Functional Mesenchymal Stem Cells Derived From Human Induced Pluripotent Stem Cells Attenuate Limb Ischemia in Mice
Background— Aging and aging-related disorders impair the survival and differentiation potential of bone marrow mesenchymal stem cells (MSCs) and limit their therapeutic efficacy. Induced pluripotent stem cells (iPSCs) may provide an alternative source of functional MSCs for tissue repair. This study aimed to generate and characterize human iPSC-derived MSCs and to investigate their biological function for the treatment of limb ischemia.
Methods and Results— Human iPSCs were induced to MSC differentiation with a clinically compliant protocol. Three monoclonal, karyotypically stable, and functional MSC-like cultures were successfully isolated using a combination of CD24− and CD105+ sorting. They did not express pluripotent-associated markers but displayed MSC surface antigens and differentiated into adipocytes, osteocytes, and chondrocytes. Transplanting iPSC-MSCs into mice significantly attenuated severe hind-limb ischemia and promoted vascular and muscle regeneration. The benefits of iPSC-MSCs on limb ischemia were superior to those of adult bone marrow MSCs. The greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo differentiation and paracrine mechanisms.
Conclusions— Functional MSCs can be clonally generated, beginning at a single-cell level, from human iPSCs. Patient-specific iPSC-MSCs can be prepared as an “off-the-shelf” format for the treatment of tissue ischemia.
- induced pluripotent stem cells
- mesenchymal stem cells
- peripheral vascular disease
Received August 2, 2009; accepted January 5, 2010.
Human mesenchymal stem cells (MSCs), also called multipotent stromal cells, have emerged as a promising cell type in regenerative medicine, including in the treatment of myocardial and limb ischemia.1,2 In most preclinical and clinical studies, MSCs were derived from bone marrow (BM). However, there are several potential shortcomings in using BM-derived MSCs (BM-MSCs). They have a limited capacity to proliferate, quickly lose differentiation potential, and reduce protective factors during ex vivo expansion before possible therapeutic use.3,4 Importantly, aging and aging-related disorders also significantly impair the survival and differentiation potential of BM-MSCs, thus limiting their therapeutic efficacy.5–7 There is therefore emerging interest in the identification of alternative cell sources for MSCs.
Clinical Perspective on p 1123
A recent breakthrough in the generation of induced pluripotent stem cells (iPSCs) from adult somatic cells using reprogramming techniques8,9 offers the possibility of generating a high yield of patient-specific MSCs. The differentiation potential of human iPSCs into functional MSCs and their therapeutic efficacy has nonetheless not been demonstrated. In this study, we derived multipotent MSCs from human iPSCs (iPSC-MSCs). These iPSC-MSCs derived from a single-cell level are highly expandable for >120 population doublings without obvious senescence and exhibit the capacity for differentiation into multiple cell types. Compared with adult BM-MSCs, transplantation of iPSC-MSCs into mice achieved a better beneficial effect in the attenuation of severe hind-limb ischemia. This greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment after transplantation to induce vascular and muscle regeneration via direct de novo vascular and muscle differentiation and paracrine mechanisms. An infinite number of functional MSCs can be directly generated from iPSCs and provide an alternative cell source for tissue repair in ischemic diseases.
