Extraction Adipose

The American Society for Cell Biology
Mol Biol Cell. 2002 December; 13(12): 4279–4295.
doi: 10.1091/mbc.E02-02-0105.

Patricia A. Zuk,^*† Min Zhu,^* Peter Ashjian,^* Daniel A. De Ugarte,^* Jerry I. Huang,^* Hiroshi Mizuno,^* Zeni C. Alfonso,^‡ John K. Fraser,^‡ Prosper Benhaim,^* and Marc H. Hedrick^*

^*Departments of Surgery and Orthopedics, Regenerative Bioengineering and Repair Laboratory, UCLA School of Medicine, Los Angeles, California 90095; and ^‡Department of Medicine and the Jonsson Comprehensive Cancer Center, Division of Hematology and Oncology, UCLA School of Medicine, Los Angeles, California 90095

MATERIALS AND METHODS

Cell Culture and Differentiation

PLA cells were obtained from raw human lipoaspirates and cultured as described in a previous study (Zuk et al., 2001). Briefly, raw lipoaspirates were washed extensively with sterile phosphate-buffered saline (PBS) to remove contaminating debris and red blood cells. Washed aspirates were treated with 0.075% collagenase (type I; Sigma-Aldrich, St. Louis, MO) in PBS for 30 min at 37°C with gentle agitation. The collagenase was inactivated with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant centrifuged for 10 min at low speed. The cellular pellet was resuspended in DMEM/10% FBS and filtered through a 100-μm mesh filter to remove debris. The filtrate was centrifuged as detailed above and plated onto conventional tissue culture plates in control medium (Table 1). Normal human osteoblasts (NHOst), normal human chondrocytes from the knee (NHCK), and a population of MSCs from human bone marrow were purchased from Clonetics (Walkersville, MD) and maintained in commercial medium. The murine 3T3-L1 preadipocyte cell line (Green and Meuth, 1974) was obtained from American Type Culture Collection (Manassas, VA). NHOst, PLA cells, and 3T3-L1 cells were treated with mesenchymal lineage-specific media as outlined in Table 1. MSCs were induced using commercial control medium supplemented with the growth factors outlined in Table 1. NHOst and NHCK cells were induced using commercially available induction media (Clonetics).

The antibodies and commercial sources used in this study are indicated in online Table S1.

PLA cells and MSCs were cultured in control medium 72 h before analysis. Flow cytometry with a FACscan argon laser cytometer (BD Biosciences, San Jose, CA) was performed according to a previous study (Zuk et al., 2001). Briefly, cells were harvested in 0.25% trypsin/EDTA and fixed for 30 min in ice-cold 2% formaldehyde. The fixed cells were washed in flow cytometry buffer (PBS, 2% FBS, 0.2% Tween 20) and incubated for 30 min in flow cytometry buffer containing fluorescein isothiocyanate-conjugated monoclonal antibodies to SH3, STRO-1, and the following CD antigens: 13, 14, 16, 31, 34, 44, 45, 49d, 56, 62e, 71, 90, 104, 105, and 106. PLA cells and MSCs were stained with a phycoerythrin-conjugated nonspecific IgG to assess background fluorescence.

Histology, Immunohistochemistry, and Indirect Immunofluorescence

PLA cells and MSCs were processed as described previously (Zuk et al., 2001) by using monoclonal antibodies to specific CD markers and lineage-specific proteins (online Table S1).

Differentiated PLA cells and clones were processed as described previously (Zuk et al., 2001) by using the following histological assays: alkaline phosphatase (AP) (osteogenesis), Oil Red O (adipogenesis), and Alcian blue (AB) (chondrogenesis). Chondrogenic PLA cells and clones were examined for collagen type 2 (CNII), keratan sulfate, and chondroitin-4-sulfate expression by immunohistochemistry as described previously (Zuk et al., 2001). Neurogenic PLA cells and clones were examined by immunohistochemistry for the expression of neural-specific proteins.

Triplicate samples of PLA cells were differentiated in osteogenic medium (OM) for up to 6 wk. Cells were washed with PBS, harvested, and AP enzyme activity was assayed using a commercial AP enzyme kit according to the method of Beresford et al. (1986). AP activity was expressed as nanomoles of p-nitrophenol produced per minute per microgram of protein. Differentiated MSCs were assayed as a positive control, whereas non-induced PLA cells were assayed as a negative control. Values are expressed as the mean ± SD. A Student's t test (paired) was performed to determine statistical significance between induced and control samples.

