At 1.2 M of L-Alanine, the viability of 201B7 cells and ehiPSCs decreases to 7.5±1.3% and 3.7±0.7%, respectively, as the concentration of L-Alanine increases. The viability of hFBs and hSkMCs, however, does not appear to have decreased. Although iCMs' vitality marginally declines as L-Alanine concentration rises, it is still considerably greater (p<0.01) than that of undifferentiated iPSCs, 201B7 cells, and ehiPSCs at 1.2 M concentration of L-Alanine (49.4±6.9%). Even after two or four hours of treatment, the vitality of 201B7 cells, hiPSCs, and ehiPSCs sharply declines. However, the viability of hFBs exhibits a slight decline at 24 hours of treatment, failing to decrease at 1, 2, and 4 hours. Following a 2-hour treatment with 1.2 M L-Alanine, the vitality of 201B7 cells in suspension culture drops to 11.8±6.0%, while that of hFBs is 72.9±14.2%[1].

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Undifferentiated human induced pluripotent stem cells (hiPSCs, lines 201B7 and ehiPSC) showed high sensitivity to treatment with medium supplemented with high concentrations of L-alanine. After a 2-hour treatment with 1.2 mol/L L-alanine, the viability of 201B7 cells dropped to 7.5 ± 1.3%, and ehiPSCs to 3.7 ± 0.7%. [1]
- Differentiated cells, including human fibroblasts (hFBs), human skeletal muscle cells (hSkMCs), and hiPSC-derived cardiomyocytes (iCMs), were more resistant to the same treatment. At 1.2 mol/L L-alanine for 2 hours, the viability was 94.9 ± 12.5% for hFBs, 87.0 ± 13.1% for hSkMCs, and 49.4 ± 6.9% for iCMs. The viability of iCMs, while slightly reduced, was significantly higher than that of undifferentiated hiPSCs (p < 0.01). [1]
- Time-course experiments with 0.6 mol/L L-alanine showed that the viability of hiPSCs decreased drastically after 2-4 hours of treatment, while hFBs maintained high viability up to 4 hours and showed only a small decrease (69.3 ± 1.3%) after 24 hours. [1]
- The selective effect of high L-alanine was also observed in suspension culture. Treatment with 1.2 mol/L L-alanine for 2 hours reduced the viability of 201B7 cells to 11.8 ± 6.0%, while hFBs remained at 72.9 ± 14.2%. [1]
- In co-culture experiments, hiPSCs (labeled with Cell Tracker Green) were selectively eliminated from a mixed population with hiPSC-derived fibroblast-like cells (iFLCs, labeled with Cell Tracker Orange) or hFBs after treatment with 1.2 mol/L L-alanine for 2 hours. Flow cytometry analysis confirmed a significant reduction in the percentage of hiPSCs after single or repetitive treatments. [1]
- The cell death mechanism appeared to involve osmotic shock. hiPSCs treated with high L-alanine at 37°C initially shrank, then swelled dramatically upon return to normal medium, leading to membrane rupture and cell death. This process was energy-dependent, as treatment at 4°C prevented the drastic swelling and cell death, with viability remaining high. [1]
- Other amino acid isomers, D-alanine and β-alanine, induced a similar decrease in hiPSC viability when used at high concentrations. [1]
- High concentrations of other osmolytes like NaCl, KCl, or D-glucose did not selectively eliminate hiPSCs; they reduced the viability of both hiPSCs and hFBs. [1]

