A Summary of Stem Cell Expansion Protocols prepared by graduate student, Paul Faduola, Panama College of Cell Science

Graduate Studies at the Panama College of Cell Science- Sample Work

From time to time we like to post particularly good work by our graduate students. In this Assignment from the course, “Stem Cell Laboratory Protocols”, students were asked to present reviews of the current state of expansion protocols for stem cells. Expansion of stem cells (multiplication of cells ex vivo) is a particularly important issue for stem cell treatments with a patient’s own stem cells (autologous therapy) because many times it is the lack of sufficient numbers of stem cells harvested from the patient which leads to a non-therapeutic result. 

The following is an excerpt from an Assignment generated by graduate student Paul Faduola of Nigeria. It is presented not only as a sample of our graduate students’ work, but also because it presents an excellent summary of the current state of research relating to stem cell expansion protocols:


Panama College of Cell Science

Stem Cell Laboratory Protocols

In your Syllabus for Stem Cell Laboratory Protocols, you were given 6 papers for download.  Please be sure you have downloaded these papers for study and also the following two additional papers which I am giving to you here for download:

Bieback et al: “Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow”, Stem Cells 27:2331-2341 (2009)

Shahdadfar et al: “In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression, and Transcriptome Stability” Stem Cells 23:1357-1366 (2005). This is in substitution for the Stute paper which was not available for free on the internet but which is mentioned in this paper. This paper builds on Stute and advances the concepts.

Assignment Part 1: Please prepare a scholarly review of each paper as if you were going to present its findings to your professors and colleagues at an informal scientific meeting. For each review, please emphasize what the investigators were trying to do from the standpoint of advancing knowledge with respect to stem cell expansion methods, and what contribution each paper made to IMPROVING expansion methodology.

1. Krawetz et al: Large Scale Expansion of Pluripotent Human Embryonic Stem Cells in Stirred-Suspension Bioreactors. Tissue Engineering, 16:573-582 (2010)

The current developments in cell therapy is focusing on identifying essential requirements for successful expansion of adult stem cells for therapeutic applications. Stem cells can be expanded from a particular cell types for specific applications if the stem cell fate is properly controlled by directing the cell differentiation to a desired lineage. The challenge in achieving this include the technical capacity in increasing expansion, honing differentiation efficiency, enhancing population purity, and improving cell viability.

This article by Roman Krawetz et al, is an attempt to translate progress made in expanding pluripotent murine ES cells using suspension bioreactor method into hESC research for the purpose of efficiently generating sufficient and viable stem cells necessary to generate a therapeutic result. Their protocol yielded a 25-fold expansion of hES cells over 6 days and enables hES cells to be expanded without feeder layers or matrices with minimal labor in a controlled and reproducible culture system. This finding demonstrate that the expansion and directed differentiation of human embryonic stem cells (hesc’s) in stirred bioreactors for large scale production of therapeutically useful hesc progeny is feasible and can facilitate the adaptation of good manufacturing process (GMP) standards to the development of hES cell therapies. This paper has open a new challenge in hESC clinical application using this protocol in terms of expansion and differentiation by creating the need to improve on the expansion and directed differentiation of human embryonic stem cells (hESCs) in stirred-suspension bioreactors (SSBs) for large-scale production of therapeutically useful hESC progeny is feasible controlling key variables like nutrient and metabolite concentrations, growth factor compositions, and physiological parameters (e.g., temperature, pH, and oxygen).To control this expansion, this study used microcarriers which offers the advantage of providing a larger surface area for the growth of anchorage-dependent cells in a suspension culture system, and overcomes several problems with static cultures, including the requirement for large quantities of culture media, space expense, inefficient gas-liquid oxygen transfer, presence of concentration gradients, and difficulties in monitoring and control. Though this article attributed the successful expansion to the microcarriers in stirred bioreactors used and also compared the outcome to be the same with other similar works using microcarriers, they fail to mention that the favourable outcome recorded against similar works with reduced expansion could have been influence by variable factors including microcarrier shape and size which could affect cell-microcarrier aggregate morphology. High attachment efficiency have been observed on uncoated microcarriers with poor cell growth and/or gradual loss of pluripotency during extended culturing, higher cell yields and stable pluripotent states have been reported in microcarriers coated with Matrigel. Also,positively charged cylindrical cellulose microcarriers and large (190μm) positively charged spherical microcarriers have been shown to exhibit a high cell expansion potential and levels of pluripotency while lower cell yields have been obtained using smaller diameter spherical (65μm and 10μm) or macroporous beads (1).

