True Donor Cell Leukemia after Allogeneic Hematopoietic Stem Cell Transplantation: Diagnostic and Therapeutic Considerations-Brief Report.

Donor cell leukemia (DCL) is a rare complication after allogeneic hematopoietic stem cell transplantation (HSCT) accounting for 0.1% of relapses and presenting as secondary leukemia of donor origin. Distinct in phenotype and cytogenetics from the original leukemia, DCL's clinical challenge lies in its late onset. Its origin is affected by donor cell anomalies, transplant environment, and additional mutations. A 43-year-old woman, treated for early stage triple-negative breast cancer, developed mixed-phenotype acute leukemia (MPAL), 12 years later. Following induction chemotherapy, myeloablative conditioning, and allo-HSCT from her fully HLA-matched brother, she exhibited multiple cutaneous relapses of the original leukemia, subsequently evolving into DCL of the bone marrow. Cytogenetic analysis revealed a complex male karyotype in 20 out of 21 metaphases, however, still showing the MPAL phenotype. DCL diagnosis was confirmed by 90.5% XY in FISH analysis and the male karyotype. Declining further intensive chemotherapy including a second allo-HSCT, she was subsequently treated with repeated radiotherapy, palliative systemic therapies, and finally venetoclax and navitoclax but died seven months post-DCL diagnosis. This case underlines DCL's complexity, characterized by unique genetics, further complicating diagnosis. It highlights the need for advanced diagnostic techniques for DCL identification and underscores the urgency for early detection and better prevention and treatment strategies.

Donor Cell Acute Myeloid Leukemia after Hematopoietic Stem Cell Transplantation for Chronic Granulomatous Disease: A Case Report and Literature Review.

The patient reported here underwent hematopoietic stem cell transplantation (HSCT) due to chronic granulomatous disease (CGD) caused by biallelic mutations of the NCF1 gene. Two years later, he developed AML, which was unexpected and was recognized via sex-mismatched chromosomes as deriving from the donor cells; the patient was male, and the donor was his sister. Donor cell leukemia (DCL) is very rare, and it had never been reported in patients with CGD after HSCT. In the subsequent ten years, the AML relapsed three times and the patient underwent chemotherapy and three further HSCTs; donors were the same sister from the first HSCT, an unrelated donor, and his mother. The patient died during the third relapse. The DCL was characterized since onset by an acquired translocation between chromosomes 9 and 11, with a molecular rearrangement between the MLL and MLLT3 genes-a quite frequent cause of AML. In all of the relapses, the malignant clone had XX sex chromosomes and this rearrangement, thus indicating that it was always the original clone derived from the transplanted sister's cells. It exhibited the ability to remain quiescent in the BM during repeated chemotherapy courses, remission periods and HSCT. The leukemic clone then acquired different additional anomalies during the ten years of follow-up, with cytogenetic results characterized both by anomalies frequent in AML and by different, non-recurrent changes. This type of cytogenetic course is uncommon in AML.

Integrated genomic and single-cell transcriptomic analyses reveal clonal evolution and immune signature in donor cell leukemia after haploidentical allogeneic hematopoietic stem cell transplantation.

The pathogenesis of donor cell leukemia (DCL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is unclear and likely multifactorial. Leukemic transformation of healthy donor HSCs in recipient's bone marrow microenvironment provides a useful in vivo model for investigating the mechanisms involved in leukemogenesis. Here, we report a rare case of late-onset DCL developing in a recipient. Whole-genome sequencing indicates that donor-derived cells harboring clonal hematopoiesis of indeterminate potential (CHIP)-associated genetic alterations expand and eventually transform to full-blown AML via acquisition of additional somatic mutations within the recipient's bone marrow microenvironment. The 10× single-cell RNA sequencing reveals the abundance of GMP-like cells with a specific transcriptional signature in DCL. Moreover, impaired immune surveillance, including dysfunction of cytotoxic T lymphocytes (CTLs) and decreased number of canonical NK cells, is discovered in DCL. Our data add valuable information to the current understanding of the mechanisms of DCL.

Anti-CD19 chimeric antigen receptor T-cell followed by interferon-α therapy induces durable complete remission in donor cell-derived acute lymphoblastic leukemia: A case report.

