Isolation and Characterization of Multipotent Progenitor Cells from the Bowman’s Capsule of Adult Human Kidneys

Isolation and characterization of CD24⁺CD133⁺ PEC. (A) Light microscopy image of cells outgrowing from seeded capsulated glomeruli. (B) Laser confocal microscopy demonstrates CD24 expression by all cells outgrowing from glomeruli (green). To-pro-3 counterstains nuclei (blue; bar = 100 μm). (C) Laser confocal microscopy demonstrates absence of green signal in all cells outgrowing from glomeruli when stained with an isotype-matched control antibody. To-pro-3 counterstains nuclei (blue; bar = 100 μm). (D) CD24⁺CD133⁺ PEC that were derived from glomerular outgrowth represent a homogeneous population that is composed of virtually 100% of cells that express CD24, CD133, CD106, CD105, and CD44, but all are negative for the endothelial markers CD31 and CD34. Flow cytometry analysis of a representative bulk culture is shown. (E) CD24⁺CD133⁺ PEC that were derived from glomerular outgrowth do not express the podocyte marker CD35, as assessed by flow cytometry. (F) Confocal microscopy demonstrates that CD24⁺CD133⁺ do not express the podocyte markers synaptopodin and WT-1 (bar = 100 μm). (G) Primary cultures of podocytes express high levels of synaptopodin at the cytoplasmic level (red) and WT-1 at the nuclear level (light blue; bar = 100 μm). (H) CD24⁺CD133⁺ PEC that were derived from glomerular outgrowth do not express the distal tubules/collecting ducts marker epithelial membrane antigen-1 (EMA-1), as assessed by flow cytometry. (I) CD24⁺CD133⁺ PEC that were derived from glomerular outgrowth lack the distal tubule marker Tamm-Horsfall glycoprotein (THG; left), as well as fluorescence staining for the proximal tubule markers Lotus Tetragonolobus lectin (LTA; middle; bar = 100 μm), as assessed by confocal microscopy. Negative histochemical staining for alkaline phosphatase (AP; right). A representative bulk culture is shown. (J) Comparison by quantitative reverse transcriptase–PCR (RT-PCR) of mRNA levels for markers of differentiated tubular cells in CD24⁺CD133⁺ versus CD24⁻CD133⁻ renal cells. Columns represent mean values ± SD as obtained from three different donors. Magnification, ×40 in A.

Growth properties of CD24⁺CD133⁺ cells and comparison with CD24⁻CD133⁻ cells. (A) Representative growth curves of the CD24⁺CD133⁺ cells (•) or CD24⁻CD133⁻ (□) cells that were obtained through immunomagnetic sorting from total renal cell suspensions. The results represent mean values ± SD of cell counts that were obtained in four different experiments from four different donors in the first 10 d of culture. (B) CD24⁺CD133⁺ cells in culture were expanded for 60 to 90 population doublings (PD) during a 4-mo period. Results are mean ± SD obtained from four different donors. (C) Flow cytometric analysis of DNA content performed on bulk cultures of CD24⁺CD133⁺ cells at 50 PD, demonstrating 100% diploid cells. One representative of four separate experiments is shown. (D) Assessment of mRNA levels for BmI-1 by real-time quantitative RT-PCR in cultures of human microvascular endothelial cells (HMVEC), human renal proximal tubular cells (HRPTEC), human mesangial cells (HMC), human glomerular visceral epithelial cells (HGVEC), human aortic smooth muscle cells (HASMC), CD24⁺CD133⁺ cells, CD24⁻CD133⁻ cells, and HEK cells. Results are expressed as mean ± SD of triplicate assessment in primary cultures from five different donors. (E) Assessment of mRNA levels for Oct-4 by real-time quantitative RT-PCR in cultures of HMVEC, HRPTEC, HMC, HGVEC, HASMC, CD24⁺CD133⁺ cells, CD24⁻CD133⁻ cells, and HEK cells. Results are expressed as mean ± SD of triplicate assessment in primary cultures from five different donors.

