**2.3. Co-culture and transwell culture experiments**

of patients are shown in **Table 1**. Twelve bone marrow aspirates were received from healthy donors (HDs) as control. Written informed consent was obtained from all patients and donors in accordance with the Declaration of Helsinki and the ethical guidelines of the Charité University

BMMSCs from patients and donors (HD-BMMSC) were isolated using adhesion method and cultivated as previously described [42–44]. The colony-forming unit fibroblast (CFU-F) assay was used to study the self-renewal capacity of BMMSC. The staining was carried out with the Hemacolor Rapid Staining Kit from Merck according to the manufacturer's instructions. The

Non-hematopoietic cell characteristics were identified by flow cytometry by the absence of CD105-FITC, CD90-FITC, CD45-PE, and CD34-PE (Miltenyi). Data were acquired and ana-

were isolated from HD mononuclear cells using magnetic-activated cell sorting (MACS) with a CD138 antibody (Miltenyi) as recommended by the manufacturer's protocol and seeded in culture flask with RPMI media with 20% of fetal calf serum and antibiotic/antimycotic.

Age, median (range) 63 (33–87) 64 (33–87) 62 (57–84) 69 (38–81) Gender (M/F, %) 66/34 67/33 66/34 62/38

**Patients at diagnosis (***n* **= 69)**

50 (10–100) 60 (10–100) 40 (5–90)

**Patients at relapse (***n* **= 47)**

plasma cells

**Donors (***n* **= 16)**

lyzed with a FACS Calibur Flow Cytometer (BD Biosciences). Control CD138<sup>+</sup>

**(***n* **= 116)**

IgG 58 60 53 IgA 13 14 13 IgD 1 0 2 Light chain 27 25 32 Non-secretary 1 1 0

I A 10 13 7 I B 6 8 2 II A 8 6 11 II B 5 3 7 III A 55 54 57 III B 16 16 16

School of Medicine, which approved this study (Votum No.: EA4/131/13).

**2.2. Isolation of BMSC and CD138+ plasma cells**

122 Stromal Cells - Structure, Function, and Therapeutic Implications

evaluation was done by counting blue colonies.

**Characteristics All patients**

*Ig expression* (*%*)

*Stage on Durie-Salmon* (*%*)

Bone marrow infiltration % median

**Table 1.** Patients and donor characteristics.

(range):

KMS12-PE cells received from DSMZ (ACC606) were cultured in enriched RPMI media. For cocultures, MM-BMMSCs were seeded in a six-well plate and incubated for 4 h. Then, KMS12-PE myeloma cells were added followed by incubation for 72 h. After incubation, KMS12-PE cells were removed. The absence of CD138+ cells was confirmed using microscopy and checked with FACS analysis. MM-BMMSCs were washed twice with PBS and applied for future analysis. Co-cultured KMS12-PE myeloma cells were suspended in TRIzol for future analysis.

For transwell cultures (0.4 μM pore size, Corning), 2 × 104 MM-BMMSCs were seeded in the lower chamber of a 12-well plate and incubated for 4 h. Then, 2 × 104 KMS12-PE myeloma cells were added to the upper chamber. Incubation was performed for 72 h. Cultures without KMS12-PE cells served as negative control for transwell cultures and co-cultures.