Donor cell-derived leukemia (DCL) is a special type of relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Patients with DCL generally have a poor prognosis due to resistance to conventional chemotherapy. Here, we report a case of donor cell-derived acute lymphoblastic leukemia after umbilical cord blood transplantation. The patient didn't respond to induction chemotherapy. She then received anti-CD19 CAR-T cell therapy and achieved MRD-negative complete remission (CR). However, MRD levels rose from negative to 0.05% at 5 months after CAR-T cell therapy. Higher MRD levels were significantly associated with an increased risk of leukemia recurrence. Afterward, preemptive interferon-α treatment was administrated to prevent disease recurrence. To date, the patient has maintained MRD-negative CR for 41 months. Our results suggested that anti-CD19 CAR-T cells followed by interferon-α therapy are effective in treating donor cell-derived acute lymphoblastic leukemia. This report provides a novel strategy for the treatment of DCL.

Adult-onset hereditary myeloid malignancy and allogeneic stem cell transplantation.

Hereditary myeloid malignancies, especially in adults or elderly persons, had been considered quite rare before the next-generation sequencing era; however, increased usage of clinical sequencing has revealed much higher prevalence of inherited myeloid malignancies. DDX41 and various pathogenic germline mutations have newly been recognized as the cause of adult-onset familial leukemia and myeloid malignancies. Although germline predisposition to myeloid neoplasms had been categorized as a provisional entity in the World Health Organization classification of hematopoietic neoplasms in 2016, methodology for the identification of hereditary myeloid malignancies has not been fully established yet. In addition, many unresolved problems, such as epidemiology, the exact pathogenic mechanisms, and ideal treatment strategy, including indications of allogeneic hematopoietic stem cell transplantation, still remain. Related donor selection for stem cell transplant is a particularly sensitive issue due to the possibility of germline mutation of the candidate relatives and the risk of donor cell leukemia after transplantation. Here, we reviewed the current evidence regarding epidemiology, diagnosis, mechanisms of progression, and transplantation strategy for hereditary myeloid malignancies.

Identifying the optimal developmental age of human pluripotent stem cell-derived midbrain dopaminergic progenitors for transplantation in a rodent model of Parkinson's disease.

Donor cell age can have a significant impact on transplantation outcomes. Despite the rapid advancement of human pluripotent stem cell (hPSC)-derived dopaminergic (DA) progenitors to the clinic for transplantation into Parkinson's Disease (PD), surprisingly limited data exists regarding the influence of cellular age on neural graft survival, composition, and integration. Here we examined the impact of transplanting ventral midbrain (VM) progenitors at varying days of differentiation (from day 13-30) into a rodent PD model, comparing two hPSC lines (an embryonic and an induced pluripotent cell line, hESC and hiPSC, respectively). Both hPSC lines expressed GFP under the promoter PITX3 enabling specific tracking of graft-derived DA neurons. Post-mortem analysis at 6 months revealed larger grafts from Day19 (D19), D22 and D25 progenitors, yet contained a higher proportion of non-DA and poorly specified (FOXA2-) cells. While D13 and D30 progenitors yielded smaller grafts. D13-derived grafts had the highest DA neuron proportion and proportionally more GIRK2+ DA neurons, the subpopulation critical for motor function. These younger progenitor grafts maintained their capacity to innervate developmentally relevant DA targets, with increased innervation capacity per DA neuron, collectively resulting in restoration of motor deficits with equal or greater proficiency than older donor cells. While donor age effects were reproducible for a given hPSC line and trends were similar between the two hPSC lines, grafts of D13 hiPSC-derived progenitors showed a 6-fold greater density of DA neurons compared to D13 hESC-derived grafts, highlighting between-line variability. These findings show that hPSC-derived VM donor age has a direct impact on graft survival, composition and maturation, and that careful assessment, on a line-to-line basis is required prior to translation.

Hypothesis: can transfer of primary neoplasm-derived extracellular vesicles and mitochondria contribute to the development of donor cell-derived hematologic neoplasms after allogeneic hematopoietic cell transplantation?

Allogeneic hematopoietic cell transplantation (allo-HCT) is an essential treatment option for various neoplastic and non-neoplastic hematologic diseases. Although its efficacy is modest, a significant proportion of patients experience relapse, graft-versus-host disease, infection or impaired hematopoiesis. Among these, the most frequent cause of post-transplant mortality is relapse, whereas the development of de novo hematologic neoplasms from donor cells after allo-HCT occurs on some occasion as a rare complication. The mechanisms involved in the pathogenesis of the de novo hematologic neoplasms from donor cells are complex, and a multifactorial process contributes to the development of this complication. Recently, extracellular vesicles, particularly exosomes, and mitochondria have been shown to play crucial roles in intercellular communication through the transfer of specific constituents, such as deoxyribonucleic acids, ribonucleic acids, lipids, metabolites and cytosolic and cell-surface proteins. Here, I discuss the potential causative roles of these subcellular components in the development of de novo hematologic neoplasms from donor cells after allo-HCT, in addition to other etiologies.