Generation of Single Cell–Derived MSC Culture From Human iPSC
Three human iPSC lines were used in the generation of MSCs. The first iPSC line, iPSC(iMR90)-5, was generated from IMR90 fibroblast cells (ATCC catalog No. CCL-186) by transduction with lentivirus-mediated Oct4, Sox2, Nanog, and Lin28 factors (see the online-only Data Supplement).9 Two additional iPSC lines, iPSC(foreskin) Clone1 and iPSC(iMR90)-4, were acquired from WiCell Research Institute (Madison, Wis). For differentiation of human iPSCs into MSCs, we optimized an established clinically compliant protocol.10 Briefly, a confluent 6-cm plate of iPSCs was trypsinized for 3 minutes at 37°C and placed on a gelatinized 10-cm dish containing knockout Dulbecco modified Eagle’s medium (DMEM GIBCO) supplemented with 10% serum replacement medium (GIBCO), 10 ng/mL basic fibroblast growth factor (bFGF; GIBCO), 10 ng/mL platelet-derived growth factor AB (Peprotech, Rocky Hill, NH), and 10 ng/mL epidermal growth factor (Peprotech) for enrichment of MSC outgrowth. After 1 week, differentiating iPSCs were harvested and incubated with CD24-phycoerythrin (PE) and CD105-FITC (BD PharMingen, San Diego, Calif). Sorting for CD24−CD105+ was performed by a fluorescence-activated cell sorting (FACS) system. The CD24−CD105+ cells were seeded in a 6-well plate beginning with 10 000 cells per well under knockout DMEM plus 10% FCS (GIBCO), bFGF (5 ng/mL), platelet-derived growth factor AB (10 ng/mL), and epidermal growth factor (10 ng/mL). When CD24−CD105+ cultures were confluent, one quarter of the cells were split for pLL3.7–green fluorescence protein–positive (GFP+) labeling, followed by limiting dilution (0.5 cell per well in a 96-well plate). We selected wells containing a single cell visualized under fluorescent microscopy and excluded those containing more. When the clones derived from a single cell were grown up to 60% to 70% of confluence, the cells from each well were reseeded into 1 well of 6-well plates and serially reseeded thereafter in 25-, 75-, and 175-cm2 tissue culture flasks at a density of 1×104/mL. When cells were confluent in 175-cm2 tissue culture flasks, they were set as passage 1 and frozen down as cell stocks; 8 clone lines were achieved in this manner. Clones of iPSC-MSC9 [from iPSC(iMR90)-4], iPSC-MSC10 [from iPSC(iMR90)-5], and iPSC-MSC11 [from iPSC(foreskin)-1] were selected for continuous culture. Characterized adult human BM-MSCs were purchased commercially and served as a control (Cambrex BioScience, Rockland, Me; catalog No. PT-2501).
Surface Antigen Analysis of iPSC-MSCs
Cell surface antigens for human iPSC-MSCs were analyzed with FACS.10 We incubated 1.5×105 cells with each of the following conjugated monoclonal antibodies: TRA-81-FITC, CD24-PE, CD29-PE, CD44-PE, CD49a-PE, CD49e-PE, CD105-PE (R&D Systems, Minneapolis, Minn), CD166-PE, CD34-FITC, CD45-FITC, and CD133-FITC (BD PharMingen). Nonspecific fluorescence was determined by incubation of similar cell aliquots with isotype-matched mouse monoclonal antibodies (BD PharMingen). Data were analyzed by collecting 20 000 events on a BD FACS Aria using FlowJo 8.8.4 software.
Mesenchymal Differentiation of Human iPSC-MSCs: Adipogenesis, Osteogenesis, Chondrogenesis
Adipogenesis, osteogenesis, and chondrogenesis of iPSC-MSCs were carried out as previously described.10 Oil Red, Alizarin Red, and Alcian Blue staining for adipocytes, osteocytes, and chondrocytes, respectively, was performed using standard techniques. Immunostaining for collagen type II was performed on paraformaldehyde-fixed, paraffin-embedded sections using a goat anti-collagen α1 type II and donkey anti-goat immunoglobulin G antibody conjugated with FITC (Santa Cruz Biotechnology, Santa Cruz, Calif).
Karyotyping, Methylation, and Telomerase Activity of iPSC-MSCs
Determination of karyotyping and methylation was performed using a standard technique (see the online-only Data Supplement). Telomerase activity was assessed by the telomeric repeat amplification protocol (Allied Biotech, Vallejo, Calif; catalog No. MT 3010).11 Each sample was analyzed twice. Telomerase activity was expressed relative to IMR90 cells.
