干细胞修复技术

d s im 5 and engraftment is present over 11 months post- associated with human ES cells are eliminated, and the possi- bility of generating patient-specific iPS cells for autologous ther- tions (Emery, 2002). Current treatment options are only palliative, and thus far there is no cure for any type of MD. Therapeutic strategies that focus on the replacement of the diseased muscle between transplantation studies involving mouse and human pluripotent stem cells. Proof-of-principle studies using human iPS cells are required in order to begin seriously considering tissue with stem cells that can give rise to healthy myofibers, as well as self-renew, are particularly attractive. This strategy has been used in the hematopoietic system for the past 40 years potential therapeutic applications of these cells. Here we describe the efficient derivation of a proliferating pop- ulation of human skeletal myogenic progenitors from both ES transplant. This study provides the proof of principle for the derivation of functional skeletal myogenic progenitors from human ES/iPS cells and highlights their potential for future therapeutic application in muscular dystrophies. INTRODUCTION Muscle wasting affects millions of individuals worldwide and is caused by a variety of conditions, including cachexia, sarcope- nia, and muscular dystrophies (MDs). The latter comprises more than 30 genetically distinct disorders that culminate in paralysis and, in many instances, cardiopulmonary complica- apies is enabled. Whereas safety issues still need to be carefully addressed before these cells can be used in the clinical setting, a critical prerequisite for a potential therapeutic application is the generation of abundant engraftable tissue-specific cell prepara- tions. Although the use of mouse iPS-derived cells to correct a disease phenotype has been documented for several models of disease through derivation of hematopoietic (Hanna et al., 2007), endothelial (Xu et al., 2009), neural (Wernig et al., 2008), pancreatic (Alipio et al., 2010), liver (Espejel et al., 2010), and myogenic (Darabi et al., 2011a; Mizuno et al., 2010) precursor cells, the human iPS field lags far behind in this regard. To date, there is only one study documenting functional improve- ment from human iPS cells, using a rat model of Parkinson’s disease (Hargus et al., 2010). Thus, there is clearly a huge gap exhibit superior strength. Importantly, transplanted cells also seed themuscle satellite cell compartment, 2008; Takahashi et al., 2007; Yu et al., 2007), ethical concerns Human ES- and iPS-Derive Restore DYSTROPHIN and upon Transplantation in Dy Radbod Darabi,1 Robert W. Arpke,2 Stefan Irion,3 John T. D and Rita C.R. Perlingeiro1,* 1Department of Medicine 2Department of Pediatrics Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 554 3iPierian, 951 Gateway Blvd, South San Francisco, CA 94080, USA *Correspondence: perli032@umn.edu DOI 10.1016/j.stem.2012.02.015 SUMMARY A major obstacle in the application of cell-based therapies for the treatment of neuromuscular disor- ders is obtaining the appropriate number of stem/ progenitor cells to produce effective engraftment. The use of embryonic stem (ES) or induced pluripo- tent stem (iPS) cells could overcome this hurdle. However, to date, derivation of engraftable skeletal muscle precursors that can restore muscle function from human pluripotent cells has not been achieved. Here we applied conditional expression of PAX7 in human ES/iPS cells to successfully derive large quantities of myogenic precursors, which, upon transplantation into dystrophic muscle, are able to engraft efficiently, producing abundant human- derived DYSTROPHIN-positive myofibers that 610 Cell Stem Cell 10, 610–619, May 4, 2012 ª2012 Elsevier Inc. Cell Stem Cell Short Article Myogenic Progenitors Improve