間充質(zhì)干細(xì)胞和再生醫(yī)學(xué)

1、© 2014 Baishideng Publishing Group Co., Limited. All rights reserved.World J Stem Cells 2014 April 26; 6(2: 94-110ISSN 1948-0210 (online TOPIC HIGHLIGHTWJSC 6th Anniversary Special Issues (2: Mesenchymal stem cells“Ins” and “Outs” of mesenchymal stem cell osteogenesis in regenerative medicineDe

2、an T YamaguchiDean T Yamaguchi, Research Service, Veteran Administra-tion Greater Los Angeles Healthcare System and David Geffen School of Medicine at University of California at Los Angeles, Los Angeles, CA 90073, United StatesAuthor contributions: Yamaguchi DT solely contributed to this review.Sup

3、ported by Veterans Administration Merit Review Award 2 I01 BX000170-05Correspondence to: Dean T Yamaguchi, MD, PhD, Research Service, Veteran Administration Greater Los Angeles Healthcare System and David Geffen School of Medicine at University of California at Los Angeles, 11301 Wilshire Blvd, Bldg

4、 114, Rm 330, Los Angeles, CA 90073,Telephone: +1-310-2683459 Fax: +1-310-2684856Received: October 20, 2013 Revised: January 15, 2014Accepted: January 17, 2014Published online: April 26, 2014etc . that may provide peculiarities to self-renewal, dif-ferentiation, and physiologic function that may dif

5、fer from non-transformed native cells. Tissue engineering approaches to use hMSCs to repair bone defects utilize the growth of hMSCs on three-dimensional scaffolds that can either be a base on which hMSCs can attach and grow or as a means of sequestering growth fac-tors to assist in the chemoattract

6、ion and differentiation of native hMSCs. The use of whole native extracellular matrix (ECM produced by hMSCs, rather than individu-al ECM components, appear to be advantageous in not only being utilized as a three-dimensional attachment base but also in appropriate orientation of cells and their dif

7、ferentiation through the growth factors that na-tive ECM harbor or in simulating growth factor motifs. The origin of native ECM, whether from hMSCs from young or old individuals is a critical factor in “rejuvenat-ing” hMSCs from older individuals grown on ECM from younger individuals.© 2014 Bai

8、shideng Publishing Group Co., Limited. All rights reserved.AbstractRepair and regeneration of bone requires mesenchymal stem cells that by self-renewal, are able to generate a critical mass of cells with the ability to differentiate into osteoblasts that can produce bone protein matrix (os-teoid and

9、 enable its mineralization. The number of hu-man mesenchymal stem cells (hMSCs diminishes with age and ex vivo replication of hMSCs has limited poten-tial. While propagating hMSCs under hypoxic conditions may maintain their ability to self-renew, the strategy of using human telomerase reverse transc

10、riptase (hTERT to allow for hMSCs to prolong their replicative lifes-pan is an attractive means of ensuring a critical mass of cells with the potential to differentiate into various mesodermal structural tissues including bone. How-ever, this strategy must be tempered by the oncogenic potential of T

11、ERT-transformed cells, or their ability to enhance already established cancers, the unknown differentiating potential of high population doubling hMSCs and the source of hMSCs (e.g. , bone marrow, adipose-derived, muscle-derived, umbilical cord blood,Key words: Mesenchymal stem cell; Telomerase re-v

12、erse transcriptase; Extracellular matrix; Osteogenesis; Regenerative medicine; Tissue engineering; Prolifera-tion; DifferentiationCore tip: When human telomerase reverse transcrip-tase (hTERT transformed human mesenchymal stem cells (hMSCs are used to prolong replicative potential and osteogenic dif

13、ferentiation, consideration should be given to using lower population doubling hTERT-transformed hMSCs to avoid potential oncogenesis. An inducible hTERT system may also avoid oncogenic transformation. Demonstration of in vivo bone forming capacity of hTERT-transformed cells should be used as standa

14、rd in determining osteogenic differentiation of such cells rather than in vitro culture mineralization; the CD146 marker may be a suggested surface marker for hTERT-transformed hMSCs that may have the capacityYamaguchi DT. Mesenchymal stem cells in bone regenerationto form bone in vivo. Native ECM f