Polymerase Chain Reaction, Western Blotting, and Cytokine Assays
Quantitative polymerase chain reaction (PCR) was performed with Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, Calif).12 The primers for genomic DNA and complementary DNA are listed in the online-only Data Supplement. Standard procedures of Western blotting were performed for Oct4 (AB3209, Millipore, Billerica, Mass), Sox2 (MAB4343, Millipore), and Nanog (SC-33759, Santa Cruz). Quantitative human cytokines were measured using customized Bio-Plex cytokine assays (Bio-Rad Laboratories, Hercules, Calif; Bio-Plex cytokine arrays, catalog No. M50007VNJK and MF00038C9E) according to the manufacturer’s instructions.
Mice tissue was paraformaldehyde fixed, paraffin embedded, and sectioned for standard immunostaining.12 To quantitatively study fibrosis, inflammation, myogenesis, and vascular and muscle differentiation, tissues were stained with Masson trichrome, rat anti-mouse CD45 (catalog No. 553081, BD PharMingen), monoclonal mouse anti-human desmin (SC-70960, Santa Cruz), monoclonal mouse antihuman CD31 (M0823, Dako, Glostrup, Denmark), and monoclonal mouse anti-human α-smooth muscle actin (α-SMA; A2547, Sigma, St Louis, Mo) respectively. Five mice from each group were analyzed. Six sections were randomly collected from each mouse and viewed at 40 viewing fields to calculate the average positive immunoactivity. To detect the fate of injected cells with GFP labeling, the tissues were double-immunostained with rabbit anti-human GFP antibody (SC-8334, Santa Cruz) and mouse anti-human CD31 antibody. The tissues were incubated with human nuclear antigen antibody (MAB1281, Chemicon, Billerica, Mass) to detect injected cells. Sections were analyzed with a deconvoluted fluorescent microscope and Metamorph software.
Limb Ischemia, Cell Transplantation, and Laser Doppler Imaging
Critical limb ischemia was induced in severe combined immunodeficient (SCID) mice.13 The femoral artery was excised from its proximal origin as a branch of the external iliac artery to the distal point where it bifurcates into the saphenous and popliteal arteries. After arterial ligation, SCID mice were immediately assigned to the following experimental groups: the iPSC-MSC group, the BM-MSC group, or the vehicle group (medium). In each animal, a total of 3.0×106 cells (200 μL) or medium (DMEM) was injected intramuscularly into 4 sites of the gracilis muscle in the medial thigh. Assessment of limb function and ischemic damage was scored as described previously.14 Tissue perfusion of the hind limbs was assessed with a laser Doppler perfusion imager (Moor Instruments, Devon, UK) on days 0, 14, and 21 after treatment.15 The digital color-coded images were analyzed to quantify blood flow in the region from the knee joint to the toe.
Quantitative data are expressed as mean±SD. Statistical analysis was performed by unpaired Student t test for comparisons between 2 groups and by 1-way ANOVA, followed by Bonferroni test for comparisons between >2 groups. Furthermore, comparisons of variables, including blood perfusion, ambulatory impairment scores, and tissue damage scores, between baseline and follow-up were performed with repeated-measures ANOVA, followed by Bonferroni multiple comparison. For analysis of limb salvage, foot necrosis, and limb loss, an extension of the Fisher test (Freeman-Halton) was used via a 3×3 contingency table. A value of P<0.05 was considered statistically significant.