Maintenance of Hypoimmunogenic Features via Regulation of Endogenous Antigen Processing and Presentation Machinery.

Universally acceptable donor cells have been developed to address the unmet need for immunotypically matched materials for regenerative medicine. Since forced expression of hypoimmunogenic genes represses the immune response, we established universal pluripotent stem cells (PSCs) by replacing endogenous ß2-microglobulin (ß2m) with ß2m directly conjugated to human leukocyte antigen (HLA)-G, thereby simultaneously suppressing HLA-I expression and the natural killer (NK) cell-mediated immune response. These modified human PSCs retained their pluripotency and differentiation capacity; however, surface presentation of HLA-G was absent from subsequently differentiated cells, particularly cells of neural lineages, due to the downregulation of antigen processing and presentation machinery (APM) genes. Induction of APM genes by overexpression of NLR-family CARD domain-containing 5 (NLRC5) or activator subunit of nuclear factor kappa B (NF-κB) heterodimer (RelA) recovered the surface expression of HLA-G and the hypoimmunogenicity of neural cells. Our findings enhance the utility of hypoimmunogenic cells as universal donors and will contribute to the development of off-the-shelf stem-cell therapeutics.

Chromatin openness of donor cells is directly correlated with the in vitro developmental capabilities of cloned buffalo embryos.

The Switch/sucrose nonfermentable (SWI/SNF) chromatin remodelling complex is closely related to chromatin openness and gene transcriptional activity. To understand if the chromatin openness of donor cells was related to the development efficiency of somatic cell cloning embryos, two buffalo fetal fibroblasts (BFF), BFF1 and BFF3, with significantly different cloned blastocyst development rates (18.4% and 30.9% respectively), were selected in this study. The expression of SWI/SNF complex genes, chromatin openness, and transcript level of these two cell lines were analysed, and the effect of ATP on the expression of the SWI/SNF complex genes was further explored. The results showed that compared with BFF1, the expression of SWI/SNF complex family genes was higher in BFF3 at the G0/G1 phase, where SMARCC1, SMARCC2 and SMARCE1 were significantly different (p < .05). Assay of Transposase Accessible Chromatin sequencing (ATAC-seq) results showed that, at the genome-wide level, BFF3 had more open chromatin, especially which having more open chromatin peaks at SMARCA4, SMARCA2, and RBPMS2 (RNA Binding Protein, mRNA Processing Factor 2) sites. In total, 2,712 differentially expressed genes (DEGs) were identified by the RNA-Seq method, with 1380 up- and 1332 down-regulated genes in BFF3. Interestingly, the ATPase-related genes ATP1B1 and ATP11A were extreme significantly up-regulated in BFF3 (p < .01). The ATP content and the expression of SWI/SNF complex genes in both BFF1 and BFF3 decreased when treated with rotenone. The above results demonstrated that the SWI/SNF complex contributed to chromatin opening, and chromatin opening of donor cells was essential for cloned embryo development.

Lessons Learned from Donor Cell-Derived Myeloid Neoplasms: Report of Three Cases and Review of the Literature.

Donor-cell derived myeloid neoplasm (DDMN), a rare complication after allogeneic hematopoietic cell transplantation (HCT), is of interest for its potential to reveal donor-derived and host-derived factors that contribute to the pathogenesis of leukemia. The accurate diagnosis of donor-derived leukemias has been facilitated by the more frequent use of molecular techniques. In this study, we describe three additional cases of DDMN; the first reported case of donor-derived chronic myelomonocytic leukemia (CMML), one acute myeloid leukemia (AML) with t(8;21)(q22;22); RUNX1-RUNX1T1 and one donor-derived MDS with deletion 5q. A review of the cytogenetic profiles of previously reported DDMN indicates a significant contribution of therapy-related myeloid neoplasms. Cases with direct evidence of donor- or recipient-dependent factors are rare; a role of direct transfer of leukemic cells, genomic instability of the donor, abnormal gene methylation in donor cells, proleukemic potential of abnormal stromal niche, and the role of immunological surveillance after transplantation has been observed. The role of additional potential pathogenetic factors that are without clinically observed evidence are also reviewed.

