*2.1. Animals*

Female Wistar rats weighing 220–250 g were purchased from BioLASCO Taiwan Co. Ltd. (Taipei, Taiwan) and housed in the Experimental Animal Center, National Taiwan University. All the surgical and experimental procedures were approved by the ethics committee "Institutional Animal Care and Use Committee of the National Taiwan University College of Science" (identification code of approval: 20100244 and date of approval: 02/11/2011) and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). To monitor fecal and urinary excretion, the rats were placed into the metabolic cage. The feces and urine samples were collected and recorded every 12 h before surgical experiments. During the experiment, the rats were given free access to food and water.

#### *2.2. Cell Preparations (MSCS Isolation, Characterization, and Culture)*

Femora from Wistar rats (BioLASCO Taiwan Co Ltd., Taipei, Taiwan), 8 to 10 weeks of age, were removed, and soft tissues were detached aseptically. Bone marrow was extruded by inserting a 23-gauge needle into the shaft of the bone and was flushed out with basal medium ( α-minimal essential medium [ α-MEM], Gibco-BRL, Gaithersburg, MD, USA). Isolation of MSCs was performed according to similar procedures as described previously [18]. Briefly, mononuclear cells were isolated from the bone marrow aspirates by a density gradient centrifugation method were suspended in complete culture medium ( α-MEM supplemented with 16.6% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine) and seeded in plastic dishes. After 24 h of the initial culture, nonadherent cells were removed by a change of medium and irrigation of the culture. The culture typically reaches 65% to 70% confluency within 6 to 8 days and reached subconfluency at 9 days, when the cells (passage 0) were harvested for further subculturing. Starting from passage 1, the cells were seeded at 100 cells/cm<sup>2</sup> and were grown in complete culture medium with a medium change twice per week. For hypoxic MSC cultures, cells were cultured in a gas mixture composed of 94% N2, 5% CO2, and 1% O2 [19], whereas in normoxic MSC cultures, cells were cultured in 95% air and 5% CO2. For maintenance of the hypoxic gas mixture, an incubator with two air sensors, one for CO2 and the other for O2, was used. O2 concentration was achieved and maintained using delivery of N2 gas from a tank containing pure N2. If the O2 concentration rose above the desired level, N2 gas was automatically injected into the system to displace the excess O2.

#### *2.3. HIF-1*α *Determination and Growth Factors Array Assay*

The hypoxic inducible factor-1 α (HIF-1 α) concentration and multiple growth factors assay from cultured condition medium and MSCs or HMSCs were determined with HIF-1 α ELISA kit (MBS2702491, MyBioSource, San Diego, CA, USA) and RayBio ® rat growth factor array (AAR-GF-1-2, RayBiotech, Peachtree Corners, GA, USA) according to the manufacturer's instructions.

## *2.4. Experimental Model and Design*

Anti-thy1.1 GN was induced by injection of 0.2 mL of phosphate-bu ffered saline containing 250 μg anti-thy1.1 monoclonal antibody (Cedarlane, Burlington, ON, Canada) into rats via a jugular vein under sodium pentobarbital anesthesia (50 mg/kg, i.p.) at day 0, and 0.2 mL of saline injection into the jugular vein as a control group. This method for induction of acute GN had been reported previously [20]. Under avertin anesthesia (400 mg/kg, Acros Organics, Morris Plains, NJ, USA), one PE10 tubing was introduced into the left renal artery from the left femoral artery via the aorta for direct MSCs or HMSCs delivery (Figure 1A). Varied numbers of MSCs or HMSCs including 1, 2, and 5 × 10<sup>5</sup> cells were administered via this catheter in the therapeutic groups, and saline was administration in control groups. The grouping and experimental design are shown in Figure 1B.

**Figure 1.** The technique for locally intra-renal arterial administration of mesenchymal stem cells (MSCs) was displayed in ( **A**). The experimental grouping and design are displayed in the eight groups (**B**). High levels of green fluorescence were visualized under UV light in the glomeruli of nephritic kidney sections (**C3–4**) but not in the normal kidney sections (**C1–2**). C1, C3 magnification 200×; C2, C4 magnification 400×.

#### *2.5. Tracking of Intrarenal Arterial Injected MSCs in Rat Kidneys*

To ascertain the MSC expression in the kidney, we infused MSCs containing a green fluorescent protein (GFP) into the left kidney and examined the GFP expression in rat kidneys one hour later. The sections were examined under UV light for the detection of fluorescence around the glomeruli, arterial lining cells and tubular cells. Immunochemical stains with primary antibodies against MSC CD44 (MCA643GA, Serotec, Kidlington, UK) were also performed for identification of MSCs in kidneys.

#### *2.6. Measurements of Proteinuria and Hydroxyproline Degree*

Twenty four hours urine samples were collected (on day 5 after anti-Thy1.1 infusion) from all experimental rats with free access to water. Urinary protein concentration was determined by a Bio-Rad protein assay (Bio-Rad Laboratories, München, Germany). Hydroxyproline content was measured with Hydroxyproline Assay Kit (STA-675, Cell Biolabs, Inc., San Diego, CA, USA).