This Critic however,can not take away the important finding of this article.In my view,the expansion method proposed by this paper is important especially because the hES cells do not have to be separated from microcarriers to be utilized in downstream applications suggesting that the microcarrier may be of bio-degradeble material thereby solving the difficulty of separating of cells from non-biodegradable microcarriers after enzyme treatment. This method can be improved upon to develop a bioreactor system which is precisely suited for expansion of specific cell types to optimize yield, purity, and quality of a desired cell product.

2.Allen Kuan-Liang Chen, Xiaoli Chen, Andre Boon Hwa Choo, Shaul Reuveny, Steve Kah Weng Oh. Stem Cell Research 2011;7 (2): 97-111 Umbilical Cord Blood Transplantation May Be Option for Adults with Hematologic Malignancies, Oncology News International Vol 9 No. 21: discusses ex vivo expansion

The expansion of cord blood stem cells will be a major breakthrough in transplantation science because these cells can be collected only once for a particular person and is limited to the quantity obtained at this single point in time.To allow for multiple uses and also to increase their capacity for transplantation which is cell dose dependent especially in adults, researchers are developing methods to stimulate stem cells to divide and increase in number while retaining their primitive state. Stem cell expansion is an important tool both for improving transplant outcomes and enabling individuals to use their own cord blood samples for more than one treatment. Investigators at Duke University Medical Center administered cord blood stem cells expanded by the Aastrom Replicell System (developed by Aastrom Biosciences) to 27 patients with malignant and nonmalignant disorders. The recipients exhibited durable long-term engraftment and demonstrated the safety of this cell expansion technique for clinical use. This study has shown that is feasible to expand cord blood.

3.Sekiya,et al Expansion of Human Adult Stem Cells from Bone Marrow Stroma: Conditions that Maximize the Yields of Early Progenitors and Evaluate Their Quality STEM CELLS, 20: 530–541 (2002).

Mesenchymal stem cells (MSCs) is an attractive option for new therapeutic approaches, due to their plasticity and differentiative potential. MSCs are multipotent stem cells that are able to differentiate into different lineages including mesodermal, ectodermal, and endodermal type cells . MSCs can be easily isolated by their ability to adhere to plastic generating single-cell-derived colonies that can be expanded to obtain high numbers of Cells but the rate of expansion and the yields of multipotential progenitors are inversely related to the plating density and incubation time of each passage. This study try to define conditions that maximize the yields of early progenitors, they found out how plating densities and incubation times can be varied to reach a compromise between the total yields of MSCs and the quality of the cells in terms of their content of early progenitors. They found a simple procedure of scoring cultures by phase-contrast microscopy which provides a rapid method of assessing the cultures. One of their important findings is that cultures enriched for the earliest progenitors (RS-1A) have the greatest potential for differentiation into adipocytes, cultures with somewhat later progenitors (RS-1B) have the greatest potential to differentiate into chondrocytes. One possible explanation for this observation they claim is that the later progenitors more readily undergo the condensation step that occurs in the initial phase of chondrogenesis. Their results show that the low plating density results in higher yields and a faster expansion of MSCs. They also observed that small spindle-shaped cells in some cultures grew more rapidly at a low plating density. MSCs isolated at the 1st passage were mostly broad and uneven, the expansion was slower and the cells were senescent at a low plating density.

4. Csaszar et al: Rapid Expansion of Human Hematopoietic Stem Cells by Automated Control of Inhibitory Feedback Signaling, Cell Stem Cell 10:218-229 (2012)