Contractility trophic Mice os,3 Marica Grskovic,3 Michael Kyba,2 5, USA with great success. A major caveat with muscle tissue is the impossibility of obtaining enough skeletal muscle stem cells (satellite cells) without causing severe and permanent damage to the muscle of the donor, in contrast to hematopoietic stem cells (HSCs), which can be harvested from mobilized peripheral blood or marrow with minimal harm to the donor. Small muscle biopsies allow for the ex vivo expansion of satellite cell progeny; however, as observed for HSCs (Guenechea et al., 1999), ex vivo expansion of myoblasts from satellite cells results in loss of engraftment ability (Montarras et al., 2005). Consistently, early clinical trials involving the transplantation of ex-vivo-expanded myoblasts failed to improve strength in patients with Duchenne’s MD (Mendell et al., 1995; Vilquin, 2005). Therefore, alternate sources of early skeletal muscle progenitors are required for the feasibility of a stem cell therapy approach for MD. One of the major advantages of pluripotent stem cells is the prospect of generating large quantities of specific cell popula- tions for regenerative purposes. In particular, with the recent breakthrough of reprogramming somatic cells (Park et al., no A VVI B sBEsllecSE9H-7XAPi EB-derived mo IIIIII + Purification of PAX7+ (GFP+) cells Cell Stem Cell Muscle Engraftment from Human ES and iPS Cells and iPS cells, which, upon transplantation into dystrophin-defi- cient mice, promote extensive and long-term regeneration that is accompanied by functional improvement. RESULTS PAX7 Induces the Myogenic Program in Differentiating Human ES and iPS Cells To assess whether PAX7, a paired-box transcription factor well known for its role in the maintenance of the adult satellite cell compartment (Oustanina et al., 2004; Seale et al., 2000), can efficiently induce the myogenic program in human ES- and iPS-derived embryoid bodies, as observed in mouse cultures (Darabi et al., 2011a; Darabi et al., 2011b), we modified the C e ll c o u n t H9 IPRN 14.57 IPRN 13.13 Days 10 10 10 10 0 4 8 12 16 8 7 6 5 C PAX7 MYOGENIN MHC D if f e r e n t ia t io n P r o li f e r a t io n iPAX7-H9 92 ± 1.7 3.6 ± 0.2 12.6 ± 2.7 89 ± 1.2 5 ± 1.2 94 ± 1.02 iPAX7-IPRN 13.13 95 ± 0.7 3.1 ± 0.5 10 ± 1.4 91 ± 1.3 2.8 ± 0.5 96 ± 1.5 D if f e r e n t ia t io n P r o li f e r a t io n D if f e r e n t ia t io n P r o li f e r a t io n iPAX7-IPRN 14.57 94 ± 1.01 2.9 ± 0.3 7.9 ± 1.01 87 ± 2.3 3.09 ± 0.8 93 ± 1.9 Expansion of PAX7 progenitors 14% GFP D E vector encoding PAX was detected by inc stream of the PAX7 g PAX7 induction in the cence analyses, whic GFP upon doxycyclin modification did not al or their ability to di (Figure 1A). In embryogenesis, P myogenic fate within p tiated iPAX7 human (h lowed by 3 days in m (Figure 1A). This time Cell Stem Cell 10, 610 differentiation by adding dox to the myogenic medium. GFP+ (PAX7+) cells emerge in these cultures and begin to proliferate. GFP+ cells are purified by FACS (IV). Representative FACS profile shows PAX7 (GFP) expression after 4 days of dox induction in H9 differentiating ES cells. The percentage indicated represents the fraction of GFP+ cells (IV). PAX7+ myogenic progenitors are layer PAX7 induction + dox Figure 1. Myogenic Induction of Human ES/ iPS Cells by PAX7 (A) Schematic of differentiation protocol with representative morphological aspects of iPAX7 H9: in the undifferentiated state as ES cell colonies in mTeSR medium (I), and in the EB stage (II). At day 7 of differentiation, EBs are collected and plated on a gelatinized flask to grow as a mono- layer (III). PAX7 induction is initiated at day 10 of expanded in myogenic induction medium sup- plemented with dox and human bFGF (V). Scale bars represent 100 mm. (B) Growth curve of PAX7-induced ES- and iPS- derived myogenic