Characterization of single clones of CD24⁺CD133⁺ PEC. (A) Cloning efficiency of CD24⁺CD133⁺ cells in comparison with CD24⁻CD133⁻ cells. Results are expressed as mean ± SD of the percentage of clones over the number of single cells plated, as obtained from eight different donors (*P < 0.05). (B) Representative image of single-cell deposition (red circle, top left) obtained by limiting dilution of CD24⁺CD133⁺ PEC, followed by its proliferation and formation of a clone (subsequent panels). (C) Coexpression of CD24 and CD133 by 100% of cells that were derived from one representative clone of CD24⁺CD133⁺ PEC, as assessed by FACS analysis. (D) Coexpression of vimentin (red) and cytokeratin (green) by a representative clone of CD24⁺CD133⁺ PEC, as assessed by confocal microscopy (merged image, yellow). To-pro-3 counterstains nuclei (bar = 100 μm). Magnification, ×50 in B.

Differentiation of CD24⁺CD133⁺ PEC–derived clones into tubular epithelial cells. (A) Representative micrographs of histochemical staining for AP activity in CD24⁺CD133⁺ PEC before (day 0) and after (day 30) culture in tubular differentiation medium. (B) Staining for LTA before (day 0) and after (day 30) culture in tubular differentiation medium, as assessed by confocal microscopy (green). To-pro-3 counterstains nuclei (bar = 100 μm). (C) Expression of THG before (day 0) and after (day 30) culture in tubular differentiation medium, as assessed by confocal microscopy (green). To-pro-3 counterstains nuclei (bar = 100 μm). (D) Double labeling for LTA (green) and THG (red) showing coexistence in the same clone of cells that coexpress markers of multiple tubule segments (merged, yellow) and of cells that express only proximal or distal tubule markers (bar = 100 μm). One representative of five experiments is shown. (E) Assessment by quantitative RT-PCR of mRNA levels fold increase for tubular markers after 30 d of culture in tubular differentiation medium compared with values that were obtained in the same cells before differentiation. Columns represent mean values ± SD as obtained after differentiation of 50 different clones. (F, left) Representative micrographs of confocal fluo-4 fluorescence images recorded at 488-nm excitation before and after angiotensin II (1 μM) treatment (bar = 20 μm). (Right) Time course of change in fluorescence intensity recorded from five single cells from 10 different clones examined is shown. Magnifications: ×65 in A, left; ×80 in A, middle; ×320 in A, right.

Differentiation of CD24⁺CD133⁺ PEC–derived clones in osteoblasts and adipocytes. (A, left) Representative micrographs of histochemical staining for Alizarin red and AP before (day 0) and after (21 d) CD24⁺CD133⁺ PEC culture in osteogenic differentiation medium. (Right) Assessment of mRNA levels of Runx2 before (day 0) and after (21 d) culture in the same medium. Columns represent mean values ± SD obtained from 50 different clones. (B, left) Representative micrographs of histochemical staining for Oil Red-O before (day 0) and after (21 d) CD24⁺CD133⁺ PEC culture in adipogenic differentiation medium. (Inset) High-power magnification of some differentiated cells. (Right) Assessment of mRNA levels of adiponectin at 0 d and after 21 d of culture in the same medium. Columns represent mean values ± SD obtained from 50 different clones. Magnifications: ×100 in A; ×200 in B; ×320 in B, inset.