PLA Cells Are Phenotypically Similar to MSCs

Characterization of MSCs has been performed using the expression of cell-specific proteins and CD markers (Bruder et al., 1998b; Conget and Minguell, 1999; Pittenger et al., 1999). Like MSCs, PLA cells expressed CD29, CD44, CD71, CD90, CD105/SH2, and SH3 and were absent for CD31, CD34, and CD45 expression (online Figure S1). Moreover, flow cytometry on PLA cells confirmed the expression of CD13, whereas no expression of CD14, 16, 56, 62e, or 104 was detected (Table 2). These results demonstrate that similar CD complements are expressed on both PLA cells and MSCs. However, distinctions in two CD markers were observed: PLA cells were positive for CD49d and negative for CD106, whereas the opposite was observed on MSCs. Expression of CD106 has been confirmed in the bone marrow stroma and, specifically, MSCs (Levesque et al., 2001) where it is functionally associated with hematopoiesis. The lack of CD106 on PLA cells is consistent with the localization of these cells to a non-hematopoietic tissue.

PLA Cells Differentiate into Bone, Fat, Cartilage, and Muscle: Multiple Mesodermal Lineage Capacity

As suggested in a previous study (Zuk et al., 2001), PLA cells seem to possess the capacity to differentiate into multiple mesodermal lineages, including bone, fat, and cartilage. This observation has led us to speculate that adipose tissue may be a source of mesodermal stem cells. The current study supports this hypothesis, characterizing the metabolic activity of several mesodermal lineages, in addition to confirming the expression of multiple lineage-specific genes and proteins.

PLA cells express a unique set of CD markers. (A) PLA cells and MSCs were processed by immunofluorescence for expression of multiple CD antigens (green). Cells were costained with 4,6-diamidino-2-phenylindole to visualize nuclei (blue) and the fluorescent images combined. The differential expression of CD49d and CD106 between PLA cells and MSCs is shown (Figure S1 for remaining CD antigens). (B) Flow cytometric analysis on PLA cells and MSCs for the expression of CD49d and CD106 was performed (red). Cells stained with a fluorochrome-conjugated nonspecific IgG were examined as a control (γPE, green). The geometric mean and median values for CD49d and Cd106 are shown below. Significant differences are shown in bold.

CD Antigen	Geometric Mean
CD13	148.88
CD14	2.43
CD16	2.38
CD31	2.22
CD34	3.55
CD44	16.92
CD45	2.52
CD49d	14.99
CD56	2.66
CD62E	2.30
CD71	3.76
CD90	25.96
CD104	2.31
CD105	8.39
CD106	2.45
SH3	8.95
STRO-1	31.26
−ve	2.59

Mol Biol Cell. 2002 December; 13(12): 4279–4295. doi: 10.1091/mbc.E02-02-0105.

Stem Cell Markers

Mesenchymal/Stromal Stem Cell Markers

STRO-1: The murine IgM monoclonal Ab STRO-1, produced from an immunization with a population of human CD34⁺ bone marrow cells, can identify a cell surface antigen expressed by stromal elements in human bone marrow.⁵⁵ From bone marrow cells, the frequency of fibroblast colony-forming cells (CFU-F) is enriched approximately 100-fold in the STRO-1⁺/Glycophorin A- population than in the STRO-1⁺/Glycophorin A⁺ population.⁵⁵ A STRO-1⁺ enriched subset of marrow cells is capable of differentiating into multiple mesenchymal lineages including hematopoiesis-supportive stromal cells with a vascular smooth muscle-like phenotype, adipocytes, osteoblasts and chondrocytes.^56-59 STRO-1 is a valuable Ab for the identification, isolation and functional characterization of human bone marrow stromal cell precursors, which are quite distinct from those of primitive HSCs.

One of the main issues regarding cancer stem cells is whether they're normal stem cells gone awry or differentiated cells that have acquired stem cell characteristics. The former scenario appeals to most scientists, although they acknowledge it's largely unproved.

Because it can replicate endlessly, a normal stem cell is a "very dangerous cell" that's poised on the edge of becoming cancerous, says Dick. The potentially endless reproduction of a stem cell also allows enough time for cancer-promoting mutations to accumulate in such a cell, he explains.

The cancer–stem-cell hypothesis could explain why many cancers are resistant to radiation and drugs. Normal stem cells are unusually hardy and possess molecular pumps similar to the ones that some cancer cells use to flush out chemotherapy agents, notes Kornblum.

The discovery of cancer stem cells is forcing scientists to reconsider how they look for tumor-fighting drugs. "Everyone has been concentrating on proliferation," says Clark. Traditionally, researchers screen for compounds that kill dividing tumor cells, but stem cells are often quiescent, only occasionally spawning progeny that then rapidly proliferate.