progenitors during in vitro expansion. Data represent mean ± SE of four independent experiments. (C–E) Immunostaining of PAX7-induced human ES-derived (C) and iPS-derived (D and E) myogenic cells for PAX7, MYOGENIN, andMHC in proliferation (top) and differentiation (bottom) conditions. With PAX7 induction under prolifera- tion conditions, most cells express PAX7 and only a few express markers of terminal differentiation (top panels), whereas under differentiation condi- tions (and dox withdrawal), almost all of the cells become positive for MYOGENIN and MHC, form- ing multinucleated myotubes (bottom panels). Cells were costained with DAPI (blue). Numbers on each panel represent the percentage of cells ex- pressing PAX7, MYOGENIN, or MHC. Data are mean ± SE. For each condition, four slides were used for quantification. Scale bars represent 100 mm. See also Figure S1. human H9 ES cell line and two well-char- acterized human iPS cell lines, IPRN13.13 and IPRN14.57 (see Figures S1A–S1F available online), generated from fibroblasts from normal donors, with a doxycycline-inducible lentiviral 7 (iPAX7). Expression of the transgene orporating an ires-GFP reporter down- ene (Figure S1G). Further confirmation of se cells was provided by immunofluores- h showed coexpression of PAX7 and e (dox) induction (Figure S1H). Genetic ter the morphology of the pluripotent cells fferentiate into embryoid bodies (EBs) AX7 and its homolog PAX3 act to confer araxial mesoderm. We therefore differen- ) ES and iPS cells for 7 days as EBs fol- onolayer before inducing PAX7 with dox point is well into the peak of mesoderm –619, May 4, 2012 ª2012 Elsevier Inc. 611 CD IPRN 13.13 98% 88% 98% 100% 100% CD56 α 7 INTEGRIN M- CADHERIN CD29 70% 98% 100% H9 IPRN 14.57 98% 93% 98% 100% A generation, as indicated by Brachyury expression (Figure S1I). Following 4 days of induction, PAX7+GFP+ cells were purified by fluorescence-activated cell sorting (FACS) and expanded in secondary monolayer culture in proliferation medium containing dox and bFGF (Figure 1A and Figure S1J). Both ES- and iPS- derived myogenic progenitors demonstrated notable expansion potential, averaging 86-fold by week 2 (Figure 1B), with a total of six to seven doublings during this period. Under these prolifera- tion conditions, iPAX7 hES and hiPS cells expressed PAX7 abundantly (Figures 1C–1E and Figures S1K and S1L). MYOGENIN and MYOSIN HEAVY CHAIN (MHC), markers of terminal muscle differentiation, were barely detectable (Figures 1C–1E). This profile changed when iPAX7 hES and hiPS cells were subjected to differentiation (5% horse serum and with- drawal of dox and bFGF; differentiation medium). In these culture conditions for 2 weeks, human myogenic progenitors differentiated into multinucleated myotubes, with abundant expression of MYOGENIN and MHC, while rare cells expressed PAX7 (Figures 1C–1E). These results were confirmed by gene Human DYSTROPHIN Human / pan-dystrophin Human DYSTROPHIN Human / pan-dystrophin I P R N 1 3 .1 3 C o n t r o l I P R N 1 4 .4 7 H 9 B 0 80 160 H9 iPS1 iPS2 Total Number of Human DYSTROPHIN Positive Fibers/TA Section A v e ra g e N u m b e r iPS1: IPRN 13.13 iPS2: IPRN 14.57 C ED F myogenic progenito (Figure S1M). Human ES- and iPS- Display Similar Surf We characterized s myogenic progenitors Our results show a r ure 2A and Figure S2A (Figure 2A and Figures showed homogenous M-CADHERIN, and a markers are associat myogenic progenitors et al., 2008; Sherwoo has not yet been defi been considered to be (Pe´ault et al., 2007). high levels of CD63, 612 Cell Stem Cell 10, 610–619, May 4, 2012 ª2012 Elsevier Inc. dystrophin (left, in green), as evidenced by the use of a pandystrophin antibody. (C–E) Engraftment of proliferating myogenic progenitors obtained from PAX7-induced human ES-derived (C) and iPS- derived (D and E) cells in