Acquisition by CD24⁺CD133⁺ PEC–derived clones of phenotypic and functional properties of neural cells. (A) Absence of the neural markers neurofilament 200 (NF200), neurofilament M (NFM), choline acetyl-transferase (ChAT), and microtubule-associated protein-2 (MAP-2) before culturing PEC in neurogenic differentiation medium, as assessed by confocal microscopy. To-pro-3 counterstains nuclei (bar = 100 μm). One representative clone is shown. (B) Strong expression of the neural markers NF200, NFM, ChAT, and MAP-2 after differentiation in the same medium (green). To-pro-3 counterstains nuclei (bar = 100 μm). One representative clone is shown. (C) High-power magnification of a representative image showing acquisition of a typical neuronal morphology and staining for ChAT (green) by CD24⁺CD133⁺ PEC cultured under neurogenic conditions (bar = 100 μm). (D) Assessment by real-time quantitative RT-PCR of mRNA levels fold increase of several neural markers after differentiation under neurogenic conditions compared with values that were obtained in the same cells before differentiation. Columns represent mean values ± SD obtained from 50 different clones. (E through H) Inward Ca²⁺ and Na⁺ currents in CD24⁺CD133⁺ PEC–derived neurons. Representative current traces recorded at a holding potential of −90 mV; 1-s step pulses from −80 to 50 mV were applied in 10-mV increments. Data were acquired with different sampling time (50 μs in the first 100 ms and 1 ms for the remaining duration of the test pulse) to highlight fast or slow phenomena. (E) Time course of L-type Ca²⁺ current (I_Ca); for clarity, only current traces that were recorded at −60, −40, −20, 0, 20, 30, and 40 mV are presented. (F) I_Ca-V curve determined at the current peak (n = 26). (G) Time course of Na⁺ current (I_Na); only current traces that were recorded at −60, −40, −30, −20, −10, 0, 20, and 30 mV are presented; red line is I_Na elicited at 0 mV in the presence of Tetrodotoxin (1 μM). (H) I_Na-V curve determined at the current peak (n = 26). (F and H) Continuous line superimposed through the data are the fitted Boltzmann function for activation: I_a(V) = G_max(V − V_rev)/{1 + exp[(V_a − V)/k_a]}, where G_max is the maximal conductance, V_rev is the apparent reversal potential, V_a is the potential that elicits the half-maximal increase in conductance and k_a is the slope factor. The best-fit parameters for I_Ca were G_max = 6 ± 1 nS, V_a = 0 ± 2 mV, k_a = 8.4 ± 2 mV, and V_rev = 79 ± 8 mV; those for I_Na were G_max = 28 ± 7 pS, V_a = −18 ± 2 mV, k_a = 6.0 ± 1 mV, and V_rev = 47 ± 4 mV. (I) I_Na inactivation evoked from holding potential of −90 mV; test pulse to 0 mV prepulsed from −90 to 30 mV in 10-mV increments, only traces without prepulse (−90 mV) and prepulsed at −70, −60, −30, −40, and −30 mV are depicted. (J) Normalized inactivation curve for I_Na; continuous line superimposed through the data are the fitted Boltzmann function for inactivation: I_h(V) = 1/{1 + exp[−(V_h − V)/k_h]}, where V_h is the potential eliciting the half-maximal current and k_h is the slope factor for inactivation. The best-fit parameters were V_h = −58 ± 5 mV and k_h = 6.0 ± 6 mV. For comparison, the curve reported on the right is that for activation.

Engraftment of CD24⁺CD133⁺ PEC in kidneys of SCID mice with acute renal failure (ARF) and generation of different types of renal tubular cells. (A) Light micrograph showing normal mouse renal tissue stained with hematoxylin and eosin (H&E; left) or with phalloidin (green, right; bar = 50 μm). (B) Tubulonecrotic injury observed after an intramuscular injection of glycerol, as assessed with H&E staining (left) or with phalloidin (right); the latter reveals the loss of brush border and the flattening of epithelial cells (green; bar = 50 μm). (C) Representative micrograph of kidney sections of control SCID mice that received injections of CD24⁻CD133⁻ cells and stained with LTA showing the absence of red-stained cells, as assessed by confocal microscopy (bar = 20 μm). (D) Representative micrograph of kidney sections of mice that had ARF and received injections of PKH26-labeled CD24⁺CD133⁺ PEC (red) and stained with LTA (green), as assessed by confocal microscopy. Small arrows point to multiple red cells. The larger arrow points to a proximal tubule (bar = 20 μm). (E) High-power magnification of the kidney section shown in D, which demonstrates regeneration of a proximal tubule structure (bar = 20 μm). (F) High-power magnification of another kidney section obtained from a SCID mouse that had ARF and received an injection of PKH26-labeled CD24⁺CD133⁺ PEC (red) and stained with Dolichos Biflorus Agglutinin (DBA) on the basal surface of two tubular structures (green), which demonstrates regeneration of a collecting duct structure (arrow). Other tubular structures that are stained with PKH26 but not with the collecting ducts marker DBA are visible (bar = 20 μm). (G) Double-label immunohistochemistry for cytokeratin (blue) and HLA-I human antigen (red) in kidneys of SCID mice with glycerol-induced ARF. (Left) Absence of red signal in tubules of a kidney section from a mouse that received an injection of CD24⁻CD133⁻ cells. (Middle and right) Human HLA class I–positive cells (red, arrows) in cytokeratin-expressing (blue) tubules of SCID mice with glycerol-induced ARF after injection of CD24⁺CD133⁺ PEC. (H) Detection of chromosome Y by the fluorescence in situ hybridization technique in control mice that received injections of saline solution (left) and in kidneys from female mice that received injections of CD24⁺CD133⁺ PEC from human men (red, middle and right; bar = 20 μm).