Generation of Single Cell Level–Derived MSCs From Human iPSCs
Using a modified step-wise protocol used in human embryonic stem cell (ESC) lines,10 we derived human MSCs from 3 different iPSC lines. A homogeneous culture of fibroblast-like cells was generated within 2 weeks of sorting with CD105+/CD24−. The cultures had a fibroblastic cellular morphology that resembled BM-MSCs and human ESC-MSCs (Figure 1A) and expressed CD105 but not CD24− (Figure 1B). A limiting dilution resulted in the formation of fibroblast-like colony-forming units (Figure 1C, i through iii). To derive monoclonal MSC culture, colonies that arose from CD105+/CD24− culture were subjected to pLL3.7-GFP+ labeling, followed by limiting dilution (0.5 cell per well in a 96-well plate). We selected wells containing a single cell visualized by fluorescent microscopy (Figure 1C, iv). Because cell proliferation did not begin within the first 48 hours, daily monitoring provided further opportunity to verify the presence of a single cell in each well. Of the wells verified to contain a single cell, a uniformed colony was formed in 15±4%. After picking up and expanding single-cell–derived MSC colonies, we subsequently performed limiting dilution and found that these MSCs were able to again form fibroblast-like colony-forming units (Figure 1C, v and vi).
Characterization of Human iPSC-MSCs
Unlike its parental iPSC, expression of pluripotency-associated genes was generally reduced in iPSC-MSCs. For example, protein levels of Oct4, Nanog, and Sox2 were undetected in iPSC-MSCs by Western blotting and immunostaining (Figure 1D and 1E). To confirm the loss of pluripotency in iPSC-MSCs, 1×106 iPSC-MSCs were subcutaneously transplanted into SCID mice. Unlike similar transplantation of parental iPSCs that invariably generated a 2- to 4-cm teratoma containing 3 germ layer cell types (Figure I of the online-only Data Supplement), iPSC-MSCs did not induce any tumor formation after 4 months of observation. The transplanted iPSC(iMR90)-MSCs did not appear to survive or engraft into the normal recipient tissue. The single-cell level–derived iPSC-MSCs were not contaminated with mouse feeder cells because these cells were undetectable for the mouse genomic repeat sequences c-mos but were positive for the human repeat sequences Alu-sx (Figure 1F). These iPSC-MSCs were also not contaminated with undifferentiated iPSCs because they are negative for iPSC surface marker TRA-81 (Figure 1G). Not only were iPSC-MSCs morphologically highly similar to BM-MSCs, but their surface antigen profiling was also similar to that defined for BM-MSCs, ie, CD44+, CD49a and e+, CD73, CD105, CD166+, and CD34−, CD45−, and CD133− as demonstrated by FACS analysis (Figure 1G).15
Adipocytic differentiation was highly efficient, with Oil Red droplets observed in >80% of the iPSC-MSCs (Figure 2A, i). The expression of important transcription factors in adipogenesis, PPARG2 and LPL,16 was increased (Figure 2B). Osteogenesis was efficient, with >80% of cells demonstrating positive staining with Alizarin Red for the detection of calcium deposition (Figure 2A, ii). Genes osteocalcin and APL, known to be expressed in osteoblasts for biomineralization, were consistently upregulated (Figure 2B). Chondrogenesis was also efficient, with >90% of cells producing proteoglycans in extracellular matrix as detected by Alcian Blue staining (Figure 2A, iii) and ≈60% of cells being immunoreactive for collagen II (Figure 2A, iv). Chondrogenetic master regulator Sox9 and extracellular matrix gene AGC were also upregulated (Figure 2B).
Compared with original IMR90 fibroblast cells used for iPSC generation, iPSC-MSCs are more dismethylated in the Oct4 promotor region (Figure 2D), suggesting that iPSC-MSCs are in a more naïve state than IMR90 fibroblast cells. The iPSC-MSCs did not differentiate spontaneously and maintained a normal karyotype during culture expansion (Figure II of the online-only Data Supplement). These iPSC-MSCs are highly expandable up to 40 passages (120 population doublings) without obvious loss of self-renewal capacity and constitutively express surface antigens of multipotent MSCs (Figure 2C). Above passage 45, random chromosomal aberrations were observed. Four of 20 metaphase nuclei had chromosomal aberrations: 1 lost 1 chromosome 18 and 2 chromosome 9, 2 lost 1 chromosome 22, and 1 gained 1 chromosome 16. Therefore, the karyotype of these iPSC-MSCs was normal and stable up to at least 120 population doublings.