The transplantation of particulated juvenile allograft cartilage and synovium for the repair of meniscal defect in a lapine model.

Background: Synovium has been confirmed to be the primary contributor to meniscal repair. Particulated Juvenile Allograft Cartilage (PJAC) has demonstrated promising clinical effect on repairing cartilage. The synergistic effect of synovium and PJAC transplant on meniscal fibrocartilaginous repair is unclear. We hypothesize that the transplantation of synovium and PJAC synergistically facilitates meniscal regeneration and the donor cells within graft tissues still survive in the regenerated tissue at the last follow up (16 weeks postoperatively). Methods: The study included 24 mature female rabbits, which were randomly divided into experimental and control groups. A cylindrical full-thickness defect measuring 2.0 â€‹mm was prepared in the avascular portion of the anterior horn of medial meniscus in both knees. The synovium and PJAC transplant were harvested from juvenile male rabbits (2 months after birth). The experimental group received synovium and PJAC transplant encapsulated with fibrin gel. The control groups received synovium transplant encapsulated with fibrin gel, pure fibrin gel and nothing. The macroscopic, imageological and histological evaluations of repaired tissue were performed at 8 weeks and 16 weeks postoperatively. The in situ hybridization (ISH) of male-specific sex-determining region Y-linked (SRY) gene was performed to detect the transplanted cells. Results: The regenerated tissue in experimental group showed superior structural integrity, superficial smoothness, and marginal integration compared to control groups at 8 weeks or 16 weeks postoperatively. More meniscus-like fibrochondrocytes filled the repaired tissue in the experimental group, and the matrix surrounding these cell clusters demonstrated strongly positive safranin O and type 2 collagen immunohistochemistry staining. By SRY gene ISH, the positive SRY signal of experimental group could be detected at 8 weeks (75.72%, median) and 16 weeks (48.69%, median). The expression of SOX9 in experimental group was the most robust, with median positive rates of 65.52% at 8 weeks and 67.55% at 16 weeks. Conclusion: The transplantation of synovium and PJAC synergistically facilitates meniscal regeneration. The donor cells survive for at least 16 weeks in the recipient. The translational potential of this article: This study highlighted the positive effect of PJAC and synovium transplant on meniscal repair. We also clarified the potential repair mechanisms reflected by the survival of donor cells and upregulated expression of meniscal fibrochondrocytes related genes. Thus, based on our study, further clinical experiments are needed to investigate synovium and PJAC transplant as a possible treatment to meniscal defects.

Effect of donor clonal hematopoiesis on allogeneic hematopoietic stem cell transplantation / 白血病·淋巴瘤

Clonal hematopoiesis (CH) refers to the clonal expansion of hematopoietic stem/progenitor cells in some individuals with normal blood indexes. The incidence of CH increases with age, reflecting the decline of the hematopoietic and potential clonal evolution to a certain extent. In recent years, an increasing number of studies have shown that donor CH is an unfavorable factor affecting transplantation, graft-versus-host disease and donor cell leukemia after allogeneic hematopoietic stem cell transplantation. Emphasis on and identification of donor CH can optimize donor selection and help transplant patients benefit more. This article introduces the relevant research progress in combination with the content of the 63rd American Society of Hematology Annual Meeting.

TDG is a pig-specific epigenetic regulator with insensitivity to H3K9 and H3K27 demethylation in nuclear transfer embryos.

Pig cloning by somatic cell nuclear transfer (SCNT) frequently undergoes incomplete epigenetic remodeling during the maternal-to-zygotic transition, which leads to a significant embryonic loss before implantation. Here, we generated the first genome-wide landscapes of histone methylation in pig SCNT embryos. Excessive H3K9me3 and H3K27me3, but not H3K4me3, were observed in the genomic regions with unfaithful embryonic genome activation and donor-cell-specific gene silencing. A combination of H3K9 demethylase KDM4A and GSK126, an inhibitor of H3K27me3 writer, were able to remove these epigenetic barriers and restore the global transcriptome in SCNT embryos. More importantly, thymine DNA glycosylase (TDG) was defined as a pig-specific epigenetic regulator for nuclear reprogramming, which was not reactivated by H3K9me3 and H3K27me3 removal. Both combined treatment and transient TDG overexpression promoted DNA demethylation and enhanced the blastocyst-forming rates of SCNT embryos, thus offering valuable methods to increase the cloning efficiency of genome-edited pigs for agricultural and biomedical purposes.