15、rom early population doubling hMSCs or hMSCs from a younger source may be best when seeking to extend the proliferative and differentiating potential of hMSCs from either young or older sources.INTRODUCTIONThe regeneration of mesodermal and neural crest-derived structural or connective tissues such

16、as bone, cartilage, muscle and tendon continues to be a widely pursued for the reason that such structural tissues are generally ho-mogeneous with either a predominantly single cell type or limited number of cells that contribute to the make-up of the tissue and that precursors to the mature cell ty

17、pes can be found in adult tissues. These precursor cells are generally multipotent, in that they can differentiate into a variety of connective tissue phenotypes. These precursor cells are generally referred to as adult mesenchymal stem cells (MSCs or bone marrow stromal cells and can be found in th

18、e bone marrow but also as similar multipotent cells in specific tissues as well as circulating cells in blood. Tissue engineering seeks to replace tissues that are either lost by traumatic events or by disease through the use of specific cell types that can recapitulate the lost or diseased tissue,

19、and generally used in combination with a three-dimensional structural scaffold, and in many instances in combination with various growth factors, cytokines, and hormones or other biological molecules to assist in either the creation of a critical mass of needed cells or to assist in differentiating

20、these cells to the re-quired tissue type.Because generating a critical mass of cells used in the regenerative process is a key to successful tissue engineer-ing followed by differentiating those cells into the specific cell type comprising the tissue, stem cells have been the preferred starting cell

21、 type in many tissue engineering trials. This minireview will focus only on human adult bone marrow MSCs (herein assumed to be synonymous with bone marrow stromal cells as much as possible and the telomerase strategy of inducing self-renewal of these cells to create a critical cell mass. Secondly, t

22、he minireview will examine the strategy of using extracellular matrix as a native scaffold upon which mesenchymal stem cells can self-renew and differentiate into bone.MESENCHYMAL STEM CELL SELF-RENEWALThe ability to self-renew is a hallmark of any stem cell1.Self-renewal is simply defined as the ab

23、ility of the result-ing daughter cells, after mitotic division of the original mother cell, to retain the ability to generate a variety of differentiated cell types identical to that of the ability of the mother cell to differentiate in to those same cell types, and for a daughter cell to be able to

24、 generate daughter cells that also maintain the ability to differentiate into the same variety of cell types as the original “grandmother” and mother cells2. The maintenance of self-renewal and pluripotency of stem cells occurs in the stem cell niche, where stem cells are able to receive cues from t

25、he stroma and other cell types either by direct contact or by secreted soluble factors within this microenvironmental niche3,4. Adult MSCs also share the ability to self-renew. This potential to self-replicate and to differentiate into connec-tive tissue phenotypes has led to the exploration to util

26、ize MSCs in the repair of injured tissues5,6. While the bone marrow has been a common site to harvest MSCs, other cell types similar to bone marrow-derived MSCs can also be found in other sites. Adipose-derived stem cells, satel-lite cells in muscle, and pericytes around blood vessels and umbilical

27、cord blood cells also may share multipotent characteristics for differentiation into connective tissue phenotypes under specific conditions which include se-lective differentiation media and growth factors7-10. In a comparison of MSCs from bone marrow, adipose tissue, and cord blood, Rebelatto et al

28、11 (2008 reported that iso-lation rate of MSCs from umbilical cord blood was only a third that of bone marrow-derived and adipose-derived MSCs. The initial growth rate of bone marrow-derived and adipose-derived MSCs was much higher than that of umbilical cord blood MSCs. However, others have shown t

29、hat the proliferation of umbilical cord tissue-derived MSCs show higher population doublings and shorter doubling times compared to adipose-derived MSCs al-though adipose-derived MSC had higher numbers of colony-forming units compared to MSCs from umbilical cord tissue12. Surface marker expression o

30、f CD34 (cluster of differentiation molecule in family of sialomucin pro-teins was significantly higher in adipose-derived MSCs compared to that of bone marrow-derived MSCs. Inter-estingly, CD117 (tyrosine-protein kinase Kit was found to be positive in about 98% of adipose-derived MSCs but positive i