To understand the molecular basis of the proliferation potential of iPSC-MSCs, telomerase activity was compared among BM-MSCs, iPSC-MSCs, and fibroblasts (IMR90). It revealed a nearly 10-fold higher level of telomerase activity in iPSC(iMR90)-MSCs (passage 36) than in BM-MSCs (passage 6) and a 100-fold higher level than in IMR90 (passage 8) (Figure 2E). This may explain the greater capacity of cell proliferation in iPSC-MSCs than BM-MSCs.
Transplantation of iPSC-MSCs in Attenuation of Severe Hind-Limb Ischemia
To determine the impact of iPSC-MSCs on tissue ischemia, we transplanted iPSC-MSCs into a hind-limb ischemia model of SCID mice. After 21 days of transplantation, the 3-group comparison showed a great difference in physiological status of ischemic limbs (Figure 3A and 3B). In 15 mice with hind-limb ischemia that received medium injection, 14 (93%) had limb loss, and 1 (7%) demonstrated extensive foot necrosis (Figure 3B). In 15 mice given adult human BM-MSC injection, limb salvage was observed in only 4 (27%). Eight (53%) eventually suffered limb loss, and 3 (20%) displayed moderate to severe necrosis from toe to knee. In contrast, in 15 mice given iPSC-MSC injection, limb salvage was observed in 9 (60%). Three mice (20%) suffered limb loss, and 3 (20%) demonstrated moderate to severe necrosis from toe to knee (Figure 3B).
Histological examination of the adductor muscle revealed extensive muscle degeneration and pronounced interstitial fibrosis in the vehicle group (Figure 3C). In contrast, remarkably reduced fibrosis and less muscle degeneration were observed in the iPSC-MSC– and BM-MSC–treated mice (Figure 3C). Compared with the BM-MSC group, iPSC-MSC mice exhibited significantly less fibrosis (Figure 3C and 5⇓D, i through iv) and more muscle regeneration (Figure 3C and 5⇓D, ix through xii). SCID mice are more susceptible than wild-type mice to bacterial infection after surgery, and this will affect recovery. To exclude this variable, specimens of the ischemic muscle were cultured on day 7 after surgery, and no bacterial growth was detected in each group.
To determine the impact of local cell transplantation on blood perfusion after femoral artery ligation, laser Doppler imaging was performed at days 0, 14, and 21 after surgery. Repeated-measures ANOVA analysis of blood perfusion in the 3-group comparison demonstrated a significant difference (P<0.001). Laser Doppler imaging showed a low ratio of blood perfusion persistent over 21 days in the vehicle group, indicating development of severe hind-limb ischemia (Figure 4A and 4B, i; P=0.31, day 0 versus 21). In the BM-MSC and iPSC-MSC transplantation groups, blood perfusion was gradually recovered over 21 days. A significant difference in blood perfusion between the iPSC-MSC and BM-MSC groups was observed at both day 14 and 21 (Figure 4A and 4B, i), indicating that iPSC-MSCs are superior to BM-MSCs in recovery of blood perfusion.
We evaluated ischemic limb function by scoring limb ambulatory impairment and tissue damage. Repeated-measures ANOVA analysis demonstrated a significant difference in scores of limb ambulatory impairment (Figure 4B, ii; P<0.001) and tissue damage (Figure 4B, iii; P<0.001) among the 3 groups. Within 3 days after surgery, a similar abrupt reduction in hind-limb active use occurred in the 3 groups (Figure 4B, ii). In the vehicle (DMEM) group, a reduction in active use of hind limb persisted from day 7 to 21 (Figure 4B, ii). Nonetheless, active use of the hind limb in the iPSC-MSCs and BM-MSC groups was gradually improved after day 7. Compared with the vehicle group, scores of ambulatory impairment and tissue damage were significantly reduced in the BM-MSC and iPSC-MSC groups at day 21 (P<0.001). Compared with the BM-MSC group, significantly lower scores of ambulatory impairment and tissue damage were observed in iPSC-MSC–treated mice at day 21 (P<0.001), indicating that iPSC-MSC transplantation achieved a better beneficial effect on improvement of ischemic limb functions.