TA muscles of NSG (n = 4 for each cell line) 2 months after intramuscular transplantation. Immunofluorescence stainingwith anti-human (in red) and anti-pandystrophin (in green) antibodies reveals presence of donor- 98% 44 98% 98% Figure 2. Phenotypic Profile and Regenera- tive Potential of Human ES/iPS-Derived Myogenic Cells (A) Representative FACS profile of PAX7-induced humanES- and iPS-derived proliferatingmyogenic progenitors. Histogram plots show isotype control staining profile (gray line) versus specific antibody staining profile (red line). Percentages represent the fraction of cells that express a given surface antigen. See also Figure S2. (B–F) Transplantation ofmyogenic progenitors into cardiotoxin-injured NSG mice. (B) PBS-injected control muscles show no staining for human- specific DYSTROPHIN (right, in red), but, as ex- pected, do show uniform expression of mouse Cell Stem Cell Muscle Engraftment from Human ES and iPS Cells derived myofibers expressing human DYSTRO- PHIN in recipient muscles (in red). Scale bars represent 100 mm. (F) Quantification of human DYSTROPHIN+ fibers in engrafted muscles shows similar engraftment of human ES- versus iPS-derived myogenic progenitors. For this, the total number of human DYSTROPHIN+ fibers in cross-sections of TA muscles (sections spanned entire muscles) was counted. Data are shown as mean ± SE. expression analyses, which showed high levels of PAX7 expression solely under proliferation conditions (in the presence of dox) (Figure S1M) and upre- gulation of MYOD and late skeletal muscle-specific markers, MYOGENIN, DYSTROPHIN, and MHC, when these rs had undergone final maturation Derived Myogenic Progenitors ace Marker Profile urface marker expression of these by FACS using a panel of antibodies. emarkable similarity between hES- (Fig- ) and hiPS-derived myogenic progenitors S2B and S2C). Cells in each preparation expression of CD56, CD29, CD44, 7-INTEGRIN. Although most of these ed with murine satellite cells and early (Cornelison and Wold, 1997; Sacco d et al., 2004), the human satellite cell ned by flow cytometry. Only CD56 has a reliable marker of human satellite cells These cells were also found to express CD146, CD105, CD90, and CD13; the Human LAMIN AC Human DYSTROPHIN Merge Control H9 IPRN 13.13 IPRN 14.57 0 50 100 H9 iPS1 iPS2 Total Number of Human DYSTROPHIN Positive Fibers/TA Section A ve ra ge N um be r s 0 20 40 60 gr am 2 6 10 14 16 H9 iPS1 iPS2NSG NSG mdx iPS1: IPRN 13.13 iPS2: IPRN 14.57 Cell PBS PBS Cell NSG NSG- mdx Keys: 0 25 50 75 1 F0 gr am * ***+ H9 iPS1 iPS2NSG NSG -mdx 0 2.5 5 7.5 1 CSA m m 2 +++ H9 iPS1 iPS2NSG NSG -mdx Specific force (sF0) K N /m 2 0 45 90 135 1 * * **+++ H9 iPS1 iPS2NSG NSG -mdx 0 10 20 30 1 Fatigue Index T im e (s ) +++ H9 iPS1 iPS2NSG NSG -mdx Weight 0 30 60 90 1 m g +++ H9 iPS1 iPS2NSG NSG -mdx A CB FED HG Figure 3. Efficient Engraftment and Functional Recovery after Transplantation of Human ES/iPS-Derived Myogenic Progenitors into Dystro- phic Mice (A) While no staining for human LAMIN AC or DYSTROPHIN is detected in PBS-injected control TA muscles of NSG-mdx4Cv mice (top), abundant expression for human LAMIN AC (in green) and DYSTROPHIN (in red) is observed (bottom) in dystrophic muscles treated with human ES/iPS-derived myogenic progenitors 1 month after the transplantation (n = 5 for H9, n = 6 for IPRN13.13, and n = 7 for IPRN 14.47). Note that nuclear LAMIN AC staining occurs predominantly within human DYSTROPHIN+ myofibers. Scale bars represent 100 mm. See also Figure S3. Cell Stem Cell Muscle Engraftment from Human ES and iPS Cells Cell Stem Cell 10, 610–619, May 4, 2012 ª2012 Elsevier Inc. 613 last three are antigens known to be present in mesenchymal stem cells (Pittenger and Martin, 2004). CD34 labeled a discrete subfraction of these cells. Other screened antigens, including