Human Pluripotent Stem Cells Made from Umbilical Cord Cells Using Small Molecules

Researchers report that they have generated human chemically induced pluripotent stem cells, or CiPSCs, from umbilical cord-derived mesenchymal stem cells using a small-molecule cocktail under ordinary oxygen conditions, eliminating the need for the specialized hypoxic equipment required by earlier protocols. The work, published as a letter in the journal Cell Regeneration, also describes an optimized version of the chemical reprogramming approach applied to adipose-derived stromal cells, and side-by-side comparisons suggesting that chemically derived and transcription-factor-derived stem cells from the same donor share broadly comparable pluripotency.

Reprogramming technology can convert somatic cells into pluripotent stem cells with self-renewal and multi-lineage differentiation potential, a capability that holds promise for regenerative medicine and cell replacement therapy. Transcription factor-based reprogramming overcomes technical and ethical difficulties associated with somatic cell nuclear transfer, but its reliance on massive genetic manipulation raises safety concerns for clinical application. Chemical reprogramming, by contrast, uses small-molecule compounds to manipulate cell fate without integrating genetic material, offering easier process control and, in principle, a path toward clinical-grade cell production.

Human chemical reprogramming was first achieved in 2022, when a research group converted human adipose-derived stromal cells into CiPSCs. A later three-stage protocol significantly increased the efficiency and robustness of that process, and more recently, inhibiting histone acetyltransferases was shown to trigger a swift epigenetic switch that overcame donor variability and shortened the reprogramming timeframe. Adjusted protocols have also been reported to work with blood cells. Even so, the range of applicable starting cell types has remained limited, and all reported protocols relied on hypoxic culture at 5% oxygen, which demands specialized equipment and adds operational complexity. The universality of chemical reprogramming across different cell types therefore still required systematic evaluation.

In the new study, the team first repeated the reported three-stage protocol on adipose-derived stromal cells from a single breast adipose tissue sample. Although OCT4-positive cells appeared in Stage 3, they were present in very low numbers, failed to form compact colonies, and died during attempts to establish a stable cell line. Because Stage 1, the step that erases somatic cell identity, is considered the most critical, the researchers focused their modifications there. They titrated the concentrations of seven key small molecules and found that raising the concentration of most of them promoted expression of LIN28A, an early reprogramming marker. They also tested serum-replacement reagents and found that either 1% ITS-X or an increased amount of B27 significantly promoted cell proliferation, drove a transition to an epithelial-like morphology, and enhanced LIN28A expression.

To improve the economy and convenience of the procedure, the team added deferoxamine mesylate, a small-molecule hypoxia mimetic, allowing the process to run under normoxic conditions. Four micromolar DFOM effectively increased LIN28A expression. Combined, these modifications raised LIN28A levels at the end of Stage 1 and pluripotency gene expression at the end of Stage 3 relative to the original protocol under normal oxygen. The researchers then observed significant CiPSC colonies and established multiple stable CiPSC lines from the same adipose tissue sample across different batches. These cells showed typical pluripotent stem cell morphology, expressed the core pluripotency markers OCT4, NANOG, SOX2 and SSEA4, and maintained a normal karyotype in high-resolution G-banding analysis.

As a benchmark, the team also generated transcription-factor-based induced pluripotent stem cells from the same adipose sample by electroporating plasmids carrying SOX2, KLF4, OCT3/4, L-MYC and LIN28A, with no exogenous plasmid DNA residue detected by PCR. RT-qPCR showed highly similar pluripotency gene expression between the chemically induced and transcription-factor-derived lines, and RNA-seq transcriptomic analysis indicated consistent gene expression patterns, with high levels of pluripotency-related genes and low levels of lineage-specific differentiation genes. No significant differences emerged in cell cycle profiles or population doubling times. Teratoma assays in immunodeficient mice showed that both cell types formed tumors containing structures representative of ectoderm, mesoderm and endoderm, confirming tri-lineage differentiation potential in both.