Differential Effects of iPSC-MSCs and BM-MSCs in Severe Hind-Limb Ischemia
To determine the differentiation potential in vivo, iPSC-MSCs were first transduced with pLL3.7-GFP vector permanently expressing GFP and then transplanted into a growing mouse ESC-induced teratoma. The rationale was that a teratoma in which ESCs are actively differentiating into tissues of all 3 germ layers is likely to provide a microenvironment conducive to iPSC-MSC differentiation into tissues of the multiple cell types, including vascular tissues. After 4 weeks, iPSC-MSCs were found to be extensively incorporated into the capillary plexuses of the teratomas and were immunoreactive for human-specific CD31 and desmin, markers for endothelial cells and smooth muscle, respectively (Figure 5A, i through vi). In addition, iPSC-MSCs were dispersed throughout the teratomas, although the associated tissue type could not be identified.
The differentiation potential of iPSC-MSCs and BM-MSCs in vitro was then compared. In vitro endothelial and smooth muscle differentiation was induced17,18 and quantitatively analyzed with immunostaining and FACS. When cells were exposed to endothelial differentiation medium for 5 days, iPSC-MSCs displayed only slightly higher potential in endothelial differentiation than BM-MSCs by expression of CD31 (17.5% versus 10.2%; P=0.07; Figure 5B). In contrast, when cells were exposed to sphingosylphosphorylcholine-induced muscle differentiation conditions, iPSC-MSCs showed more potential in smooth muscle differentiation than BM-MSCs, as shown by expression of α-SMA (54.4% versus 34.7%; P=0.03; Figure 5C).
Differential effects of iPSC-MSCs and BM-MSCs in hind-limb ischemia were also compared. Because muscle function and blood perfusion significantly recovered around day 14 after iPSC-MSCs treatment, histological analysis of gastrocnemius muscle was performed at day 14. The iPSC-MSC group had significantly less muscle fibrosis (Figure 5D, i through iv) and inflammation (Figure 5D, v through viii) than the BM-MSC group. Positive desmin immunostaining, which is known to be strongly positive in small regenerating myogenic cells, revealed that muscle regeneration in the iPSC-MSC group was also much more pronounced19 (Figure 5D, ix through xii). Three times as many α-SMA+ cells were observed in the iPSC-MSC group, and many of them were intercalated between muscle cells (Figure 5D, xv and xvi). In contrast, many α-SMA+ cells engrafted in connective and fat tissues outside muscle in the BM-MSC group (Figure 5D, xiv). At day 14, anti-human CD31 staining revealed overall neovascularization that was not significantly different between the iPSC-MSC and BM-MSC groups (Figure 5D, xvii through xx).
Retention of iPSC-MSCs and BM-MSCs in Hind-Limb Ischemia
The retention ability of iPSC-MSCs or BM-MSCs in ischemic limbs after transplantation was determined up to day 35. Immunostaining showed that antihuman α-SMA+ cells were detected in both iPSC-MSC– and BM-MSC–treated tissues from day 7 to 21 (Figure 6A, i through iv). Cells were distributed between muscle fibers or among muscle tissues, and cell densities decreased over time (Figure 6A, i through iv). Some α-SMA+ cells were also found in small arteries, suggesting that iPSC-MSCs/BM-MSCs contributed to arteriole formation (Figure 6A). Nonetheless, α-SMA+ cells were detectable only in the iPSC-MSC group, not the BM-MSC group, 35 days after transplantation (Figure 6A, v and vi). Reverse-transcription PCR analysis indicated that gene expression of human α-SMA decreased over time in both groups and ceased in BM-MSC–treated tissues at day 35 (Figure 6B, i). Staining with anti–human nuclear antigen revealed that a few human nuclear antigen–positive cells were dispersed in iPSC-MSC–treated tissues but were undetectable in BM-MSC–treated tissues at day 35 (Figure 6B, ii). Staining with anti-human CD31 for GFP+-labeled iPSC-MSCs showed that sporadic GFP+ iPSC-MSCs expressed CD31 at day 35, indicating endothelial differentiation of iPSC-MSCs in ischemic limbs (Figure 6B, iii).