31、n only 52% and 39% of bone marrow-derived and umbilical cord blood-derived MSCs. Additionally, while osteogenic and chondrogenic differentiation was similar in MSCs from all three sources, umbilical cord blood-derived MSCs showed a lesser propensity for adip-ogenic differentiation. Others have also

32、noted differences in marker expression between bone marrow-derived and adipose-derived MSCs. For instance, CD106 (vascular cell adhesion molecule-1 is expressed in bone marrow-derived MSCs but its expression in adipose-derived MSCs is either low or non-existent while CD49d (integrin 4 subunit is exp

33、ressed in adipose-derived MSCs but not in bone-marrow-derived MSCs13. Culture conditions such as the use of fetal bovine serum, human serum, or serum-free medium have been shown to influenceYamaguchi DT. Mesenchymal stem cells in bone regenerationnot only the expression of surface markers for adipos

34、e-derived MSCs e.g. , CD117, CD166 (activated leucocyte cell adhesion molecule and bone marrow-derived MSCs but also in differentiation potential of such MSCs. As an example, fetal bovine serum has a stronger influence on osteogenic differentiation of adipose-derived MSCs than it does on adipogenic

35、differentiation while allogeneic human serum and serum-free conditions have greater propensity to drive adipose-derived MSCs towards adipo-genic differentiation than towards either osteogenic or chondrogenic lineages14. Thus while adipose tissue and perhaps umbilical cord tissue sources may provide

36、ample sources for MSCs compared to that of bone marrow and umbilical cord blood, differences in some specific surface markers for MSCs, proliferative potential, and differentia-tion potential in vitro occur based on the source of start-ing material to isolate MSCs, tissue culture supplements and con

37、ditions, and even human individual heterogeneity. Whether non-bone marrow-derived MSCs favor dif-ferentiation into specific connective tissue types or even non-mesodermal cell types as in the case of umbilical cord blood MSCs and adipose-derived MSCs in an in vivo environment is still a ripe area of

38、 investigation13-15.Age of the organism is a determinant of the number of bone marrow MSCs present as well as in vitro tissue culture conditions that are critical for MSCs to retain their ability to self-renew yet demonstrate plasticity in their ability to differentiate into various mesodermal tissu

39、es16. The number of cells from human bone marrow that are MSCs as determined by colony forming unit-fibroblastic (CFU-f assay are less than 0.1% of total bone marrow mononuclear cells, thus demonstrating a minimal number of hMSCs that can be used in bone regeneration17. The numbers of CFU-f and the

40、capacity of CFU-fs that can differentiate into osteoblasts further decrease as a func-tion of age of the bone marrow donor up to age 40; after age 40, there does not appear to be any further diminish-ing of CFU-fs that can differentiate into osteoblasts18. It was suggested that hMSCs have decreased

41、proliferative capacity as a function of age19. Thus hMSCs from young individuals ages 18-29 years achieved an average popula-tion doubling level of 41 whereas hMSCs from older in-dividuals ages 66-81 years achieved an average population doubling level of 24 with about a 55% lower population doubling

42、 rate than in hMSCs from the younger individu-als. However, no difference in in vivo bone formation was noted as a function of donor age with early passage cells from either age group. Thus, once placed in primary culture, hMSCs have a limited lifespan (average 20 to 40 population doublings, but the

43、 number of popula-tion doublings may differ depending on growth medium or any added growth factors19-21 under environmental conditions normally used for in vitro cell culture (humidi-fied 5% CO2 and 95% air (21% O2 and when grown on tissue culture plastic. hMSCs grown in such conditions attain the H

44、ayflick limit where cell division ceases, and the usual hMSC size becomes larger and the usual spindle shape of normal hMSCs becomes more polygonal orwith a variety of shapes and sizes, at times with multi-nucleation, and overall with less cell density per culture than cells undergoing cell division

45、22. As the number of population doublings for such cells is limited practically in primary culture, slower cell division and finally lack of cell division ensues and the above morphological changes are noted, and the expression of senescence-associated -galactosidase, and p16, markers of cellular se