The team then turned to human umbilical cord-derived mesenchymal stem cells, one of the most widely used somatic cell types in translational and clinical applications because of their robust proliferation, low immunogenicity and abundant banking resources. Distinct from the adipose cell case, lower concentrations of small molecules proved more beneficial for umbilical cord cells. DFOM and ITS-X together with B27 or G27 enhanced LIN28A expression at the end of Stage 1. Because reprogramming involves massive epigenetic changes, the researchers also tested chemicals targeting histone modifications and found that SGC-CBP30, an inhibitor of the KAT3A/KAT3B catalytic enzymes, enhanced LIN28A expression, consistent with a recent report identifying KAT3 as a key epigenetic obstacle to reprogramming. A-485, another KAT3 inhibitor, showed a similar promotive effect. These optimizations constituted the improved protocol for umbilical cord cell reprogramming.

Under normoxic conditions, the improved protocol significantly increased LIN28A expression, produced more LIN28A-positive cells by immunofluorescence and flow cytometry, and accelerated morphological changes: cells appeared more regular, reached high confluence in as little as 10 days, and formed a more pronounced multi-layered accumulation structure at Stage 2, along with a significant increase in SALL4-positive cells. The protocol yielded a markedly higher number of CiPSC colonies at Stage 3, and the team established several stable CiPSC lines. These cells showed typical pluripotent morphology, a population doubling time comparable to the H9 embryonic stem cell line, expression of core pluripotency markers, transcriptional profiles comparable to H9 by RNA-seq, tri-lineage differentiation potential in teratoma assays, and normal karyotypes. Applying the protocol to umbilical cord cells from additional donors consistently produced CiPSC colonies and upregulation of pluripotency-associated genes, supporting robustness across donors.

The authors note that a recent preprint independently reported CiPSCs from frozen human umbilical cord tissue using the original four-stage system, and that the successful induction of stable lines from multiple umbilical cord donors together supports the adaptability of chemical reprogramming across distinct somatic cell types. Their optimization results suggest that modulation of core small-molecule conditions and KAT3-associated epigenetic barriers plays an important role in efficient umbilical cord cell reprogramming, while DFOM and ITS-X mainly served supportive roles during early-stage induction and epithelial-like cell generation. The lower small-molecule concentrations effective in umbilical cord cells may reflect their relatively anti-inflammatory intrinsic profile, whereas JNK-associated pro-inflammatory signaling has been identified as a barrier to human chemical reprogramming; distinct epigenetic landscapes of different tissue origins may also contribute to differential small-molecule sensitivity.

The researchers caution that the current protocol still relies on research-grade reagents such as fetal bovine serum and Matrigel, and that further optimization of chemically defined, GMP-grade culture conditions will be required before the approach can support future translational applications. Within those limits, the study provides an optimized strategy for generating human chemically induced pluripotent stem cells from adipose-derived and umbilical cord-derived cells under normal oxygen conditions, and adds evidence that chemical and transcription-factor reprogramming can yield pluripotent cells of broadly comparable quality from the same donor.

Subject of Research: Chemical reprogramming of human umbilical cord-derived mesenchymal stem cells into induced pluripotent stem cells

Article Title: Generation of human chemically induced pluripotent stem cells from UC-MSCs

Article References: Gong, G., Wen, S., Huang, S., Liu, R., Zheng, R., & Jiang, W. (2026). Generation of human chemically induced pluripotent stem cells from UC-MSCs. Cell Regeneration, 15(1), Article 20. https://doi.org/10.1186/s13619-026-00295-9

Image Credits: AI Generated

DOI: 10.1186/s13619-026-00295-9

Keywords: chemical reprogramming, CiPSCs, pluripotent stem cells, UC-MSCs, small molecules, regenerative medicine, stem cells, hypoxia mimetic, KAT3 inhibitor, teratoma assay, Cell Regeneration, normoxic culture