Cell Viability Assay (High L-Alanine Treatment): hiPSCs (201B7 and ehiPSC) and differentiated cells (hFBs, hSkMCs, iCMs) were seeded in 96-well plates. The next day, the culture medium was replaced with medium containing various concentrations of L-alanine (0-1.2 mol/L) for different treatment times (1-24 h). After treatment, the medium was replaced with normal culture medium. Cell viability was assessed 24 hours later using a Cell Counting Kit-8 (WST-8 assay), following the manufacturer's instructions. Absorbance was measured, and relative cell viability was calculated. [1]
- Suspension Culture Viability Assay: Cells were suspended in normal medium or medium supplemented with 1.2 mol/L L-alanine (both containing a ROCK inhibitor) and incubated for 2 hours. Cells were then collected by centrifugation, resuspended in normal medium, and seeded in 96-well plates. Viability was measured 24 hours later using the WST-8 assay. [1]
- Co-culture and Flow Cytometry Analysis: hiPSCs were fluorescently labeled with Cell Tracker Green. hFBs were seeded into plates containing the labeled hiPSCs at different initial ratios (1:1, 1:2, 1:4). After 12 hours, the medium was replaced with medium containing 1.2 mol/L L-alanine for 2 hours, then switched back to normal medium. After 12 hours, cells were dissociated, collected, and analyzed by flow cytometry to determine the remaining percentage of green fluorescent (hiPSC) cells. Repetitive treatments were performed by reapplying the high-L-alanine medium. [1]
- Co-culture and Fluorescence Microscopy: hiPSCs and hiPSC-derived fibroblast-like cells (iFLCs) were fluorescently labeled with Cell Tracker Green and Cell Tracker Orange, respectively. They were co-cultured and then treated with 1.2 mol/L L-alanine for 2 hours. After 24 hours in normal medium, the cells were observed under a fluorescence microscope. [1]
- Dead Cell Staining: hiPSC colonies cultured in 35 mm dishes were treated with normal medium or 1.2 mol/L L-alanine medium for 2 hours. The medium was then replaced with normal medium containing propidium iodide (PI). Cells were observed under a fluorescence microscope to visualize dead cells (PI-positive). [1]

The study demonstrates that high concentrations (e.g., 1.2 mol/L) of L-alanine are toxic to undifferentiated hiPSCs, inducing cell death through an osmotic shock mechanism. This toxicity is selective, as differentiated cells like fibroblasts and skeletal muscle cells are much more resistant. The process is energy-dependent, as it is inhibited at 4°C. [1]
- No systemic toxicity data (e.g., LD50) for Alanine were provided. The study notes that L-alanine is a component of proteins in the human body and a common ingredient in cell culture media, suggesting that any residual amount after treatment might be non-toxic. [1]

[1]. Selective Elimination of Human Induced Pluripotent Stem Cells Using Medium with High Concentration of L-Alanine. Sci Rep. 2018 Aug 20;8(1):12427.

L-Alanine is the L-enantiomer of alanine. It is an EC 4.3.1.15 (diaminopropionic acid lyase) inhibitor and an important metabolite. It belongs to the pyruvate family of amino acids, is a protein-forming amino acid, and is also an L-α-amino acid, belonging to the alanine family. It is the conjugate base of L-alanine, the conjugate acid of L-alanine, an enantiomer of D-alanine, and a zwitterion tautomer of L-alanine. Alanine is a non-essential amino acid, existing in high concentrations in its free state in plasma. It is produced from pyruvate via transamination. It participates in the metabolism of sugars and acids, enhances immunity, and provides energy for muscle tissue, the brain, and the central nervous system. L-Alanine is a metabolite found in or produced by Escherichia coli (K12 strain, MG1655 strain). Alanine is an amino acid.
It has been reported that alanine is found in plants of the genus Euphorbia (such as creeping euphorbia and angelica) and other organisms with relevant data.
Alanine is an essential small-molecule amino acid for the human body and one of the most widely used amino acids in protein synthesis, participating in the metabolism of tryptophan and vitamin pyridoxine. Alanine is an important energy source for muscles and the central nervous system, enhancing the immune system, aiding in the metabolism of sugars and organic acids, and having a cholesterol-lowering effect in animals. (NCI04)
Alanine is a non-essential amino acid synthesized in the body from the conversion of carbohydrate pyruvate or DNA breakdown, as well as from dipeptides carnosine and anserine. It is highly concentrated in muscles and is one of the most important amino acids released from muscles, serving as a primary energy source. Plasma alanine levels are typically lower when branched-chain amino acids (BCAAs) are deficient. This finding may be related to muscle metabolism. Alanine is found in high concentrations in meat products and other high-protein foods such as wheat germ and cheese. Alanine is an important participant and regulator of glucose metabolism. In diabetes and hypoglycemia, alanine levels are positively correlated with blood glucose levels, and alanine can alleviate severe hypoglycemia and diabetic ketoacidosis. It is an important amino acid for lymphocyte proliferation and immunity. Alanine therapy has helped dissolve kidney stones in laboratory animals. Like other amino acids, normal alanine metabolism is highly dependent on enzymes containing vitamin B6. Alanine, along with GABA, taurine, and glycine, is an inhibitory neurotransmitter in the brain. It is produced from pyruvate via transamination. Alanine participates in the metabolism of sugars and acids, enhances immunity, and provides energy to muscle tissue, the brain, and the central nervous system.
Alanine is a non-essential amino acid, existing in high concentrations in its free state in plasma. It is produced from pyruvate via transamination. Alanine participates in the metabolism of sugars and acids, enhances immunity, and provides energy to muscle tissue, the brain, and the central nervous system.
See also: Glatiramer acetate (monomer); Glatiramer (monomer). Amliximod (monomer)...See more...
Drug Indications
For protein synthesis.

