标题: Human Umbilical Cord Perivascular (HUCPV) Cells: A Source of Mesenchymal Progeni [打印本页] 作者: 江边孤钓 时间: 2009-3-5 10:52 标题: Human Umbilical Cord Perivascular (HUCPV) Cells: A Source of Mesenchymal Progeni
Institute for Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada6 _& `- W- F5 ^/ F! Z' B
- E% T/ `' {* F9 ]+ XKey Words. Mesenchymal progenitors ? Umbilical cord ? Allogeneic cells ? Major histocompatibility complexes ? Cryopreservation ? Therapeutic dose 1 M2 J- x9 s; S9 e1 i J) m U U5 C# N, _& M5 i5 TCorrespondence: J. E. Davies, B.D.S., D.Sc., Institute of Biomaterials and Biomedical Engineering, University of Toronto, 4 Taddle Creek Road, Room 407, Toronto, ON M5S 3G9, Canada. Telephone: 416-978-1471; Fax: 416-946-5639 ; e-mail: davies@ecf.utoronto.ca; Website: http://www.ecf.utoronto.ca/~bonehead ( h" F! m* C! j. O ( I* t5 ^" E* p% p9 z9 i- |* xABSTRACT" j/ d: z% \( ^4 o4 x" |7 b
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Since it was first used to treat a patient with Wiskott-Aldrich syndrome , bone marrow (BM) has been the most common source of cells for cell-based therapies. The mesenchymal population of BM is targeted for a variety of therapeutic approaches affecting a wide range of tissues, including those of the musculoskeletal system: bone , cartilage , and tendons and ligaments . BM cell therapy has also been suggested for repair of the myocardium and is being pursued clinically for applications in hematology and oncology such as aplastic anemia and malignant lymphoma . Following encouraging results in nonobese diabetic/severe combined immunodeficient mice , Ko? et al. have shown beneficial clinical outcomes by coinfusion of culture-expanded mesenchymal cells with hematopoietic stem cells in patients treated with high-dose chemotherapy for solid tumors. Other promising therapeutic approaches include mesenchymal stem cells (MSCs) as carriers of the therapeutic genes or the infusion of allogenic BM for the treatment of osteogenesis imperfecta (OI) . In the latter, BM was from an HLA (human leukocyte antigen)–identical or single mismatched sibling. However, since immune rejection and donor number limitations are major constraints to common use, there is an acute need to find alternative cell sources for such cell-based therapies. As cells are a fundamental requirement for tissue engineering , cell sourcing also remains a major challenge for human tissue-engineering strategies.& I0 B/ P* U3 ^/ c) k8 S
) s# @3 G1 [# {; L6 m- u0 K; tOne potential alternative source of mesenchymal cells became feasible with the report by McElreavey et al. of the culture of cells from Wharton’s jelly (WJ), the primitive connective tissue of the human umbilical cord (UC), first described by Thomas Wharton in 1656 . Thus, Naughton et al. and Purchio et al. derived "prechondrocytes," from explant cultures of UC WJ, and Mitchell et al. , using a similar approach, reported that the fibroblast-like cells of WJ could be induced to differentiate into "neural-like" cells expressing neuron-specific enolase (NSE), as well as other neural cell markers. Romanov et al. , using a different approach, enzymatically digested mesenchymal precursor cellsfrom the UC vasculature endothelial surface, and Kadner et al. minced either UC vessels or whole cord to derive an autologous cell source of myofibroblasts for cardiovascular tissue engineering. Chacko and Reynolds described the cells residing in WJ as "smooth muscle cells," but Takechi et al. refined the description to "myofibroblasts" after in situ labeling of vimentin, desmin, -actin, and myosin, which has been recently confirmed by Kadner et al. .. u0 J6 B$ o: f" J& s! G2 ~
9 G& ~5 O$ [$ i4 o" S# kThe human UC is embryologically derived at day 26 of gestation, and it grows to form a 30- to 50-cm-long helical organ at birth. Given this expansion, during the 40 weeks of gestation, there must be a mesenchymal precursor cell population within the UC that gives rise to the WJ connective tissue. We postulated that these cells would most likely be located closest to the vasculature, and thus to their source of oxygen and nutrients. Consequently, we reasoned that human umbilical cord perivascular (HUCPV) cells, which were either discarded, or not specifically isolated, in the previously described studies, should contain a subpopulation that, when isolated, would be capable of exhibiting a functional mesenchymal phenotype. 8 k% K+ e( a6 z7 `8 v1 D" ], l1 k( ?1 K3 P' Q+ u