Contribution of iPSC-MSC Paracrine Factors to Hind-Limb Ischemia
The relative contribution of a paracrine effect of iPSC-MSC transplantation in hind-limb ischemia was also investigated. Supernatants of iPSC-MSCs (5×105 cells/5 mL) or BM-MSCs (5×105 cells/5 mL) in 1% FBS culture medium were harvested after exposure to hypoxia (5% O2) for 48 hours. The MSC-released cytokines in supernatants were measured with the Bio-Plex cytokine arrays. There were many shared characteristics and differences in the cytokine profiling. Stromal-derived factor 1α, stem cell factor, bFGF, hepatocyte growth factor, β-nerve growth factor, and vascular endothelial growth factor were detected in both groups and are important for cell survival and differentiation. The amounts of stromal-derived factor 1α, stem cell factor, and bFGF were significantly higher in iPSC-MSCs, whereas hepatocyte growth factor and nerve growth factor were higher in BM-MSCs (Figure 6C). Compared with 1% FBS culture media, supernatants of iPSC-MSCs or BM-MSCs significantly increased tubular formation of iPSC-MSCs on Matrigel-coated plates (Figure 6D), indicating that iPSC-MSC– and BM-MSC–secreted cytokines are functional and at least partially contribute to improved limb ischemia.
We have derived a novel multipotent MSC from human iPSCs and demonstrated its therapeutic effects in the treatment of hind-limb ischemia. This study had 4 main findings. First, iPSC-MSCs can be clonally isolated starting at the single-cell level, and self-renewal in culture for >120 population doublings is possible without obvious loss of plasticity or onset of replicative senescence. Second, intramuscular injection of iPSC-MSCs increases myogenesis and neovascularization and restores limb function. Third, attenuation of tissue ischemia with iPSC-MSC transplantation is attributable not only to their vascular and muscular differentiation but also to their trophic factors that protect endangered cells after ischemic injury. Fourth, compared with adult BM-MSCs, transplanting iPSC-MSCs into an ischemic hind limb is more beneficial. This greater potential of iPSC-MSCs may be related to their ability to remain longer and to induce vascular and muscle regeneration via direct de novo vascular and muscle differentiation or paracrine mechanisms.
Multipotent MSCs, especially those derived from BM, have recently emerged as an attractive cell type for treating myocardial ischemia20 and peripheral vascular disease.21 The survival and differentiation potential of BM stem cells obtained from aged patients or age-related disorders are nonetheless impaired,5 thus limiting their therapeutic efficiency. With the novel ability to generate iPSCs,8,9 we sought to establish a high yield of MSCs from human iPSCs. Compared with conventional adult BM-MSCs, there are great advantages in using iPSCs. First, iPSCs are customized and infinitely expandable ex vivo and thus offer an unlimited source for MSCs generation. Second, a high yield of MSCs can be generated from iPSCs and serve as an “off-the-shelf” format. Third, patient-specific iPSC–generated MSCs can be used for autologous transplantation without the need for immunosuppression. The differentiation potential of human iPSCs into MSCs nonetheless remains unclear, and the biological functions of iPSC-MSCs remain to be evaluated.
