Human-induced pluripotent stem cells from blood cells of healthy donors and patients with acquired blood disorders

Abstract

Human induced pluripotent stem (iPS) cells derived from somatic cells hold promise to develop novel patient-specific cell therapies and research models for inherited and acquired diseases. We and others previously reprogrammed human adherent cells, such as postnatal fibroblasts to iPS cells, which resemble adherent embryonic stem cells. Here we report derivation of iPS cells from postnatal human blood cells and the potential of these pluripotent cells for disease modeling. Multiple human iPS cell lines were generated from previously frozen cord blood or adult CD34(+) cells of healthy donors, and could be redirected to hematopoietic differentiation. Multiple iPS cell lines were also generated from peripheral blood CD34(+) cells of 2 patients with myeloproliferative disorders (MPDs) who acquired the JAK2-V617F somatic mutation in their blood cells. The MPD-derived iPS cells containing the mutation appeared normal in phenotypes, karyotype, and pluripotency. After directed hematopoietic differentiation, the MPD-iPS cell-derived hematopoietic progenitor (CD34(+)CD45(+)) cells showed the increased erythropoiesis and gene expression of specific genes, recapitulating features of the primary CD34(+) cells of the corresponding patient from whom the iPS cells were derived. These iPS cells provide a renewable cell source and a prospective hematopoiesis model for investigating MPD pathogenesis.

Figure 1

Reprogramming of human CB and adult BM CD34⁺ cells to iPS cells. (A) Live culture was stained by the TRA-1-60 antibody, 3 to 4 weeks after transduction of CB CD34⁺ cells. TRA-1-60⁺ colony shown on right was first seen at week 3 and was picked at week 4 (after restaining). (B) An illustration of various colonies 4 weeks after transduction of BM CD34⁺ cells, after live staining with TRA-1-60 and a secondary fluorescent reagent. A smaller fraction of formed colonies are TRA-1-60⁺. TRA-1-60⁺ colonies were individually picked and gave rise to iPS clones. (C) Immunofluorescence staining images of expanded iPS cells from CB are shown here (BM-derived iPS cells are shown in supplemental Figure 1). In addition to TRA-1-60, they also express other pluripotency markers NANOG and SSEA4. (D) Gene expression of undifferentiated (OCT4 and NANOG) and differentiation markers in undifferentiated iPS cells (U) derived from CB and teratoma cells (T) after in vivo differentiation. The expression of α-fetoprotein (AFP, endoderm), CD34 (mesoderm), and PAX6 (ectoderm) and a housekeeping gene GAPDH was measured by RT-PCR analysis. (E) Differentiation potential of CB-derived iPS cells after in vitro differentiation by EB formation (10 days). After immunofluorescence staining, differentiated cells expressing AFP, CD34, and β-III-tubulin (an ectoderm marker) were seen. The image of specific staining is overlaid by 4,6-diamidino-2-phenylindole staining of nuclei. (F) In vivo differentiation potential after teratoma formation from CB-derived iPS cells. Hematoxylin and eosin staining of various slides after sectioning show various tissues from the 3 embryonic germ layers: gut epithelium (endoderm), cartilage (mesoderm), and glycogenated epithelium (ectoderm). Scale bar represents 200 μm.

Figure 2

Human iPS lines containing the JAK2-V617F mutation from PB CD34⁺ cells of 2 MPD patients. (A) Immunostaining of different colonies from a representative iPS line (clone 8) derived from MPD183 shows the expression of undifferentiated cell markers TRA-1-60, SSEA4, and NANOG. (B) A pluripotency test for the iPS clone 8 from MPD183 (iMPD183.C8) after EB formation (day 10) as we did for human ES cells and other (normal) iPS cells, showing that JAK2-V617F iPS cells can also differentiate into various cell types expressing markers of 3 embryonic germ layers. Similar results were obtained from the iPS clone 3 of the second MPD patient (iMPD562.C3) before and after EB-mediated differentiation, as shown in supplemental Figure 4. (C) In vivo differentiation potential after teratoma formation from MPD183-derived iPS cells. Hematoxylin and eosin staining of various slides after sectioning shows various tissues from the 3 embryonic germ layers: gut epithelium (endoderm), cartilage (mesoderm), and glycogenated epithelium (ectoderm). (D-E) Expanded iPS lines from the 2 female MPD patients (D: MPD183; E: MPD562) retained a normal karyotype (46,XX), after 10 and 11 passages, respectively. Scale bar represents 200 μm.

Figure 3

Hematopoietic potential of CD34⁺ cell–derived iPS cells after directed differentiation. Human iPS cells derived from normal control (NC, A) adult CD34⁺ cells or the PV CD34⁺ cells (D) were plated in microtiter wells and aggregated for EB formation and directed hematopoietic differentiation. After 10 to 14 days, substantial numbers of small round cells resembling immature hematopoietic cells surrounding EBs were found and increased in the next several days (A,D). Total cells were subsequently harvested and assayed for the presence of hematopoietic markers (supplemental Figure 4) and of hematopoietic colony-forming units (CFUs) formed in semisolid methylcellulose media (B-C: normal control iPS; E-F: PV-iPS). CFU-granulocyte/monocyte (B,E) and CFU-erythroid (C,F) colonies were observed after additional 10 to 14 days in culture. A similar CFU assay using purified CD34⁺CD45⁺ cells from an NC and PV sample is shown in supplemental Figure 5. (G) Wright-Giemsa staining after cytospin of individually picked myeloid and erythoid colonies generated from iPS cells derived from normal CD34⁺ cells. Cells resembling erythroblasts and multiple lineages of myeloid cells were observed. Scale bar represents 100 μm.

Figure 4

Increased erythroid differentiation of hematopoietic progenitor cells generated from PV iPS cells. To assess erythroid differentiation potential, purified CD34⁺CD45⁺ cells from both normal control (NC) and PV iPS cells after EB-mediated hematopoietic differentiation were plated into a liquid culture medium (A,C), or a serum- and methylcellulose-containing medium (B,D). (A) Fold of cell expansion after 7 days of the liquid culture from the purified CD34⁺CD45⁺ cells derived from NC (13.5 ± 3.6-fold) or PV iPS cells (27.6 ± 3.3-fold). (B) Fold of cell expansion of the CD34⁺CD45⁺ cells after 14 days of the methylcellulose culture, 69.5 ± 24.7-fold of NC versus 127 ± 28.3-fold of the PV iPS cells. Data in panels A and B are presented as mean ± SD (n = 2). (C) FACS analysis of the 7-day cultured cells for the erythroid phenotype (CD235a⁺CD45⁻). The percentages of such cell population are indicated in the top left quadrant based on the gating and comparison with background staining. (D) FACS analysis of the 14-day cells harvested from the methylcellulose-containing medium.

Figure 5

Human iPS generated hematopoietic progenitor cells exhibit unique gene expression pattern similar to the primary CD34⁺ cells from the PV patient and a normal control. (A) Total RNA was isolated from primary CD34⁺ cells from a healthy donor as an NC or from the PV patient (MPD183) where the PV-iPS cell lines were derived from. Gene expression of NFI-B, HBG, and HBB as well as β-actin (as a control) was analyzed by real-time quantitative PCR after reverse transcription of RNA. The normalized level (relative to that of β-actin) is plotted. (B) An identical analysis of purified CD34⁺CD45⁺ cells generated from PV iPS (iMPD183) cells and NC iPS cells derived from normal adult CD34⁺ cells. Data are presented as mean ± SD (n = 2).