CD45, CD33, KDR, and CD31, were undetectable in these myogenic progenitor populations (Figure S2), indicating the absence of hematopoietic and endothelial cells. The adhesion Functional Improvement in Dystrophic Mice To determine the regenerative potential of these myogenic progenitors in the context of muscular dystrophy, we trans- planted them into mdx mice engineered to lack B, T, and NK cells. These mice were generated by crossing mice carrying the mdx4Cv mutation, an ENU-induced stop codon in exon 53 ice se jur Cell Stem Cell Muscle Engraftment from Human ES and iPS Cells molecules CXCR4 and CD106 were also not detected. We examined MAJOR HISTOCOMPATIBILITY COMPLEX (MHC) expression because the lack of MHC class I expression on other embryonic and ES-derived cells has limited engraftability, even in immunodeficient mice, due to NK cell-mediated responses (Rideout et al., 2002; Tabayoyong et al., 2009). This analysis revealed that, regardless of ES or iPS origin, proliferating myogenic progenitors express MHC class I mole- cules (Figure S2). This pattern is beneficial from the perspective of avoiding an NK-mediated lack of self-MHC response but indicates the importance of HLA matching. In Vivo Regenerative Potential of HumanES/iPS-Derived Myogenic Progenitors Next we examined the in vivo skeletal muscle regenerative potential of iPAX7 hES- and hiPS-derived myogenic progenitors by transplanting these cells directly into the tibialis anterior (TA) muscles of NOD/SCID gamma-c (NSG) mice, an immune-defi- cient strain commonly used as a recipient of human hematopoi- etic cells. The gamma-c mutation (IL2Rg) ablates NK cells, rendering NSG mice unable to reject human cells due to lack of self-MHC presentation, resulting in better hematopoietic engraftment than in mice bearing the NOD/SCID mutation alone (Shultz et al., 2005). NSG mice were injured with cardiotoxin (CTX) 24 hr prior to cell transplantation. The contralateral TA muscle, which served as a control, was also preinjured with CTX but injected only with PBS. Two months after transplanta- tion, muscle sections were harvested and evaluated for engraft- ment by immunostaining with both pandystrophin and human- specific DYSTROPHIN antibodies. No expression of human DYSTROPHIN could be detected in PBS-injected control muscles (Figure 2B); staining was only observed with a pandy- strophin antibody (Figure 2B). On the other hand, muscles that had been treated with iPAX7 hES- (Figure 2C) and hiPS-derived (Figures 2D and 2E) myogenic progenitors demonstrated engraftment of human-derived myofibers, as evidenced by the clear expression of human-specific DYSTROPHIN in recipient muscles (Figures 2C–2E). We did not observe major differences in terms of engraftment between ES- and iPS-derived myogenic progenitors (Figure 2F). No tumor formation was observed in transplanted mice, even in a long-term (46 weeks) cohort. (B) Quantification of human DYSTROPHIN+ fibers in NSG-mdx4Cv engrafted m progenitors. For this, the total number of human DYSTROPHIN+ fibers in cross- shown as mean ± SE. (C) Representative example of force tracings in TAmuscles of nontreated, nonin 4Cv NSG-mdx mice that had been injected with PBS (control, red line) or human E (D and E) Effect of iPAX7 human ES/iPS-derivedmyogenic cell transplantation on nontreated, noninjured NSG (purple) and NSG-mdx4Cv (brown) mice are shown f (F and G) Weight and CSA of control and transplanted muscles, respectively. Valu shown for reference. See also Figure S4. Data are shown as mean ± SE. (H) Fatigue index: time for force to decline to 30%of itsmaximal value shows no sig as mean ± SE. *p < 0.05, **p < 0.01 compared to its PBS control. +p < 0.05, +++p 614 Cell Stem Cell 10, 610–619, May 4, 2012 ª2012 Elsevier Inc. (Im et al., 1996) with very low reversion frequency (Danko et al., 1992), t