46、nescence, are increased23. However, it has been shown that if en-vironmental conditions simulate the MSC niche in the bone marrow, specifically low oxygen tension, that self-renewal of hMSCs can be prolonged. DIppolito et al24 (2004 developed a multilineage inducible MSC model from human cadaveric v

47、ertebral body marrow (MIAMI cells and propagated them in 3% O2/5% CO2/92% N2. They reported that more than 50 cell doublings beyond the Hayflick limit for primary cells could be achieved from hMSCs from at least 3 of 12 donors and at least 30 population doublings could be achieved from all of their

48、donors. In a follow-up communication, they reported that MIAMI cells grown in 3% O2 doubled more quickly than those grown at 21% O2 and maintained the embry-onic transcription factors OCT-4, REX-1, and hTERT and had suppressed osteoblastic differentiation when exposed to osteogenic differentiation m

49、edium. At higher O 2 concentrations of 21%, these embryonic transcrip-tion factors were lost and osteogenic differentiation was enhanced 25. The mechanism by which hypoxia regulates stem cell self-renewal appears to be via hypoxia inducible factor-1 (HIF-1. Low oxygen concentrations stabilize HIF-1

50、by inhibiting its degradation by the proteasome. Mazumdar et al26 (2010 reported that hypoxia induced canonical Wnt/-catenin signaling and increased tran-scription of Lef/Tcf genes which have hypoxia response elements in their promoter regions that bind HIF-1. Canonical Wnt/-catenin signaling thus c

51、an induce in-creased cell proliferation.HTERT TRANSFORMATION OF HMSCS-THE “INS” FOR SELF-RENEWALIn lieu of special resources needed to grow hMSCs in a hypoxic environment to maintain a proliferative state, a self-renewal strategy, engineering of hMSCs to over express telomerase has been an alternati

52、ve means to maintain a longer proliferative lifespan of such cells. Telomerase, which is a multi-subunit ribonucleoprotein found in the cell nucleus and perhaps closely associated with nucleoli, allows for the addition of non-coding telomere DNA at the 3 end of linear chromosomes27-29. Maintenance o

53、f telomere length by the addition of TTAGGG repeats onto the ends of telomeres allows for cells to continue to divide30. Telomerase is expressed in human embryonic cells and in fetal, newborn, and adult testes and ovaries but not in mature spermatozoa or oo-cytes. Moreover, expression of telomerase

54、disappears in human somatic cells in the neonatal period and later in life 31. Thus lacking telomerase, telomeres shorten withYamaguchi DT. Mesenchymal stem cells in bone regenerationeach cell division leading to replicative senescence once cells reach a critical shortened telomere length. Specifi-c

55、ally, with respect to MSCs, a number of laboratories have reported that hMSCs from bone marrow do not express telomerase activity or have activity below detect-able levels by telomeric repeat amplification protocol (TRAP assay when hMSCs are asynchronously divid-ing 20,32-34. However, human telomera

56、se reverse transcrip-tase (hTERT expression and telomerase activity could be detected when cells were synchronized to S-phase34. Others have found that telomere length in hMSCs is short upon initial isolation and tend to further shorten with cell passage in vitro and appear to correlate with low to

57、undetectable levels of hTERT35. Thus theoreti-cally, maintaining telomerase expression should prevent replicative senescence. Additionally, the decrease in telo-mere length correlates with CFU-f numbers suggesting that telomere length and telomerase activity could also be related to the ability of h

58、MSCs to differentiate along various cell lineages including the osteogenic lineage35. Gronthos et al36 (2003 reported that expression of hTERT in human bone marrow-derived MSCs not only increased proliferative capacity by up-regulating G1 to S phase transition cell cycle genes but also increased the

59、 expression of osteogenic genes for cbfa-1, osterix, and osteocalcin and induced bone formation earlier and to a much larger degree in an in vivo ectopic bone forma-tion assay of hTERT-transformed hMSCs. Saeed et al37 (2011 demonstrated that in telomerase-deficient mice (Terc -/-, there was delayed ossification in occipital bone, sternum, vertebrae, and metatarsals. Overall bone vol-ume was decreased compared to wild type controls, and trabecular bone paramet