Thus, we report herein a novel harvesting protocol designed to isolate HUCPV cells and show that the resultant cell population possesses a high frequency of colony-forming unit-fibroblast (CFU-F)–deriving cells that proliferate and differentiate rapidly to form bone nodules (BNs). Furthermore, we show that the isolated cell population includes an expanding subpopulation that expresses neither class I nor class II major histocompatibility (MHC) antigens, suggesting a potential role as a human allogeneic cell source for cell-based therapies.# z5 ?3 ~( v% Q4 A6 I) T$ R7 R
; E; ^6 T/ a- |3 w, [8 OMATERIALS AND METHODS: @5 U) K2 z2 r/ s; O9 b
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Figure 1A shows the SEM appearance of the perivascular WJ matrix which, by routine hematoxylin and eosin light microscopy (not shown), was seen to possess a relatively homogeneous distribution of cells. The harvested cells exhibited a morphologically homogeneous "fibroblast-like" appearance (Fig. 1B) with a stellate shape and long cytoplasmic processes extending between 100 and 300 μm. These cells labeled positively for -actin, desmin, vimentin, and the 3G5 monoclonal antibody (not shown), but we found no evidence of NSE. % o7 P2 p( y' |! I6 A+ b: ?4 ~# I% q3 c, i4 D" H- o% K& Z2 h" I
Figure 1. (A): Scanning electron microscopy of an umbilical artery that has been excised from a human umbilical cord as part of the HUCPV cell harvesting procedure. The white dotted line represents the outer margin of the vessel and thus illustrates the perivascular tissue from which the HUCPV cells are harvested. (B): HUCPV cells display a fibroblastic morphology (field width = 660 μm). Abbreviation: HUCPV, human umbilical cord perivascular. ; p4 }- H( q; w$ Z8 V- K0 B5 K# L- W0 [& P
CFU-F and CFU-O Expansion+ x: }% r- r o
% f) ]( N/ L6 U' Y# V9 D0 u! lThe digestion procedure yielded an average of 2–5 x 106 HUCPV cells per UC (depending on the length of UC harvested, which can vary from 10–30 cm). Normalized to a unit length of cord, this represents a harvesting yield of 2.5–25 x 104 cells/cm of cord and a harvesting efficiency of 100% since every cord yielded cells (n = 72). Counting the number of cell colonies at passage 0 (P0) established a CFU-F frequency of 1:333 (±0.83). Seeding multiples of this number of cells demonstrated an increase in CFU-F frequency with increasing cell-seeding densities (Fig. 2), indicative of some paracrine signaling between HUCPV cells, which may potentiate CFU-F formation. 4 s* G, s9 m* |$ P $ v! k1 x4 Z0 RFigure 2. A nonlinear increase in CFU-F frequency is observed with serially increasing cell-seeding densities compared with the expected linear CFU-F frequency. This difference may indicate a paracrine signaling mechanism between human umbilical cord perivascular cells (n = 6). Error bars denote standard deviation. Abbreviation: CFU-F, colony-forming unit-fibroblast.$ @2 F% z1 L8 w+ V
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P0 through P7 HUCPV cells demonstrated a decreasing doubling time of 59.4 ± 42.4 hours (P0) to 19.71 ± 12.4 hours (P2), and this remained approximately constant until P8 (Fig. 3), by which time over 50 population doublings had already been achieved. The HUCPV cells demonstrated a growth curve with an initial lag phase (0–24 hours) and subsequent log phase (24–120 hours) (Fig. 3 insert). Figure 4 shows that from day 0 to the end of the second passage (30 days of culture) the number of HUCPV cells increased from 6.6 x 103 to 1.4 x 1010. Within this CFU-F population, frequencies of CFU-O were determined to be 2.6/105 CFU-F and 0.75/105 CFU-F in the absence of OSs, and 1.20/104 CFU-F and 1.29/104 CFU-F at P1 and P2, respectively, with the addition of OS. No BNs were found in P0 cultures in either osteogenic or nonosteogenic conditions. Thus, after 30 days of culture, 1.8 x 106 CFU-O cells were resident in the whole CFU-F population in OS conditions.% {6 n9 A: S+ P( V! d
7 _$ I* \/ C) } K' [! } S! b$ ?0 OFigure 3. Doubling time of HUCPV cells with successive passaging, demonstrating increasing proliferation to a 20-hour doubling time from P2 to P7, and increase after P8 (n = 3). Insert: Proliferation of P2 HUCPV cells from 0–120 hours, illustrating a normal growth curve with a lag phase of 0–24 hours and a log phase of 24–120 hours (n = 3). Error bars denote standard deviation. Abbreviation: HUCPV, human umbilical cord perivascular.5 B2 H; @6 d2 I( y: g3 e
; Z1 S6 b$ ]% t HFigure 4. Frequency of CFU-F and CFU-O cells with successive passaging of human umbilical cord perivascular cells in the presence and absence of OSs (n = 4). Error bars denote standard deviation. Abbreviation: CFU-F, colony-forming unit-fibroblast; CFU-O, colony-forming unit-osteogenic; OS, osteogenic supplement.; \, H! P, ^ o# Y