Growing of HSC cultures produces secreted factors that, if not neutralized have an overall inhibitory effect upon growing cultures. This article is an effort to improve the expansion of culturing conditions to better favor self-renewal by using automated feeding methods to limit the effects of inhibitory molecules (TGF-β, MCP-1, MIP-1α, MIP-1β, and IP-10) that accumulate within growing HSC cultures. By continually adding media at an optimized rate, an automated feeding method is reported to dilute inhibitory factors below a threshold so that the negative feedback effects on HSC expansion are sufficiently reduced. This method has led to an 80-fold increase in CD34+cells and an 11-fold increase in blood stem cells, which was better than simple media exchange of the cultures. This fed-batch method continually increases the volume of cultures so as to optimally expand cells and dilute away the inhibitory factors is better than perfusion driven media exchanges that maintain the same volume of the cultures by removing an equal amount of media that is added. The report claims that the fed-batch method simplifies HSC culturing conditions by eliminating the need for dosing the cultures with multiple compounds that independently target and inhibit the negative feedback ligands produced in culture. However, this benefit may be cancelled by the increased complexity over basic culturing methods required with an automated feeding system. Moreover, the authors found that the fed-batch method works synergistically when combined with small molecular factors that have demonstrated successful HSC expansion capabilities, suggesting that the optimal means of expanding HSCs involves combining fed-batch with small molecular therapeutics and cytokines. For instance, both the small molecule aryl hydrocarbon receptor (AhR) antagonist StemRegenin (SR1) and the soluble TAT-HOXB4 were shown to expand HSCs more robustly when the inhibitory molecules were diluted away using the fedbatch culture feeding system.

5. Quantum Cell Expansion System: Automated expansion of Human mesenchymal stem Cells from precultured Cells Using the Quantum Cell Expansion System

The investigators were trying to develop a closed, automated technology with a platform for culturing cells which may offer an ideal solution for companies wanting to expand to therapeutic doses of adherent and suspension cell types without compromising cell quality with a reduced risk of errors and labor. Cells grown in the Quantum system’s bioreactor achieved an average viability of 94% with 7AAD flow cytometry. The experiment demonstrated MSC trilineage differentiation potential into osteoblasts, adipocytes, and chondroblasts for all 12 precultured MSC expansion runs.

6. Stemgenix:  Ex-Vivo Expansion of CD34+ Stem Cells in HSC GEM/StemlineTM Medium Leads to Increased Levels of Total Nucleated Cells and CD34+ Cells (Sigma Labs)

Stem cell sources for example,cord blood products contain low numbers of total cells and progenitor cells which have limited their use primarily to smaller pediatric patients. In order to obtain optimal numbers of HSCs for transplantation in adults, invitro- expansion has been explored to ensure successful engraftment and minimize the short- term effects of neutropenia and thrombocytopenia. One of those methods that attempts to exploit the expansion of stem cells is this study by F.J. Swartzwelder et al, that seek to promote the serum-free medium, HSC GEM/StemlineTM which they develop for the optimal expansion of HSC. They evaluated this medium in the 3 primary cell sources, in both the traditional cell culture well plate format and in a clinical-scale expansion format, with both generating promising results for invitro expansion and viability which indicate that HSC GEM/StemlineTM provides a significant benefit over other commercially available serum-free formulations for the expansion of TNC, committed progenitor and primitive progenitor compartments . This suggests that in the clinical stem cell transplant setting, HSC GEM/StemlineTM may provide significant benefit toward reducing time-to-engraftment and may also result in the reduction in frequency and/or severity of neutropenia and thrombocytopenia. In the microplate culture system, use of HSC GEM/StemlineTM, when compared to other serum-free media, provides a significantly increased expansion of TNC from cultures of CD34+ cord blood cells, bone marrow and mobilized peripheral blood. Flow cytometric data indicates increased specific expansion of CD34+ cells and clinical scale data also supports the overall greater expansion of TNC and CD34+ cells in HSC GEM/StemlineTM, as well as the expansion of both committed and primitive progenitor compartments.

A criticism of this medium is that the human serum albumin is from animal-origin unlike HSA from human which has ultra low endotoxin levels and is ideal for use as a carrier protein. The use of animal source like bovine serum albumin has the disadvantage of being of Increasing the endotoxin level of the resulting solution.

7. Bieback et al: “Human Alternatives to Fetal Bovine Serum for the Expansion of Mesenchymal Stromal Cells from Bone Marrow”, Stem Cells 27:2331-2341 (2009) 

Mesenchymal stromal cells (MSCs) have successfully been expanded in FBS because of it potential clinical applications . Translating the potentials of MSC to the clinic will required substitution of FBS to that of human products .This study is an attempt to compare human serum (HS) ,thrombin-activated platelet releasate in plasma (tPRP) and pooled human platelet lysate (pHPL) to FBS to validate whether they are suitable alternative to FBS . This study showed that both HS and tPRP are comparable to FBS supported isolation and expansion of MSC but pHPL significantly accelerated BM-MSC proliferation to yield clinically relevant numbers within the first two passages. MSC quality and functionality including cell surface marker expression, adipogenic and osteogenic differentiation, and immunosuppressive action were similar in MSCs from all culture conditions. They did not find any spontaneous cell transformation in any of the culture conditions. Telomerase activity was not detected in any of the cultures at any passage. This study disagree with a previous data from adipose tissue-derived MSCs where pHPL was found to be the most suitable FBS substitute in clinical scale BM-MSC expansion.