"The biology of the tumor you see may not be the same as the biology of the stem cell. You're never going to cure someone unless you hit the stem cell," says Matsui.

Scientists battling leukemia, the disease in which a cancer stem cell was first isolated, have been focusing on this new target for a few years, says Dick. As one example, he points to a 2002 study in which Craig T. Jordan of the University of Kentucky Medical Center in Lexington and his colleagues identified compounds that specifically kill leukemia stem cells derived from patients.

The research on cancer stem cells also threatens to upend thinking on how cancers spread, or metastasize. Conventional theories hold that metastasis is an evolutionary process in which a small number of cells within a primary tumor gradually accumulate the genetic mutations that enable them to spread to other tissues and establish new tumors. An alternative model now being put forth is that many cells in a primary tumor spread in the body, but a second tumor arises only when a rare stem cell reaches a new site.

Scientists have proposed that identifying cancer stem cells from various types of tumors will help them isolate the long-sought normal stem cells in tissues such as the prostate gland and the breast. "Tracing back from the tumor to that cell population will allow us to identify these critical cells in normal tissue," says Jacks, who is a Howard Hughes Medical Institute investigator at MIT.

University of Pittsburgh
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Tel: 412-692-3239(O)
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In recent years, scientists have discovered a wide array of stem cells that have unique capabilities to self-renew, grow indefinitely, and differentiate of develop into multiple types of cells and tissues. My works focus the isolation of adult stem cells from a variety of tissues and organs. In the present study, we characterized a population of potential adipose-derived adult stem (ADAS) cells isolated from the visceral fat of the abdominal cavity of C57BL/10J mice. We used flow cytometry to examine the marker profile (CD34 and Sca-1) of these cells. The isolated cells were CD45-negative, which excludes any possible contamination by hematopoietic cells, and were partially positive for Sca-1 (38%) and CD34 (7%), two stem cell markers. After induction in conditioned medium, the ADAS cells differentiated into adipogenic, osteogenic, chondrogenic, and myogenic lineages. This finding supports previous reports that indicate the existence of multipotent stem cells in a variety of adult tissues. This finding is consistent with the cells isolated from human and rat fat tissue. These findings suggest that adipose tissue may be a novel, easily accessible, and replenishable source of pluripotent stem cells suitable for cellular therapies.

International Fat Applied Technology Society: One pint of liposuctioned fat or one pound of whole fat removed in a tummy tuck, for example, can yield up to 200 million stem cells, which in culture can be expanded by 10 times over the course of two weeks.

Although he offers cell-harvesting services to all of his patients, Dr. Ersek says, "Not that many are interested because they've never heard of it. New ideas take awhile to grab hold, even really good ones. So I decided to store my own stem cells. And it occurred to me that in order to convince my patients that (the procedure) is very safe and simple, I decided to harvest the stem cells myself. So I put in some local anesthesia and performed the procedure entirely awake, just using Xylocaine with epinephrine. This was not a liposuction procedure for changing my size. We took out about one pound of fat just for the stem cell purpose."

StemSource later processed Dr. Ersek's tissue down to 150 cc of usable material, which yielded 4 million viable stem cells. The company stores such materials at minus 320 degrees Fahrenheit, charging patients $1,675 for five years' storage (or $600 initially plus $175 annually after the first year). For more information about cell preservation and banking contact:

MacroPore Biosurgery, Elizabeth Scarbrough
Vice President Marketing and Development – Biologics

740 Top Gun, San Diego California 92121
Phone: 858-458-0900, Toll Free: 877-470-8000

CD133⁺ cells budding from the adherent cell surface by asymetric cell division. Cells were stained with CD133/1 (AC133)-PE.

CD133 is expressed on immature hematopoietic stem and progenitor cells, and is not found on mature blood cells. In contrast to the CD34 antigen, CD133 is not expressed by late progenitors, such as pre-B cells, CFU-E, and CFU-G.
The antibodies specific for the CD133 antigen (clones AC133, 293C3 and AC141) stain 35–75% of the CD34⁺ population including CD34^bright, CD38^neg/dim, HLA-DR^–, CD90⁺ and CD117⁺ cells. Moreover, a small population of CD133⁺CD34^– cells with long-term repopulating potential was identified.

Functional studies on MACS-isolated CD133⁺ cells confirmed that CD133 is expressed on long-term culture initiating and long-term repopulating hematopoietic stem cells.
Further, certain acute myeloid leukemias have been reported to be positive for CD34, but negative for CD133. Therefore, CD133 selected cells have already been used for autologous transplantation ALL.