Donor cell leukemia/myelodysplastic syndrome after allogeneic stem cell transplantation: a rare phenomenon with more challenges for hematologists.

Donor cell leukemia (DCL) or Donor cell myelodysplastic syndrome (DC-MDS) is a rare type of leukemia or MDS and originates from the donor cells. It has very low frequency, but it seems to be steadily increasing as the physiopathology and mechanisms remain largely unknown. Here we report one case of acute lymphocytic leukemia (ALL) and one case of MDS of donor origin after allogeneic hematopoietic stem cell transplantation (HSCT). We also collated and reviewed literatures regarding DCL, and discuss the morbidity of DCL, the methods used to confirm donor origin and the etiology and pathogenesis of DCL. A new proposed theory will help us to better understand the mechanism for the oncogenesis of DCL.

Bladder mesenchymal stromal cell-derived exosomal miRNA-217 modulates bladder cancer cell survival through Hippo-YAP pathway.

BACKGROUND: Donor cell-derived exosomes regulate recipient cell functions. The aim of this study was to investigate the effect of human normal bladder stromal cell (hBSC) derived exosomal miR-217 on bladder cell cancer proliferation and migration. METHODS: Human BSCs were transfected with miR-217 mimic or inhibitor and hBSC-derived exosomes were isolated. Human bladder cancer cell lines (T24 and 5367) were co-cultured with hBSC-derived exosomal miR-217 mimic or inhibitor. Proliferation, migration, and apoptosis of the bladder cancer cells were assessed by Edu assay, Transwell migration assay, and Annexin V assay. RESULTS: Expression of miR-217 was significantly higher in the T24 and 5367 cell lines (P < 0.01). Exosomal miR-217 mimic enhanced proliferation and migration of T24 and 5367 cells, but inhibited apoptosis of the cells (P < 0.01); in contrast, exosomal miR-217 inhibitor suppressed proliferation and migration but stimulated apoptosis of the two cancer cell lines (P < 0.01). Moreover, exosomal miR-217 mimic stimulated YAP and its target proteins including Cyr61, CTGF, and ANKRD1 (P < 0.01), and in contrast, exosomal miR-217 inhibitor suppressed YAP and its target proteins (P < 0.01). CONCLUSION: These findings suggested that hBSC-derived exosomal miR-217 may act as oncogene in bladder cancer cells, and that Hippo-YAP signaling pathway maybe the target for miR-217 in the bladder cancer cell lines.

Donor cell memory confers a metastable state of directly converted cells.

Generation of induced oligodendrocyte progenitor cells (iOPCs) from somatic fibroblasts is a strategy for cell-based therapy of myelin diseases. However, iOPC generation is inefficient, and the resulting iOPCs exhibit limited expansion and differentiation competence. Here we overcome these limitations by transducing an optimized transcription factor combination into a permissive donor phenotype, the pericyte. Pericyte-derived iOPCs (PC-iOPCs) are stably expandable and functionally myelinogenic with high differentiation competence. Unexpectedly, however, we found that PC-iOPCs are metastable so that they can produce myelination-competent oligodendrocytes or revert to their original identity in a context-dependent fashion. Phenotypic reversion of PC-iOPCs is tightly linked to memory of their original transcriptome and epigenome. Phenotypic reversion can be disconnected from this donor cell memory effect, and in vivo myelination can eventually be achieved by transplantation of O4+ pre-oligodendrocytes. Our data show that donor cell source and memory can contribute to the fate and stability of directly converted cells.

Volunteer unrelated donor cell-derived acute myeloid leukemia with RUNX1-RUNX1T1.

A 15-year-old male was diagnosed with acute myeloid leukemia with t(6;9)(p23;q34), a chimeric DEK-NUP214 fusion gene. He underwent allogeneic bone marrow transplantation (allo-BMT) from an unrelated volunteer donor at first molecular remission. Approximately 5 years after allo-BMT, multiple bone marrow aspirations showed increased blasts to 63%, which were positive for myeloperoxidase, CD13, CD33, CD56, and CD34. Surprisingly, t(8;21)(q22;q22.1), a chimeric RUNX1-RUNX1T1 (not DEK-NUP214) fusion gene, was detected with full donor chimerism. To our best knowledge, this is the first case of a volunteer unrelated donor cell-derived acute myeloid leukemia harboring a chimeric RUNX1-RUNX1T1 fusion gene.