8. Shahdadfar et al: “In Vitro Expansion of Human Mesenchymal Stem Cells: Choice of Serum Is a Determinant of Cell Proliferation, Differentiation, Gene Expression, and Transcriptome Stability” Stem Cells 23:1357-1366 (2005).

The plasticity and differentiative potential of Mesenchymal stem cells (MSCs) has made it an attractive option for new therapeutic approaches. This study is an attempt to investigate if autologous serum (AS) or allogeneic human serum (alloHS) could serve as an effective substitute for fetal bovine serum(FBS) which has the risk of transmitting viral and prion diseases and proteins that may initiate xenogeneic immune responses for the expansion of hMSCs in vitro. Their Study reveals that the choice of serum could affect hMSCs expansion with hMSCs proliferating faster in AS than in FBS with growth arrest and death in alloHS. In term of differentiation, hMSCs in FBS differentiated more rapidly toward mesenchymal lineages compared with hMSCs in AS. They study claimed that hMSCs may be expanded rapidly and with stable gene expression in AS in the absence of growth factors while FBS induces a more differentiated and less stable transcriptional profile.

Assignment Part 2: Now, take the papers as a group, and synthesize from the papers what methodology would probably work best (which can be a new method suggested by you, but based on your thinking about these papers) in expanding autologous stem cells taken from a tube of patient’s blood or from a few cc’s of adipose tissue. To “synthesize” means to read and understand a previous paper(s) and from that understanding generate or put together the best technique you can come with for how to expand the cells better or faster or in greater quantity.

Integrated system for stem cell expansion

Expansion of adult stem cells in bioreactors could be a powerful method to generate large numbers of stem cells for clinical applications. Bioreactor expansion that allow continuous monitoring and control of the physical and chemical environment of the culture for studying the effect of dynamic temporal nutrient,oxygen or pH profiles and culture parameters known to have crucial influence over stem cell fate is extremely necessary for a successful clinical scale expansion protocol.This can be achieved by optimizing bioreactors through the integration of micro-nanotechnologies which will open a new opportunities to move the old cell culture dish to an integrated bioreactor, mimicking more closely the human body complexity. This will allow for example mimicking the in vivo vasculature through implementing fluid flow and cell perfusion cell culture devices using microfluidic systems. Such devices have been termed “Lab-on-a Chip”, and integrate reaction chambers, sensors, and fluid control on one chip. Lab-on- a-chips are powerful tools to control the soluble and mechanical parameters of the cell culture environment. In this way, they can be configured to work like the automated feeding method in order to regulate culture environment for example neutralizing the concentrations of secreted factors produced in the culture environment to prevent their inhibitory effect upon growing cultures by continually adding media at an optimized rate so that the negative feedback effects on HSC expansion are sufficiently reduced.

Miniaturization of cell culture platforms allows cell culture to be monitored in real-time to observe cellular behavior at the scale found in living systems with high-resolution imaging modalities ,this could lead to the development of a more effective method of assessing culture than they phase-contrast microscopy previously suggested. Stem cell expansion in bioreactors can be translated into clinical settings by addressing issues like the use of chemically define culture media without animal-derived products and pathogen free. In this case a more focus research to adapt human serum(especially autologous which is promising) and thrombin-activated platelet releasate in plasma into the bioreactor trials.


The Panama College of Cell Science, located in the Commonwealth of Dominica, offers the only 3 year online doctoral program in stem cell biology leading to  a PhD degree. Sponsored in part by the Drake Biomedical Institute, the program can be completed entirely online and features a USA style curriculum that meets the equivalency standards for foreign education. Containing 72 trimester credits,  graduates may for a nominal fee obtain a certificate from a foreign credential evaluation service approved by the US Department of Education to the effect that the program is equivalent to a doctoral degree from a regionally accredited USA college or university.