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Mechanism of Action
L-alanine is a non-essential amino acid that exists in large quantities in the blood plasma in a free state. It is produced from pyruvate via transamination. It participates in the metabolism of sugars and acids, enhances immunity, and provides energy for muscle tissue, the brain, and the central nervous system. Branched-chain amino acids (BCAAs) are the energy source for muscle cells. During prolonged exercise, BCAAs are released from skeletal muscle; their carbon skeleton is used as fuel, while their nitrogenous portion is used to synthesize another amino acid—alanine. Alanine is then converted into glucose in the liver. This energy production pathway is called the alanine-glucose cycle, which plays a crucial role in maintaining blood glucose balance in the body.

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L-Alanine is a naturally occurring amino acid and a fundamental building block of proteins in the human body. It is also a common, inexpensive, and safe ingredient in standard cell culture media. [1]
- This study proposes a novel application for L-alanine: the selective elimination of tumorigenic residual undifferentiated hiPSCs from a population of differentiated cells intended for regenerative medicine therapies. This is a critical safety step to prevent teratoma formation after transplantation. [1]
- The proposed mechanism involves an energy-dependent process where hiPSCs, but not differentiated cells, accumulate L-alanine intracellularly during exposure to a high-concentration medium. Upon returning to normal, isotonic medium, water rushes into the hiPSCs, causing them to swell and rupture. The mechanism of resistance in differentiated cells is hypothesized to involve systems that prevent the excessive accumulation of L-alanine. [1]
- The method using high-concentration L-alanine is presented as a potentially low-cost, simple, and safe strategy for improving the safety of hiPSC-based cell therapies. [1]

C3H7NO2

89.09

89.047

56-41-7

25191-17-7

5950

White to off-white solid powder

1.2±0.1 g/cm3

212.9±23.0 °C at 760 mmHg

297 °C (decomposes) ; 300 °C

82.6±22.6 °C

0.1±0.9 mmHg at 25°C

1.460

-0.68

2

3

1

6

61.8

1

C[C@@H](C(=O)O)N

QNAYBMKLOCPYGJ-REOHCLBHSA-N

InChI=1S/C3H7NO2/c1-2(4)3(5)6/h2H,4H2,1H3,(H,5,6)/t2-/m0/s1

(2S)-2-aminopropanoic acid

Alanine AlaninumL-(+)-Alanine Alanina NSC-206315 NSC206315NSC 206315

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~150 mg/mL (~1683.69 mM)
DMSO :< 1 mg/mL

Solubility in Formulation 1: 50 mg/mL (561.23 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)