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Bone Nodule Formation ; L8 u* s' w. y! g4 l+ Q7 F* o4 s+ i+ z* {
Passaged HUCPV cells in the presence of OS demonstrated markers of osteogenic expression within 4–5 days of culture. Colonies of cells with high alkaline phosphatase (ALP) expression that was positive for mineralization with von Kossa staining were indicative of osteogenic differentiation. The colonies were characterized by an accumulation of fibroblast-like cells in direct contact with one another. The colonies expanded in size, to between 300 and 800 μm in diameter (Fig. 5A) and approximately 100 μm in height (Fig. 5B). The cells bordering the nodules (Fig. 5C) were of a fibroblastic morphology, while those toward the interior of a nodule were more polygonal. Ultraviolet fluorescence of the tetracycline-labeled nodules (Fig. 5D) illustrated the variation of mineralization associated with their structure. Mineralization appeared to be relatively heavy in the middle of the nodule, as seen by an intense fluorescence, while the periphery of the nodule had less fluorescence intensity.* e. U: N+ m, c7 F
" k2 v# M, T4 XFigure 5. Tetracycline-labeled bone nodules observed by (A) phase microscopy and (B) fluorescence microscopy (FW = 832 μm). (C): A similar nodule seen by scanning electron microscopy (FW = 590 μm). (D): A bone nodule sectioned horizontally (parallel to culture dish surface) and stained with Masson trichrome (FW = 720 μm). Note the cells surrounded by abundant collagenous extra-cellular matrix. Abbreviation: FW, field width. " i$ P c8 p/ x: ?) ?, w7 Y: P" |! v+ t
Figure 5D illustrates a demineralized Masson trichrome-stained transverse section of a BN. The areas in blue represent the collagen that makes up the bulk of the BN in which were embedded round nucleated cells, putatively identified as osteocytes.# J0 q. L3 O) e3 E d
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Flow Cytometric Analysis # p3 `! b/ E- T4 M) X ! A1 D/ Q* M0 ^All analyzed HUCPV cells labeled positively for CD105 (SH2), CD73 (SH3), CD90 (Thy-1), and CD44, but negatively for CD45, CD34, CD235a (glycophorin A), CD106 (VCAM1), CD123 (IL3), SSEA-4, HLA-DR, DP, DQ (MHC II), HLA-G, and Oct4 (Table 1). HUCPV cells did not label with the hybridoma-derived STRO-1 antibody, although the latter did label a 35% subpopulation of a human BM positive control. Subpopulations of HUCPV cells labeled positively for other cell-surface proteins, including 15% CD117 (c-kitlow) and 75% HLA-A, B, C (MHC Ilow).+ X9 h @: }2 v* B3 U) Y
# W- Q, f1 |% T8 KTable 1. Flow cytometry results of human umbilical cord perivascular cells labeled for several cell-surface and intracellular markers. Data gained from a total of 11 umbilical cords in which n 3. 9 Y& d+ O4 B# x/ k, ` 2 |7 F3 ^6 k. mFigure 6 illustrates the MHC I/II (MHC– /–)expression of serially passaged HUCPV cells and cryopreserved HUCPV cells. The input cell population contained 20.8% ± 3.1% which were MHC–/–. This subpopulation increased to 31.2% ± 1.7% at P5. Following cryopreservation, HUCPV cells demonstrated an increased MHC–/– population, rising from 65.2% ± 5.4% at P0 to 96.0% ± 3.9% at P5. Upon rapid thawing of the frozen aliquots of cells in a 37～C water bath, cell survival at P0 was 49.2% ± 23.8 (n = 12), while thawing of cells from P1 through P9 resulted in a survival of 62.6% ± 19.7 (n = 30). ; d) z3 Z/ N2 A5 L9 Q: @" q& M/ `' g2 a
Figure 6. Flow cytometry results of MHC–/– expression on HUCPV cells with serial passaging, and the change of MHC–/– expression with cryopreservation of serially passaged HUCPV cells, which reached 95% at P5 (n = 3). Error bars denote standard deviation. Abbreviations: HUCPV, human umbilical cord perivascular; MHC, major histocompatibility complex.4 r) o7 L: e# j$ S
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DISCUSSION1 u4 ?- K$ m# D
; o3 s$ X/ j3 t, p# a) C8 UAlthough the in vivo function of HUCPV cells still needs to be studied, we believe these cells represent a population of normal, rapidly expandable, MHC–/– cells that can potentially generate multiple therapeutic doses of cells for cell-based therapies, and thus they represent a significant alternative to BM in the treatment of pathologies associated with the connective tissues of the human body. ! P: S# T* ~6 y# A4 X9 l ?+ ^; m7 d/ P2 r1 V4 _
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Donovan PJ, Gearhart J. The end of the beginning for pluripotent stem cells. Nature 2001;414:92–97.(Rahul Sarugaser, David Li)作者: xuguofeng 时间: 2015-5-31 09:18