Donor cell-derived chronic myelomonocytic leukemia presenting after allogeneic hematopoietic cell transplantation for T-cell acute lymphoblastic leukemia.

Donor cell leukemia is a very rare cause of relapse after allogeneic hematopoietic cell transplantation (alloHCT). Herein, we describe an unprecedented case of donor cell-derived chronic myelomonocytic leukemia (CMML) presenting seven years after a 51-year-old man received a matched-related alloHCT from his 59-year-old brother for T-cell acute lymphoblastic leukemia.

Case Report: Late Onset of Myelodysplastic Syndrome From Donor Progenitor Cells After Allogeneic Stem Cell Transplantation. Which Lessons Can We Draw From the Reported Case?

BACKGROUND: Myelodysplastic syndromes and acute leukemias after allogeneic stem cell transplantation (allo-SCT) are mainly caused by recurrence of the primitive leukemic clones. More rarely, they originate from donor hematopoietic stem cells, developing the so-called donor cell leukemia (DCL) or myelodysplastic syndromes (DC-MDSs). DCL and DC-MDS can be considered as an in vivo model of leukemogenesis, and even if the pathogenetic mechanisms remain speculative, a genetic predisposition of donor progenitor cells, an altered host microenvironment, and the impairment of immune surveillance are considered the main causes. CASE PRESENTATION: We report a case of DC-MDS diagnosed 5 years after an allo-SCT from a matched related donor (patient's sister) in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). The sex-mismatch allowed us to identify the donor cell origin. At the onset, the DC-MDS was characterized by chromosome seven monosomy and NRAS, RUNX1, and BCOR mutations. Because of a familiar history of colorectal neoplasia and the variant allele frequency (VAF) of NRAS mutation at the onset, this mutation was searched on germline DNA in both the donor and the recipient, but the result was negative. Moreover, after transplant (+4 months), the patient developed severe and long-lasting chronic graft-versus-host disease (cGVHD), requiring multiple lines of treatments. Because of the severe immunosuppression, recurrent infections occurred and, lately, the patient died due to septic shock. CONCLUSION: This case report highlights the need, whenever possible, to evaluate the donor origin of the posttransplant myelodysplasia and acute leukemias. The potential key role of the impaired immune surveillance and of long-lasting immunosuppression appears to be emerging in the development of this case of DC-MDS. Finally, this case reminds the importance to investigate the familiar genetic predisposition in donors with a familiar history of neoplasia.

Transcriptome heterogeneity of porcine ear fibroblast and its potential influence on embryo development in nuclear transplantation.

There is heterogeneity among donor cells of the same source. Many studies have shown that donor cell affects the efficiency of somatic cell nuclear transfer (SCNT). However, the potential influence of donor cell heterogeneity on the efficiency of nuclear transplantation were rarely analyzed at the single-cell level. In this study, single-cell transcriptome sequencing was performed on 52 porcine ear fibroblasts randomly selected from the same source to compare their gene expression patterns. The results showed that 48 cells had similar gene expression patterns, whereas 4 cells (D11_1, D12_1, DW61_2, DW99_2) had significantly different gene expression patterns from those of other cells. There were no two cells with identical gene expression patterns. The gene expression patterns of D11_1, D12_1, DW61_2 and DW99_2 were analyzed, using the 48 cells with similar gene expression patterns as controls. Firstly, we used the R language statistics to select the differentially expressed genes in the 4 single cells, and identified the top 50 most significant differentially expressed genes. Then GO enrichment analysis and KEGG pathway analysis were performed on the differentially expressed genes. Enrichment analysis revealed that the main molecular functions of the differentially expressed genes included energy metabolism, protein metabolism and cell response to stimulation. The main pathways from KEGG enrichment were related to cell cycle, cell metabolism, and DNA replication. Finally, based on the above results and in consideration with the SCNT research progress, we discussed the potential effects of differential gene expression patterns of the 4 single cells on the embryonic development efficiency of nuclear transplantation. This study revealed transcriptional heterogeneity of porcine ear tissue fibroblasts and provided an effective method to analyze elite donor cells, thereby providing new ideas on improving the cloning efficiency of SCNT.