What are iPSCs and MSCs and how are stem cells cultivated?

As regenerative medicine advances, stem cell cultivation is essential for scaling up iPSCs and MSCs, ensuring their availability for therapeutic applications.

Stem cells are at the forefront of regenerative medicine, offering groundbreaking potential for tissue repair, disease treatment, and advanced cell therapies.

Successful cultivation of high-quality stem cells is key for developing groundbreaking advanced cell therapies and changing the lives of many patients in the future. Stem cells are unique due to their potential to self-renew (to make copies of their cells) and differentiate (to become a specialized cell like a brain cell or a heart cell). Pluripotent stem cells such as induced pluripotent stem cells (iPS cell, iPSC) are a powerful tool in regenerative medicine because they can be directed into every cell of the human body. In contrast, multipotent stem cells such as mesenchymal stem cells (MSC) are more specialized and can differentiate into a limited range of cells including bone, cartilage, and fat. MSCs are naturally occurring and play a major role in tissue repair and immune regulation, making them highly attractive for cell therapy applications.

Maintaining genetic stability and pluripotency is essential for effective iPS cell cultivation

A cell from the body, such as skin or blood cells, can be reprogrammed to an iPSC with properties similar to embryonic stem cells. This discovery, made in 2006 (1) and awarded with the Nobel Prize in 2012, revolutionized research by providing a way to generate patient-specific stem cells circumventing the ethical concerns associated with embryonic stem cells. Today, iPSCs are widely used in scientific research and have the potential to reshape medicine, serving as a valuable model in disease modeling, drug testing, and cell replacement or tissue engineering. Despite their vast potential, iPS cells come with challenges. They require careful monitoring to ensure genetic stability, as mutations can occur during reprogramming and cultivation, influencing their functionality and identity.

Cultivating iPSCs is expensive and technically demanding (2) because they need strict conditions to keep them stable and undifferentiated (pluripotent). Following reprogramming, iPSC colonies can be picked and further expanded, yielding a stable cell line with compactly packed small cells. Previously, iPS cell culture relied on a feeder cell layer usually provided by non-dividing mouse embryonic fibroblast cells and a DMEM/F12 basal media supplemented with essential nutrients like L-Glutamine and growth factors such as fibroblast growth factor FGF. Current protocols for adherent cultivation rely on feeder-free culturing conditions that use a coating consisting of extracellular matrix proteins such as fibronectin, vitronectin, or laminin. Further, chemically defined xeno-free media can ensure strictly controlled culturing conditions to prevent unwanted spontaneous differentiation into other cell types and the loss of stemness.

Various culturing systems are available for maintaining cells, which can be enzymatically lifted and passaged either as single cells or in small clumps. To ensure the stability and research applicability of iPSCs, it is essential to regularly test for key pluripotency markers, including OCT4, SOX2, and NANOG, as well as their ability to differentiate. Additionally, assessing genetic stability and screening for potential contaminants, such as mycoplasma infections, is crucial, as these factors can impact their viability and suitability for research and therapeutic applications.

Mesenchymal stem cell cultivation faces challenges of limited lifespan, standardized properties, and loss of regenerative function

Unlike iPSCs, MSCs can be isolated from sources such as bone marrow, fat tissue, and umbilical cord blood (3). These cells are of particular interest due to their immunomodulatory regenerative and angiogenic properties. However, there are challenges associated with their use because MSCs have a limited lifespan in the lab and may lose their regenerative abilities over time. Another concern is that their properties can vary significantly depending on their donor source, making standardization difficult.

Growing MSCs in the lab is relatively straightforward compared to iPSCs (4): These cells are cultured under serum-containing or serum-free conditions using specialized media, such as Mesenchymal Stem Cell Expansion Medium, supplemented with growth factors like FGF-2. In serum-free systems, surface coatings may be required to promote cell attachment and maintain adherent growth. MSC differentiation protocols often incorporate specific supplements, such as epidermal growth factor (EGF), along with coatings to support adherence and proliferation. Regular passaging is necessary to preserve MSC viability and functionality. While MSCs require less intensive monitoring compared to iPSCs, quality assessments, marker characterization, and differentiation tests are still essential to ensure their stability and therapeutic potential.

Comparison of iPSCs and MSCs: iPSCs are derived through reprogramming and can differentiate into any cell type, while MSCs are naturally occurring and specialize in mesodermal lineages such as bone, cartilage, and fat.

Overcoming bottlenecks in scaling stem cell cultivation

Both iPSCs and MSCs initially require a surface to grow on, making scalability a critical challenge in meeting industrial demand. Traditional large-scale expansion relies on:

• Multiple small vessels, such as T-flasks or multi-layered cell stacks

• Fixed-bed and hollow fiber bioreactors

• Microcarrier stirred-tank bioreactors
  

These methods are labor-intensive, costly, and require complex bioreactor processes to maintain cell quality at scale. As the demand for stem cell-derived therapies increases, the development of efficient, automated, and scalable cultivation systems is crucial. Innovations in bioreactor design and process automation will play a key role in making stem cell manufacturing more cost-effective and widely available for therapeutic applications.

Both iPSCs and MSCs are shaping the future of medicine, with applications ranging from regenerative therapies to disease modeling. While iPSC technology is still in its early stages for clinical use (5), MSCs are already showing promise in treatments for immune-related and degenerative conditions (6). As research advances, scientists continue to refine stem cell cultivation techniques and vessels, making these cells more accessible and reliable for future therapies.

References

(1) Yamanaka S. Pluripotent Stem Cell-Based Cell Therapy-Promise and Challenges. Cell Stem Cell. 2020 Oct 1;27(4):523-531. doi: 10.1016/j.stem.2020.09.014. PMID: 33007237.

(2) Moy AB, Kamath A, Ternes S, Kamath J. The Challenges to Advancing Induced Pluripotent Stem Cell-Dependent Cell Replacement Therapy. Med Res Arch. 2023 Nov;11(11):4784. doi: 10.18103/mra.v11i11.4784. Epub 2023 Nov 29. PMID: 38188933; PMCID: PMC10768945

(3) Bieback K, Schallmoser K, Klüter H, Strunk D. Clinical Protocols for the Isolation

(4) Nikolits I, Nebel S, Egger D, Kreß S, Kasper C. Towards Physiologic Culture Approaches to Improve Standard Cultivation of Mesenchymal Stem Cells. Cells. 2021 Apr 13;10(4):886. doi: 10.3390/cells10040886. PMID: 33924517; PMCID: PMC8069108.

(5) Scudellari M. How iPS cells changed the world. Nature. 2016 Jun 16;534(7607):310-2. doi: 10.1038/534310a. PMID: 27306170.

(6) Pittenger, M.F., Discher, D.E., Péault, B.M. et al.Mesenchymal stem cell perspective: cell biology to clinical progress. npj Regen Med 4, 22 (2019). https://doi.org/10.1038/s41536-019-0083