We found no major differences between differentiation of MSCs from iPSCs and those from human ESCs in terms of time course, potential, and efficiency.10 The iPSC-MSCs display typical mesenchymal characteristics, ie, a large capacity for self-renewal while maintaining their multipotent differentiation potential, and expression of common MSC surface markers, which is similar to that of BM-MSCs. Notably, iPSC-MSCs were more robust in proliferation than BM-MSCs. The iPSC-MSCs can also be expanded up to 40 passages (120 doubling populations) while maintaining a normal diploid karyotype and a stable gene expression and surface antigen profile.
The clonally expandable iPSC-MSCs share many characteristics with those derived from human ESCs and BM but appear distinct from previously described populations of adult stem cells, multipotent adult progenitor cells,22 or a clonally isolated human BM multipotent stem cell,23 which are beneficial for attenuation of tissue ischemia. In contrast to multipotent adult progenitor cells, iPSC-MSCs expressed no genetic markers of ESCs such as Oct4 that are believed to be essential for their function. Similarly, unlike a rare subpopulation of multipotent stem cells derived from human BM,23 iPSC-MSCs express well-known MSC markers such as CD29, CD44, and CD73.
Our results demonstrate that iPSC-MSCs remarkably attenuate tissue ischemia and provide a better therapeutic effect than adult BM-MSCs. The greater potential of iPSC-MSC may be related to their ability to survive longer after transplantation, as well as being more robust in enhancing vascular and muscle regeneration via direct de novo vascular and muscle differentiation or paracrine mechanisms. Although cell survival obviously decreased over time in ischemic limbs, iPSC-MSCs were able to engraft and survive at least 5 weeks after transplantation. In contrast, transplanted BM-MSCs could persist for only a month and were extinguished after 5 weeks. This is in accordance with earlier findings of poor long-term retention of BM-MSCs in injured heart.24
There are several challenges to overcome before iPSCs and their derivatives can be used clinically such as is the low efficiency of iPSC generation and viral integration of transgenes. These technical issues have been rapidly improved by the use of technology without viral integration such as chemicals, plasmids,25,26 or direct reprogramming protein delivery assays.27 Nevertheless, the present study provides proof of concept that functional MSCs can be generated from human iPSC with a robust proliferation and differentiation potential and can be used for tissue repair and engineering.
Multipotent MSCs can be directly generated from human iPSCs and are superior to adult BM-MSCs in the attenuation of tissue ischemia. The generation of human iPSC-MSCs offers the promise to treat ischemic disease in a patient-specific, cost-effective, and batch-to-batch–consistent manner.
We thank Dr Tongming Liu (GIS, Singapore) for pLL3.7-GFP vector. We also thank Ethel S.K. Ng for technique assistance with the laser Doppler imaging.
Sources of Funding
This research was supported by Seed Funding for Basic Research (10208618 to Dr Lian) and HKU Strategic Research Theme on Healthy Ageing (Drs Lian and Tse) from the University of Hong Kong and in part by a Hong Kong Research Grant Council General Research Fund (HKU 763306M and HKU 7747/08M to Drs Siu and Tse).
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Despite the initial encouraging results of preclinical and clinical studies, mesenchymal stem cells (MSCs) derived from adult tissue such as bone marrow have a limited proliferation and differentiation potential for tissue repair in ischemic disease. More important, aging significantly impairs their survival and differentiation potential and thus limits their therapeutic efficacy. Recent breakthrough in the generation of induced pluripotent stem cells (iPSCs) offers the possibility to obtain a high yield of patient-specific MSCs. In this study, we demonstrate that functional MSCs can be directly generated from iPSCs (iPSC-MSCs). Transplantation of iPSC-MSCs into mice achieved a more beneficial effect than adult bone marrow–derived MSCs in the attenuation of severe limb ischemia. This greater potential of iPSC-MSCs may be attributable to their superior survival and engraftment via de novo vascular and muscle differentiation and paracrine mechanisms. Our study suggests that functional MSCs can be generated from human iPSCs and have the potential to treat ischemic disease in a patient-specific, cost-effective, and batch-to-batch–consistent manner.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.109.898312/DC1.