cells for clinical applications . lastly , although the degree of dedifferentiation of frep cells may not be as complete as that of other reported pluripotent stem cells at least with respect to proliferation and teratoma formation , frep cells may be a 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some detail by way of illustration and example for purposes of clarity of understanding , it is understood that certain adaptations of the invention are a matter of routine optimization for those skilled in the art , and can be implemented without departing from the spirit of the invention , or the scope of the appended claims .